Search

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.

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.

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.

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.

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.

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.

Specific cell-mediated cytotoxicity in fish was investigated using both iso-geneic (clonal) ginbuna crucian carp,Carassius auratus langsdorfii, and three cell lines established from the three different clones of this fish. Specific cell-mediated cytotoxicity in ginbuna was induced as a result of intravenous immunisation with an allogenic cell line, but not a syngeneic cell line. As the cytotoxic activity tended to accelerate in proportion to the number of immunisations, specific cytotoxic cells in ginbuna resembled mammalian cytotoxic T lymphocytes (CTL) in some respects. In addition, it was unlikely that antibody-dependent cell-mediated cytotoxicity (ADCC) was involved in the cytotoxicity observed in this study because ADCC activity of unimmunised fish effector cells was not detected using the immunised ginbuna plasma or the supernatant of cultured effector cells from immunised ginbuna. These results demonstrate that specific cell-mediated cytotoxicity is inherent in fish and a set of clonal fish and their cell lines may be useful for further studyin vivoandin vitro.

By utilizing non-destructive synchrotron radiation-excited X-ray fluorescence (SR-XRF), we observed the distribution of lead (Pb) in both ontogenic and regenerating scales of lead-administrated carp, Cyprinus carpio. The fish in the Pb-administered group were fed pellets containing 1 mg/g of Pb at a rate of 1.5% body weight per day for 30 days. In the ontogenic scales, Pb was highly accumulated near the basal edge of the scale and the accumulated amount decreased toward the focus of the scale. On the other hand, in the regenerating scales, high accumulation was observed near the basal edge and the accumulated amount remained high toward the focus. The present results of Pb accumulation correspond well with the region which is calcifying in the ontogenic and regenerating scales, and indicate that the distributions of Pb show when and how long Pb was administered.

A monoclonal antibody (MAb; TCL-BE8) for carp peripheral blood leucocytes (PBL) was produced, and its reactivity was analysed by electron microscopy and flow cytometry. Electron microscopy using immunogold labelling showed that this MAb reacted with neutrophils and monocytes. The antibody reacted with 98% of the cells in a fraction of granulocytes separated by flow cytometry. The antigen detected by this was MAb defined as a membrane protein of Mr of 112 kDa. Using a magnetic separator, cells reactive with this antibody were separated from leucocytes with a density of 1·08 g ml −1 or 1·08–1·09 g ml −1 . MAb-positive cells in the PBL with a density of 1·08 g ml −1 consisted of a mixture of neutrophils and monocytes, whilst in PBL with a density of 1·09 g ml −1 they consisted of only neutrophils. The MAb-negative fractions contained mainly lymphocytes and thrombocytes. The MAb-positive cells showed strong phagocytosis, which is a characteristic function of neutrophils and monocytes, but this was absent from the MAb-negative fraction. Purification of monocytes and neutrophils, or isolation of neutro-phils, can be achieved by using this MAb allowing more effective analysis of the carp leucocyte functions.

A monoclonal antibody against carp peripheral blood leucocytes was produced, and its reactivity analysed by flow cytometry and electron microscopy. The antibody reacted positively with 10-35% of the cells in a fraction of lymphocytes and thrombocytes that was separated by flow cytometry. Electron microscopy using immunogold labelling showed that this antibody reacted strongly with thrombocytes, but not with other leucocytes. By using a magnetic separator, leucocytes that were positive and negative for this antibody were separated. The positive cells were uniform, spindle-type cells that aggregated in the presence of collagen, while the negative cells did not aggregate. Light and electron microscopy showed that many positive cells changed to a spherical form after the addition of collagen and then 40-60% of these cells aggregated.