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Interferon (IFN)γ is a pivotal cytokine that promotes and orchestrates innate cellular and adaptive cell-mediated immunity against intracellular pathogens. The capacity of T cells in mammals to produce IFNγ has been measured using specific antibodies in order to analyze cell-mediated immune responses against infection or immuno-stimulants. In fish, however, measurement of IFNγ protein levels has not been possible due to a lack of research tools. In the present study, therefore, we established antibodies that react with endogenous amberjack IFNγ. An enzyme-linked immunosorbent assay (ELISA) for IFNγ in amberjack species was developed using these antibodies. The ELISA could detect endogenous IFNγ at concentrations less than 100 pg/mL in PMA/ionomycin-stimulated leukocytes culture supernatant. IFNγ production was enhanced and lasted a long time following intracellular bacterial infection with Nocardia seriolae, which is thought to be targeted by cell-mediated immunity. These results demonstrate that quantification of IFNγ using the reported ELISA can be used to estimate the status of cell-mediated immunity in amberjack species.

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

We report the draft genome sequence of a novel member of the order Picornavirales that was obtained from the gills of farmed Japanese eel (Anguilla japonica). A putative polyprotein encoded by the genome was similar to that of other picornaviruses and shared 31% amino acid identity with eel picornavirus 1., We report the draft genome sequence of a novel member of the order Picornavirales that was obtained from the gills of farmed Japanese eel (Anguilla japonica). A putative polyprotein encoded by the genome was similar to that of other picornaviruses and shared 31% amino acid identity with that of eel picornavirus 1.

We report the draft genome sequence of a novel member of the order Picornavirales that was obtained from the gills of farmed Japanese eel ( Anguilla japonica ). A putative polyprotein encoded by the genome was similar to that of other picornaviruses and shared 31% amino acid identity with eel picornavirus 1.

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

The market for Pacific bluefin tuna (Thunnus orientalis) is expanding as global consumption increases. Meeting this demand has led to overfishing and consequent depletion of wild populations, resulting in a focus on aquaculture as an alternative to fishing. However, under the crowded conditions typical of tuna aquaculture settings, infectious diseases are a common problem associated with considerable economic losses, especially with regard to Pacific bluefin tuna, which is a high-priced species. Studying diseases in Pacific bluefin tuna requires techniques that enable experimental infection; however, no promising methods are available due to the difficulty of handling these fish, including transportation, retention, and injection. In the present study, therefore, we developed a method for experimental infection of juvenile Pacific bluefin tuna with red seabream iridovirus (RSIV), which causes an acute and highly contagious disease in this species. The route of experimental infection was validated using Pacific bluefin tuna and greater amberjack (Seriola dumerili), which is used as a model species susceptible to the disease. Oral infection with the virus was found to be suitable for reproducing red seabream iridoviral disease (RSIVD). A high copy number of the RSIV genome was detected in Pacific bluefin tuna that died following RSIV experimental infection, indicating that the virus efficiently replicated in the infected fish. In addition to virus detection, a histopathologic abnormality specific to RSIV infection was detected in the infected fish. These results indicate that experimental infection via the oral route is effective for reproducing RSIVD in Pacific bluefin tuna.

Coho salmon (Oncorhynchus kisutch) is a Pacific salmon species that is commercially farmed in Japan, and is susceptible to erythrocyte inclusion body syndrome (EIBS), which is characterized by anemia, erythrocyte inclusion bodies, and cardiovascular muscle necrosis. At present, no vaccine for this disease is available. Piscine orthoreovirus-2 (PRV-2) is the causative agent of EIBS in Japan, and is one of three genotypes of PRV that induce the formation of erythrocyte inclusion bodies. However, due to a deficiency in cell lines able to culture PRV-2, the development of vaccines for diseases caused by PRV-2 has been difficult. A recent strategy involved the combination of a DNA vaccine, a σ1, and a non-structural protein gene for moderate PRV-1 protection. In the present study, analysis of antiserum obtained from PRV-2 infection survivors identified σ1 as a PRV-2 antigenic protein. Since a DNA vaccine incorporated with only σ1 did not prove efficacious against PRV-2 infection in a preliminary study, the present study tested the enhanced protective effect of the DNA vaccine with various sequences linked to the σ1 gene. Antigenic PRV-2 gene products were first screened using recombinant PRV-2 proteins and serum from coho salmon with a history of EIBS. Six vaccine groups of DNA vaccines were then created by the linkage of several sequences to the PRV-2 gene. Two trials were conducted to identify effective vaccines and a third vaccination trial confirmed the reproducibility of an effective vaccine. Trial I showed insignificant differences in the mean antibody level after 42 days. However, an outlier test indicated significantly higher antibody levels in coho salmon vaccinated in signal-linked σ1 gene DNA vaccine group and chaperone-linked σ1 gene DNA vaccine group. Trial II evaluated the effects of individual DNA vaccines and showed that individuals in the Sec-σ1, TF-σ1, and mixed vaccine group had significantly higher antibody levels and significantly lower virus loads. Trial III confirmed the suppression of viral replication and increase in antibody levels of the TF-σ1 DNA vaccine. However, this DNA vaccine was only effective for increasing antibody levels and inhibiting viral growth in some individuals, indicating the necessity for further experimentation involving analyses of the effect of individual differences on the efficacy of the vaccine.

A novel Gram-stain-negative, rod-shaped (0.3 × 4–6 μm), non-flagellated, aerobic strain with gliding motility, designated JBKA-6T, was isolated in 1991 from a yellowtail fish, Seriola quinqueradiata, showing symptoms of bacterial haemolytic jaundice. 16S rRNA gene sequence analysis showed that strain JBKA-6T was related most closely to members of the family Flavobacteriaceae in the phylum ‘Bacteroidetes’. Furthermore, based on gyrB gene sequence analysis, JBKA-6T was classified into a single clade within the order Flavobacteriales, which was distinct from the known clades of the families Flavobacteriaceae, Blattabacteriaceae and Cryomorphaceae. The predominant isoprenoid quinone was identified as MK-6 (97.9 %), and the major cellular fatty acids (>10 %) were C14 : 0 and iso-C15 : 0. The main polar lipids were phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. The DNA G+C content of JBKA-6T, as derived from its whole genome, was 33.4 mol%. The distinct phylogenetic position and phenotypic traits of strain JBKA-6T distinguish it from all other described species of the phylum ‘Bacteroidetes’, and therefore it was concluded that strain JBKA-6T represents a new member of the phylum ‘Bacteroidetes’, and the name Ichthyobacterium seriolicida gen. nov., sp. nov. is proposed. The type strain of Ichthyobacterium seriolicida is JBKA-6T ( = ATCC BAA-2465T = JCM 18228T). We also propose that Icthyobacterium gen. nov. is the type genus of a novel family, Ichthyobacteriaceae fam. nov.

Ichthyobacterium seriolicida is the causative agent of bacterial hemolytic jaundice (BHJ) in Japanese amberjack, Seriola quinqueradiata. Fish recovering from BHJ acquire protective immunity against reinfection. In this study, fish were passively immunized to determine whether serum antibody is involved in protection against BHJ. The susceptibility of I. seriolicida to the bactericidal activity of Japanese amberjack serum was also investigated. In passive immunization tests, significantly lower mortality was noted in fish that received convalescent serum. Bacteria were killed when exposed to convalescent serum but not serum from naive fish. Electron microscopic analyses showed that I. seriolicida cells were morphologically altered by reaction with convalescent serum. Naive fish serum became bactericidal upon addition of purified IgM from convalescent serum. Involvement of the classical complement pathway in the bactericidal mechanism was confirmed because bactericidal activity was lost upon heating convalescent serum or chelation treatment using EDTA. Convalescent fish serum thus protects against reinfection by I. seriolicida via humoral immunity mediated by activation of the classical complement pathway.

Partial sequences of cytokine genes IFNγ, IL-1β, IL-8, IL-10, and TNFα were identified from next-generation sequencing data for three amberjack species: Seriola quinqueradiata, S. lalandi and S. dumerili. The cytokine genes had more than 90 % nucleotide identity among the three species. Quantitative PCR (qPCR) primers were designed from the consensus sequences to detect cytokine genes. The specificities of consensus qPCR primers were investigated in the head kidney of three amberjack species treated with formalin-killed cells (FKC) of Vibrio anguillarum and polyinosinic-polycytidylic acid [poly(I:C)] at 3 h post injection (hpi) using qPCR. The primers detected up-regulation of certain cytokine genes after FKC of V. anguillarum and poly(I:C) stimulation in all species examined. Consequently, the primers are suitable for studying and detecting cytokine genes after vaccination and pathogen infection in these three amberjack species.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Although vaccine trials with formalin-killed cells (FKC) have been reported, the vaccinations failed in protect against E. tarda infection. On the other hand, a live attenuated vaccine strategy is effective against edwardsiellosis; however, the mechanism underlying its effectiveness in fish is unclear. In the present study, we compared the adaptive immune responses in fish vaccinated with FKCs and live attenuated vaccines to elucidate the induction of adaptive immune responses following vaccination. After challenge with E. tarda, live cell (LC)-vaccinated fish showed high survival rates, high IFN-g and T-bet gene expression levels, and increased cytotoxic T lymphocytes (CTLs). In contrast, all FKC-vaccinated fish died following E. tarda infection. In addition, FKC vaccination induced high IL-4/13A and IL-10 expression levels and increased antibody titers, whereas Th1-like responses were suppressed. These results indicate that LC vaccination contributes to protection against E. tarda infection by inducing cell-mediated immunity (CMI). Thus our study findings could contribute to the development a vaccine that induces CMI against edwardsiellosis.

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents.

Ichthyobacterium seriolicida is a fish bacterial pathogen that causes hemolytic jaundice in farmed yellowtail in Japan. To understand more about the characteristics of this bacterium, we determined its complete genome sequence. Two hemolysin genes which may be important for its pathogenicity were identified in the I. seriolicida genome.

Ichthyobacterium seriolicida is a fish bacterial pathogen that causes hemolytic jaundice in farmed yellowtail in Japan. To understand more about the characteristics of this bacterium, we determined its complete genome sequence. Two hemolysin genes which may be important for its pathogenicity were identified in the I. seriolicida genome.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis altivelis and is responsible for substantial economic losses in ayu culture in Japan. To develop effective vaccines for the disease, we identified antigenic proteins of F. psychrophilum using immunoglobulin from ayu that had recovered from BCWD. The whole protein extracted from the bacterium was separated using 2-dimensional polyacrylamide gel electrophoresis and was transferred to a polyvinylidene fluoride membrane. Subsequently, antigenic proteins of the bacterium were detected using western blotting and ayu antisera against F. psychrophilum. Each protein spot showing antigenicity was subjected to tandem mass spectrometry (MS/MS) analysis using a MALDI-QIT-TOF mass spectrometer. Protein identification based on the MS/MS data was performed using the genome database for F. psychrophilum JIP02/86, and the subcellular localization for each identified protein was predicted with web-based tools (LipoP and PSORTb). In total, 62 antigenic proteins were identified: of these, 46 were putative cytoplasmic proteins (e.g. elongation factor Tu and heat shock protein 90). The remaining 21 proteins were identified as putative membrane-bound or secreted proteins and are potential vaccine candidates. These proteins include OmpA, Omp 121, M13 family metallopeptidase, and M48 family metalloprotease.

Since the 1990s, Akoya oyster disease (AOD) associated with reddish browning of the soft tissues and mortality of cultured pearl oyster, Pinctada fucata (Gould), has been occurring in the western part of Japan. Although transmission experiments suggested that the disease is infectious, the causative agent has not been identified. Previous studies suggested that a member of the Spirochaeta cause AOD, and Candidatus Maribrachyspira akoyae was proposed as the putative causative agent. However, difficulties in culturing the putative causal bacterium have hindered research on AOD. In this study, we developed a quantitative PCR assay to examine the spatial-temporal colonization patterns of Spirochaeta in pearl oysters. Spirochete genes were abundant in the mantle, adductor muscle, and hemolymph of oysters but scarce in the gill, crystalline style, hemocytes, heart, and digestive glands. Spirochete genes were detected in the hemolymph 2 weeks after infection, and their number increased with time and mortality. The Spirochaeta genes identified in the hemolymph of pearl oysters reared in culturing fields changed with variations in water temperature, and the number of Spirochaeta genes increased or decreased at approximately 20 °C. Hence, the bacterium was thought to grow at water temperatures above 20 °C. These results are consistent with those of infection tests and epidemiologic studies of the distribution and seasonal changes in AOD pathogens and support the hypothesis that a spirochete causes AOD.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Although cell-mediated immunity and innate immunity play a major role in protection against intracellular bacterial infection in mammals, their importance in protecting fish against E. tarda infection remain unclear. In this study, we examined cell-mediated and humoral immune responses in ginbuna crucian carp (Carassius auratus langsdorfii) after E. tarda infection. Innate immunity was observed to be the principal immune system for eliminating the majority of E. tarda, while a proportion of the bacteria might be resistant to its bactericidal activity. Bacterial clearance in kidney and spleen was also observed following higher cytotoxic activities of cytotoxic T lymphocytes (CTLs) and increased numbers of CD8α(+) cells, suggesting that CTLs might contribute to the elimination of E. tarda-infected cells with specific cytotoxicity. On the other hand, E. tarda-specific antibody titers did not increase until after bacterial clearance, indicating that induction of humoral immunity would be too late to provide protection against infection. Overall, these data suggest that both cell-mediated immunity and innate immunity may play important roles in the protection against intracellular bacterial infection, as they do in mammals. Our study would also contribute toward the understanding of immune responses that provide protection against other intracellular pathogens.

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain.

Enteric redmouth disease (ERM), which is caused by Yersinia ruckeri, has not been reported in Japan. In the present study, virulence of the bacterium were assessed by experimental chal­ lenges with the rainbow trout Oncorhynchus mykiss and 4 indigenous fish species in Japan: yamame (masu salmon parr) O. masou masou, amago O. masou macrostomus, whitespotted char Salvelinus leucomaenis and ayu Plecoglossus altivelis. When intraperitoneally (ip) injected with the bacterium at a dose of 7.1 x 10 2 CFU/g fish body weight (BW), rainbow trout, char, yamame, and amago showed the cumulative mortalities of 100%, 60%, 30% and 30%, respectively. The cumulative mortalities of ip­ injected ayu with doses of 1.5 x 10 6 , 1.5 X 10 5 , 1.5 X 10 4 and 1.5 x 10 3 CFU/g BW were 87%, 47%, 13% and 0%, respectively. In addition, the cumulative mortalities of ayu immersed in water containing 1.7 x 10 8 and 1.7 x 10 7 CFU/mL of the bacterium were 60% and 6.7%, respectively. Typical clinical signs of ERM, such as reddening of the wall of the oral cavity, tongue, upper jaw, lower jaw, operculum, bases of fins, or skin around the anus, were observed in all dead salmonids but not in ayu. Instead, exophthal­ mos, hemorrhage in the eyes, ascites or hypertrophy of the kidney were often observed in dead ayu. These results indicate that Yersinia ruckeri is a potentially dangerous bacterium to freshwater aquacul­ ture in Japan.

In this study, we aimed to identify the leukocyte population that expresses Fas ligand (FasL) in the Japanese flounder (Paralichthys olivaceus). The transcriptional activity of FasL was examined for the first time in the fish leukocytes. Transcription of the FasL gene in flounder leukocytes was significantly increased by phytohemagglutinin (PHA) treatment. All the leukocyte populations we tested possessed binding activity for PHA, but this was especially high in the lymphocyte population. However, the lymphocytes consisted of two subsets showing heterogeneity with respect to PHA binding, with the high-binding subset being surface IgM-negative. We also found that only the lymphocyte population showed a significant increase in the expression of the FasL gene after stimulation with PHA. In addition, only the lymphocyte subset showing high binding to PHA showed conspicuous expression of the FasL gene. This subset also had a CD3γ/δ+, CD8α+ and IgM heavy-chain (-) phenotype. These results suggested that lymphocytes with T-cell-like properties are FasL-expressing cells in the Japanese flounder.

A recombinant fimbrial protein of Edwardsiella tarda was designed to detect the specific antibody in Japanese flounder Paralichthys olivaceus. The specific antibody was detected from the fish intraperitoneally injected with E. tarda expressing the fimbria on LB agar containing 3% NaCl, but not from those injected with E. tarda without the fimbria, Streptococcus iniae, S. parauberis or viral hemorrhagic septicemia virus. In a flounder farm suffered with edwardsiellosis, the antibody-positive fish increased in ration with the occurrence of mortality. These results suggested that E. tarda with the fimbria was important for epidemics of edwardsiellosis at flounder farms.

We evaluated the prevalence of the akoya oyster disease (AOD) in Japan and the pathogenicity of the unidentified causative agent. We obtained naive Japanese pearl oysters Pinctada fucata martensii from a site with no history of AOD. In separate experiments, the oysters were either challenged by intramuscular injection with the hemolymph of infected oysters or reared in several major pearl oyster farms throughout Japan. Both groups of oysters experienced high mortality and exhibited discoloration of the adductor muscle and histopathological changes characterizing AOD. Our data suggest that AOD is prevalent in pearl oyster culture fields throughout Japan and that the causative pathogen remains highly pathogenic to P. fucata martensii. When groups of oysters produced by repeated selective breeding against AOD were reared in different pearl oyster fields alongside uninfected naive oysters, the selectively bred oysters had significantly lower mortality than naive oysters. The recent decline in outbreaks of AOD can be attributed, in part, to the introduction of selectively bred oysters that are resistant to this disease.

We evaluated the tissue persistence and live vaccine efficacy of five avirulent Edwardsiella tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1β (IL-1β), type 1 interferon (IFN), and IFN-γ in head-kidney of the Japanese flounder.

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.

The role of leukocytes from the kidney of Japanese flounder Paralichthys olivaceus in the encapsulation response was investigated using an in vitro model. The aggregation response of leukocytes to the target fish pathogen Ichthyophonus hoferi was observed over 12 h. The cellular aggregates mainly comprised granulocytes, which were characterized by intracellular peroxidase expression. Moreover, studies of isolated granulocytes demonstrated their adhesion to I. hoferi and formation of an initial layer in the encapsulation response. We observed that mRNA expression levels of three CC-chemokines (CCL3, CCL4 and CC-CLM) and one CXC-chemokine (IL-8) increased during the encapsulation process. Among these chemokines, CC-CLM and IL-8 were actively produced by granulocytes upon stimulation by I. hoferi. Flounder granulocytes therefore appeared to play an important role in the process by releasing specific chemokines upon pathogen recognition, thereby inducing subsequent cellular recruitment leading to encapsulation.

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Ig+ leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5–7.4, 23.7–50.1, 25.2–26.1 and 5.2–8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5–36.3, 1.9–2.8, 6.4–8.3 and 1.9–3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.

Viral hemorrhagic septicemia virus (VHSV) infection has been one of the major obstacles for Japanese flounder Paralichthys olivaceus culture both in inland tank systems and open sea net-pens in Japan. The mortality due to the disease in cultured Japanese flounder occurs in the cold-water season at temperatures mostly below 15 °C. We examined the effect on the disease by shifting the rearing-water temperature to a higher temperature (20 °C) as a potential control measure. Japanese flounder reared in aquariums were infected with a VHSV isolate JF00Ehi1, and the water temperature was shifted from 14 °C to 20 °C (Experiment I) and from 20 °C to 15 °C (Experiment II). In Experiment I, earlier shifting resulted in lower cumulative mortality rates. In Experiment II, the group of fish reared for a longer period at 20 °C showed lower cumulative mortalities. In a third set of experiments, fish inoculated with the virus were reared at 20 °C or at 25 °C (a viral non-permissive temperature), observing no mortality in either group. Viral multiplication was detected at a low level in the fish at 20 °C but not in those at 25 °C. At 21 dpi, these fish were challenged with the virus and reared at 14 °C, yielding no mortality in the 20 °C group, and 92% in the 25 °C group. These results indicate that rearing at 20 °C reduced mortality in the Japanese flounder experimentally infected with VHSV, probably inducing protective immune response against subsequent infection.

Teleost IL-4/13B is a cytokine related to mammalian IL-4 and IL-13, of which hitherto the function had not been studied at the protein level. We identified an IL-4/13B gene in common carp (Cyprinus carpio) and expressed the recombinant protein (rcIL-4/13B). RcIL-4/13B was shown to stimulate proliferation of IgM(+) B cells, because after four days of stimulation the IgM(+) fraction of carp kidney and spleen leukocytes had formed many cell colonies, whereas such colonies were not found in the absence of rcIL-4/13B stimulation. After nine days of incubation with rcIL-4/13B these cells had proliferated to more than 3-to-7-fold higher numbers when compared to untreated cells. The proliferating cells contained a majority of IgM(+) cells but also other cells, as indicated by FACS and RT-PCR analyses. The important conclusion is that in fish not only IL-4/13A has B cell stimulating properties, as a previous publication has shown, but also IL-4/13B.

Ayu Plecoglossus altivelis altivelis are one of the most economically important fish for freshwater aquaculture in Japan. We conducted expressed sequence tag analyses of three leukocyte subpopulations, thrombocytes, neutrophils, and B lymphocytes in ayu using a next generation sequencer. The sequencing and de novo assembly yielded 22,494, 22,733, and 16,505 contigs from the thrombocyte, neutrophil, and B lymphocyte cDNA libraries, respectively. Pathways involving endocytosis, phagosomes, and lysosomes, were found in all three cDNA libraries using pathway analysis. The thrombocyte cDNA library contained 2894 unique sequences, including CXC chemokine receptor 4 and MHC class II. Cytokine and cytokine receptor genes such as interleukin (IL)-1β, IL-8, IL-1 receptor (IL-1R), IL-8RA, and IL-8RB were found among the 3056 unique sequences of the neutrophil cDNA library. Typical B lymphocyte related genes such as B cell linker protein, immunoglobulin (Ig) M, IgD and transforming growth factor β were found in the 1590 unique sequences of the B lymphocyte cDNA library. In summary, a large number of immune-related genes were identified from the three leukocyte cDNA libraries. Our results represent a valuable sequence resource for understanding the immune system function in ayu.