倉田 修

INTRODUCTION: Temporal elevation of water temperature positively affects immune activity and disease resistance in poikilothermic teleost fish. The ayu, Plecoglossus altivelis, an important fish species for Japanese freshwater fisheries, is usually produced under higher water temperatures than the natural conditions to facilitate rapid growth. However, it has been reported that rearing fish at higher water temperatures inhibits the development of the thymus, suggesting that resistance to infectious diseases is reduced in ayu reared at higher water temperatures. Here, we show that decreased resistance to bacterial cold-water disease and excessive inflammatory responses occurred in ayu reared at 22°C compared with those reared at lower temperatures. METHODS: Ayu larvae were reared at 12°C, 15°C and 22°C for 77 days and fed 3% of their body weight. Thymus index and condition factor was calculated after the fish rearing. Then, ayu reared at the different temperatures were challenged with Flavobacterium psychrophilum and the fish were sampled for histopathology and gene expression analyses. Further, the fish were vaccinated with formalin-killed F. psychrophilum and continuously reared at the three different water temperatures. Serum antibody titer was determined by ELISA and cumulative mortality in each group was recorded after the bacterial challenge. RESULTS: Ayu reared at 22°C showed a significantly lower thymus index and higher condition factor than those reared at lower temperatures. Infiltrated leukocytes and many melanin pigments were frequently observed in the adipose tissues and spleens of ayu reared at 22°C, respectively, but not in those reared at 12°C. The gene expression levels of inflammatory cytokines such as IL-1β, IL-8 and TNFα in the spleen were significantly higher in the 22°C group than in the 12°C group. The cumulative survival rate after challenge with Flavobacterium psychrophilum was 51.7%, 40.0% and 13.3% in the 12°C, 15°C and 22°C groups, respectively. The relative percent survival values of vaccinated fish reared at 15°C and 22°C groups were lower than those reared at 12°C. Moreover, the specific antibody titer of the vaccinated fish was the lowest in the 22°C group and the highest in the 12°C group. DISCUSSION: These results suggest that rearing the fish under high water temperature causes excessive inflammatory responses similar to metabolic inflammation in human obesity, resulting in a decrease of disease resistance. In addition, thymic involution induced by higher water temperature probably leads the poor response to vaccination. The present study provides insights into the physiological and immunological changes of fish under global warming.

無機物の食品添加物で構成されるオキシリンクSPのサケ科卵のミズカビ病に対する防除効果を調べた。室内試験においてSaprolegnia属菌5種に対するオキシリンクSPの殺菌効果を検討した結果，30分間の薬浴では10,000倍以下の希釈で殺菌作用を示した。野外試験では，Saprolegnia属菌以外の菌類が発育したサクラマス卵を除き，ニジマスおよびイワナ卵での菌糸発育抑制効果を確認した。魚卵毒性については，生産上大きな被害はないが，ニジマス卵の発眼率および孵化率に影響を与えた。一方，イワナ卵およびサクラマス卵に対する影響は認められなかった。オキシリンクSPによる薬浴はサケ科卵を対象としたミズカビ病の防除に有用であるが，薬浴条件について検討する必要がある。

The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8. © 2014 Elsevier Ltd.

We developed a long-term culture system for Japanese flounder Paralichthys olivaceus leukocytes supported by JFF07-1 feeder cells established from Japanese flounder fin tissue. Kidney leukocytes were seeded onto a monolayer of the feeder cells in enriched RDF medium supplemented with 20% fetal bovine serum and 2.5% flounder serum. Several colonies adhered to the feeder cells after 7 days of cultivation, demonstrating leukocyte proliferation. Increasing numbers of floating cells, which signified colony growth, were observed as the length of the culture period increased. The optimum culture conditions consisted of an incubation temperature of 25 degrees C, the addition of 2.5% flounder serum to the medium and the inoculation of kidney leukocytes at a density of 2 x 10(6) cells/mL. The proliferated cells were grouped into three types based on May-Grunwald Giemsa staining: basophilic cytoplasmic cells (65%), neutrophilic cytoplasmic cells (30%) and large cells containing many vacuoles (5%). The cells showed acid-phosphatase activity (90%), peroxidase activity (31%) and non-specific esterase activity (57%). Electron microscopy revealed that many of the cells contained endoplasmic reticula and mitochondria, but not specific granules with the fibrillar structure that characterizes flounder granulocytes. A monocyte lineage thus appeared to be the dominant population among the proliferated cells in the culture system. The composition of growing cells was also kept after 20 passages.

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. © 2010 Elsevier Ltd.

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. © 2010 The Japanese Society of Fish Pathology.

A dematiaceous fungus was isolated from Japanese flounder Paralichthys olivaceus with ulceration and erosion of the skin surface. The fungus was identified as an Exophiala species, with different morphological, biological and molecular characteristics from three previously described pathogenic Exophiala species. Fungal hyphae extended laterally in the dermis, and were absent from the epidermis and musculature of the skin lesions and kidneys of the diseased fish. An inflammatory response with granuloma occurred in the dermis involving accumulations of epithelioid cells around the hyphae. The granulomas were surrounded by lymphocyte-like cells. Epidermal degeneration was observed above the inflamed dermis, suggesting that the inflammatory response caused epidermal damage. Experimental infection reproduced hyphal extension and infiltration of inflammatory cells in the dermis of the flounder, confirming the pathogenicity of the fungus. © 2008 The Japanese Society of Fish Pathology.

We developed an in vitro model to study the cellular and molecular mechanisms of granulomatous inflammation in response to invading pathogens. Ichthyophonus hoferi was used as a target for encapsulation by cultivated leukocytes from the kidney of the rainbow trout (Oncorhynchus mykiss). The encapsulation process was observed over 1 week. The leukocytes were identified as either macrophages in the inner layer, or neutrophils and lymphocytes in the outer layer. The encapsulation response was inhibited by treatment with heat, but not formalin or methanol. The recognition of heat-unstable molecules on the pathogen surface could induce encapsulation. Increased expression of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-8 and tumor necrosis factor-α2, was observed during encapsulation. These cytokines might play crucial roles in the encapsulation process. In particular, IL-8, which was expressed at a late phase, might recruit specific cell populations, such as the lymphocytes comprising the outer cellular layer around the target. © 2007 Elsevier Ltd. All rights reserved.

Aphanomyces piscicida is an important pathogen that plays a role in the morbidity and mortality of various fish species around the world. The poor quality of current identification techniques for this fungus led to the development of highly sensitive and specific polymerase chain reaction (PCR) techniques. Primers derived from the ITS1 and ITS2 regions of A. piscicida NJM 0204 were designed for the detection and identification of fish-pathogenic A. piscicida, with the potential for the diagnosis of mycotic granulomatosis (MG). Polymerase chain reaction amplification showed that the primer set was specific only to fish-pathogenic A. piscicida, not to nonfish-pathogenic Aphanomyces, Saprolegnia spp., Achlya spp., Dictyuchus spp., and Lagenidium spp. In addition, PCR revealed an improved sensitivity sufficient to detect A. piscicida in artificially infected goldfish Carassius auratus. Results demonstrated that the PCR method established in this study is effective for the detection and identification of A. piscicida with MG.

We describe a polymerase chain reaction (PCR)-based approach to the detection and identification of Aphanomyces piscicida (=Aphanomyces invadans). Three primer sets were designed based on the sequences of cloned expression genes of A. piscicida NJM 9803 (accession numbers: AB 104634, AB 104635 and AB 104636). They were used to detect the genomic DNA of 42, 18, 10 and 1 isolates of Aphanomyces spp., Saprolegnia spp., Achlya spp. and Dictyuchus sp., respectively. Out of 3 primer sets, a primer set (1APM 1F, 1APM 6R) detected only fish pathogenic A. piscicida but not the other non-fish pathogenic Aphanomyces species. In addition, the PCR revealed sensitivity sufficient for detecting A. piscicida in artificially infected goldfish. From the results, it was demonstrated that the PCR method with the primer set is effective for detection of A. piscicida from infected fishes and diagnosis of mycotic granulomatosis.

Aphanomyces piscicida is a water mold that is pathogenic to several species of fish. Here we describe that a galactose-binding protein (GBP) purified from A. piscicida has a hemagglutinating activity. The GBP was separated from the supernatant of homogenized hyphae of A. piscicida by affinity chromatography using an immobilized D-galactose agarose gel. The GBP agglutinated goldfish erythrocytes. The molecular weight of the GBP was estimated at approximately 40 kDa by SDS-PAGE. A specific antiserum was produced against the GBP and the 40 kDa protein reacting with the antiserum was observed in only Aphanomyces strains isolated from diseased fish. These results suggest that the 40 kDa GBP is closely associated with Aphanomyces infections, such as mycotic granulomatosis, epizootic ulcerative syndrome, red spot disease and ulcerative mycosis.

This study demonstrated that a galactose-binding protein (GBP) produced by a fish pathogenic water mold, Aphanomyces piscicida, activates carp leukocytes. Leukocytes were separated from the head kidney and peripheral blood using Percoll density centrifugation. A flow cytometric analysis revealed that GBP binds with many cells and a variety of cell types including lymphocytes, granulocytes and thrombocytes. Intracellular calcium flux of the peripheral blood leukocytes induced by stimulation with GBP was confirmed by counting the fluo-3 loaded cells whose fluorescence increased after the stimulation using flow cytometry. The percentage of cells in which a calcium flux was induced peaked 1min after the stimulation. Approximately 6% of the cells specifically responded 1min after the stimulation. The proliferation response was determined by the level of BrdU uptake by the leukocytes after the stimulation. Cell proliferation was observed 2, 4 and 6 days after stimulation with GBP. The expression of cytokines IL-1β and TGF-β1 in the peripheral blood leukocytes, after the stimulation was evaluated by a semi-quantitative reverse-transcriptase polymerase chain reaction. Increased expression of IL-1β was observed 4h after stimulation with GBP. Variation of TGF-β1 expression under the same conditions was not observed. The kinetics of intracellular calcium flux and the level of IL-1β expression induced by GBP stimulation were different from those induced by phytohemagglutinin stimulation. These results confirmed that GBP is a pathogenic microbial component that can induce cell activation. GBP seems to induce the inflammatory response observed in the Aphanomyces infection. Copyright © 2002 Elsevier Science Ltd.

This study demonstrates that the fish pathogenic Aphanomyces piscicida (= A. invadans) possesses hemagglutinating and hemolytic activities. These activities were observed against goldfish erythrocytes using supernatant of the homogenized hyphae of A. piscicida. The hemagglutinating activity was not inactivated by heat treatment at 60°C for 30 min or by the addition of EDTA. D-galactose or lactose inhibited the hemagglutinating activity. D-galactose-binding substances which were isolated by affinity chromatography showed hemagglutinating activity. On the other hand, heat treatment at 60°C for 30 min reduced the hemolytic activity. It was demonstrated that a sample that did not absorb to D-galactose in affinity chromatography was responsible for the hemolysis. The factors that have hemagglutinating and hemolytic capacities may affect the blood cells of infected fish in vivo, and may play a role in the occurrence of anemia in infected fish.

This study observed the adaptability of carp neutrophilic granulocytes possessing spontaneous cytotoxic activity to different environmental temperatures. To study the adaptability of neutrophilic granulocytes, two different temperatures (25° C and 10° C) were selected, both for rearing and for in vitro assays, in which the cytotoxicity and the adherent rate against K562 target cells were measured. The cytotoxicity and adherent rate of neutrophilic granulocytes from carp kept at 25° C for 30 days were higher when assayed at 25° C than when assayed at 10° C. On the other hand, in carp acclimated from 25° C to 10° C, the cytotoxicities and adherent rates, when assayed at 25° C, decreased with increasing acclimation times, eventually becoming smaller than the values obtained when assayed at 10° C. After the fish kept at 10° C for a long period were re-acclimated to 25° C, these activities assayed at 25° C again became higher than the activities assayed at 10° C. These results indicated that carp neutrophilic granulocytes adapt their cytotoxic activity and adherent activity to different environmental temperatures. A change in cellular composition in the head kidney was also observed in carp kept at different environmental temperatures. The percentage of neutrophilic granulocytes became higher and lymphocytes became lower in carp that were kept at 10° C for a long period compared with carp that were kept at 25° C for a long period. © 1997 Academic Press Limited.

To assess cytotoxic activity of carp (Cyprinus carpio), head kidney leukocytes were examined with special reference to the effects of rearing water temperature and of assay temperature. Leukocytes as effector cells were separated from head kidney using Histopaque 1077 and cytotoxic activities were measured by the release of 51Cr from target cells which were K562, human chronic myelogenous leukemia cells. Cytotoxic activity of leukocytes from carp kept at higher temperature (25 °C) was lower at lower assay temperature (10 °C) than at higher assay temperature (25 °C). However, activity from carp acclimated to low temperature (10 °C) increased on 10 °C assay and decreased on 25 °C assay. Cellular composition of effector cells changed in connection with the above phenomenon. In carp acclimated to 10 °C, small lymphocytes decreased while the others, e.g. large lymphocytes, granulocytes and macrophages, increased. Accommodation of carp natural killer-like cells to environmental temperatures may be regulated by changing the cellular composition of effector cells. © 1995.

This study demonstrates for the first time that carp (Cyprinus carpio) neutrophilic granulocytes from the head kidney possess potent spontaneous cytotoxic activity against several human tumor cell lines. Carp head kidney cells isolated at a density of 1.09 g/mL contained more than 90% neutrophilic granulocytes. These cells were round and approximately 10 μm in diameter with reniform or polymorphic nuclei and slightly eosinophilic cytoplasm when stained with Giemsa. Electron microscopy revealed that the cytoplasm contained numerous oval granules, some of which contained a dense rod-shaped core. The neutrophilic granulocytes readily formed conjugates with the human target cells and rapidly killed them. The neutrophilic granulocytes killed human derived target cells better than murine derived target cells. Inhibition of cytotoxicity by catalase suggested that the production of H2O2 is involved as a mediator in the cytotoxic reaction. The size and granularity of the carp effector cells indicate that they are different from the small agranular nonspecific cytotoxic cells (NCC) described in the channel catfish. © 1995.