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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.

Abalone amyotrophia is a viral disease that causes mass mortality of juvenile Haliotis discus and H. madaka. Although the cause of this disease has yet to be identified, we had previously postulated a novel virus with partial genome sequence similarity to that of African swine fever virus is the causative agent and proposed abalone asfa-like virus (AbALV) as a provisional name. In this study, three species of juvenile abalone (H. gigantea, H. discus discus, and H. diversicolor) and four species of adult abalone (the above three species plus H. discus hannai) were experimentally infected, and their susceptibility to AbALV was investigated by recording mortality, quantitatively determining viral load by PCR, and conducting immunohistological studies. In the infection test using 7-month-old animals, H. gigantea, which was previously reported to be insusceptible to the disease, showed multiplication of the virus to the same extent as in H. discus discus, resulting in mass mortality. H. discus discus at 7 months old showed abnormal cell masses, notches in the edge of the shell and brown pigmentation inside of the shell, which are histopathological and external features of this disease, while H. gigantea did not show any of these characteristics despite suffering high mortality. Adult abalones had low mortality and viral replication in all species, however, all three species, except H. diversicolor, became carriers of the virus. In immunohistological observations, cells positive for viral antigens were detected predominantly in the gills of juvenile H. discus discus and H. gigantea, and mass mortality was observed in these species. In H. diversicolor, neither juvenile nor adult mortality from infection occurred, and the AbALV genome was not increased by experimental infection through cohabitation or injection. Our results suggest that H. gigantea, H. discus discus and H. discus hannai are susceptible to AbALV, while H. diversicolor is not. These results confirmed that AbALV is the etiological agent of abalone amyotrophia.

Aquaculture is an important industry in Japan for the sustainable production of fish. It contributes to the diversity of Japanese traditional food culture, which uses fish such as "sushi" and "sashimi". In the recent aquaculture setting in Japan, infectious diseases have been an unavoidable problem and have caused serious economic losses. Therefore, there is an urgent need to overcome the disease problem to increase the productivity of aquaculture. Although our country has developed various effective vaccines against fish pathogens, which have contributed to disease prevention on fish farms, infectious diseases that cannot be controlled by conventional inactivated vaccines are still a problem. Therefore, other approaches to developing effective vaccines other than inactivated vaccines are required. This review introduces the vaccine used in Japan within the context of the current status of finfish aquacultural production and disease problems. This review also summarizes the current research into vaccine development and discusses the future perspectives of fish vaccines, focusing on the problems associated with vaccine promotion in Japan.

The Gillespie algorithm, which is a stochastic numerical simulation of continuous-time Markovian processes, has been proposed for simulating epidemic dynamics. In the present study, using the Gillespie-based epidemic model, we focused on each single trajectory by the stochastic simulation to infer the probability of controlling an epidemic by non-pharmaceutical interventions (NPIs). The single trajectory analysis by the stochastic simulation suggested that a few infected people sometimes dissipated spontaneously without spreading of infection. The outbreak probability was affected by basic reproductive number but not by infectious duration and susceptible population size. A comparative analysis suggested that the mean trajectory by the stochastic simulation has equivalent dynamics to a conventional deterministic model in terms of epidemic forecasting. The probability of outbreak containment by NPIs was inferred by trajectories derived from 1000 Monte Carlo simulation trials using model parameters assuming COVID-19 epidemic. The model-based analysis indicated that complete containment of the disease could be achieved by short-duration NPIs if performed early after the import of infected individuals. Under the correctness of the model assumptions, analysis of each trajectory by Gillespie-based stochastic model would provide a unique and valuable output such as the probabilities of outbreak containment by NPIs.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.

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.

A mass mortality of cultured kuruma prawn Penaeus japonicus occurred in Kagoshima Prefecture in June, 2005. Affected prawns displayed a sluggish behavior or swimming on their side. The mean water temperature during the disease outbreak was 27°C. A black pigment-producing bacterium was isolated from the diseased prawns and was identified as Vibrio nigripulchritudo by bacteriological tests, 16S rDNA sequence analysis and DNA array for discrimination of Vibrio species. High pathogenicity of the isolated bacterium to healthy kuruma prawn was confirmed by intramuscular injection. This is the first report of V. nigripulchritudo infection in kuruma prawn.

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.

Mass mortalities of the pen shell Atrina pectinata occurred in the fishing grounds of Ariake Bay, in southwestern Japan, during late spring and summer in 2003 and 2004. Histological examination revealed extensive necrosis in the epithelial cells of the kidney and gill, and impairment of the endothelial cells of the mantle arteria. Although cestode larvae belonging to the genus Tylocephalum were found in the mantle, adductor muscle, kidney, and digestive gland, their prevalence and the intensity of infection were low. Examinations of moribund pen shells for Haplosporidium spp. infection using PCR analysis and for Perkinsus spp. infection using Ray's fluid thioglycollate medium were negative. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of affected kidney and gill cells of moribund pen shells. They were icosahedral spherical and 50 to 55 nm in diameter. These virus-like particles found in moribund pen shells are different from those described in other marine mollusks, and may be the causative agent of the mass mortalities of pen shells.

Bacteriological examination was conducted on the disease of cultured marine fish, three-line grunt Parapristipoma trilineatum (Japanese common name: isaki). The affected fish characteristically showed white spots of granulomas containing an intracellular bacterium in the kidney and spleen. Total DNA was extracted from the kidney of the diseased fish. The coding region of small subunit ribosomal RNA (16S rDNA) of eubacteria was amplified from the DNA by PCR, and nucleotide sequence was determined. The sequenced 16S rDNA revealed high similarity (97.3-98.5%) to those of Francisella species. On the basis of phylogenetic analysis, the closest relative organism was revealed to be Francisella philomiragia. A bacterium was isolated from the spleen of one affected fish using cystine heart agar medium with 1% hemoglobin. The 16S rDNA sequence of the isolated bacterium was consistent with that determined from the kidney tissue of the affected fish. An experimental infection with the isolate exhibited the same disease signs in the injected fish, and the bacterium was reisolated from these fish. These results demonstrate that the intracellular bacterium is pathogenic and is identified to the genus Francisella This is the first report of pathogenic Francisella from marine fish.

The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances.

Edwardsiella tarda causes high mortality infections in fish. To determine the efficacy of a vaccine against E. tarda, a formalin-killed preparation of atypical E. tarda FPC503 was intraperitoneally injected into red sea bream Pagrus major with or without the oil adjuvant, Montanide ISA 763 A VG. Both vaccine regimens showed protective efficacy against experimental infection with E. tarda FPC503 at 28 and 56 days post-vaccination. An enzyme-linked immunosorbent assay (ELISA) revealed the specific antibody production in vaccinated fish but the relationship between protective efficacy and antibody titer was not clear. The present results indicate that the formalin-killed vaccine is useful for the prevention of atypical E. tarda infection in red sea bream.

Protective efficacies of three antigenic proteins (3-hydroxyacyl-CoA dehydrogenase (HCD), ATP synthase beta subunit (atpD), and glutamate dehydrogenase (gdhA)) against Flavobacterium psychrophilum were investigated in ayu (Plecoglossus altivelis). Recombinant proteins of HCD, atpD, and gdhA were expressed in Escherichia coli BL21 cells. Ayu were then vaccinated with inactivated cells via the intraperitoneal route. Compared with the empty BL21- and PBS-injected groups, the vaccinated group had a significantly longer survival time after challenge with F. psychrophilum. The antibody titers against each recombinant protein were significantly higher in serum from vaccinated fish, compared with serum from control fish. Results of indirect immunofluorescence assays using serum indicated that the HCD, atpD, and gdhA proteins are located on the surface of F. psychrophilum. These results suggest that these three surface proteins are protective antigens and are good candidates for development of vaccines against bacterial cold-water disease in ayu.

Kinetics of vascular permeability was determined by measuring the amount of Evans blue leaked into the tilapia mast cell (tMC)-lysate injection site. Injection with tMC lysate enhanced the vascular permeability. The response consisted of three distinct phases, the first started immediately after the injection, the second started at about 2 h, reaching its maximum at 4 h, and the third response started at 12 h and continued for more than 24 h. Heating of the tMC lysate at 100 degrees C for 10 min had no effect on the first response, while the second response was significantly reduced by heating at 50 degrees C for 10 min. The tMCs seem to have two kinds of factors that enhance vascular permeability. The tMC lysate induced Ca2+ uptake by cultured tilapia endothelial cells, indicating that tMC products directly activate the endothelial cells and increase vascular permeability similar to products of mammalian mast cells. These results indicate that with respect to influence on vascular permeability tilapia mast cells resemble the mast cells of mammals.

Mass mortality occurred in Manila clam Ruditapes philippinarum under a hanging cultivation condition from April to July 2008 in Japan. A bacterium was isolated from the affected clams showing brown conchiolin deposits on the inner shell surface and identified as Vibrio tapetis, the causal agent of brown ring disease (BRD) in clams, by bacteriological characterization and genetic analysis. Experimental infection of clams confirmed pathogenicity of the isolated bacterium. These results indicate that the present mortality of cultured clams was caused by BRD. This is the first report on isolation of V. tapetis from diseased R. philippinarum in Japan.