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IntroductionTemporal 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.MethodsAyu 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.ResultsAyu 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. DiscussionThese 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.

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.

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.

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.

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.

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.

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.

The ontogeny of the teleost innate immune system was studied in carp using cellular, histological and quantitative molecular techniques. Carp myeloid cells first appeared ventro-lateral of the aorta at 2 days post fertilization (the start of hatching), and subsequently around the sinuses of the vena cardinalis (or posterior blood islet), head kidney and trunk kidney. In addition, the hematopoietic tissue around the sinuses of the vena cardinalis transformed into that of the trunk kidney, which is the first description of the ontogeny of the trunk kidney hematopoietic tissue in teleosts. The mAb's used in this study reacted with carp myeloid surface molecules that are already transcribed and processed from the first appearance of myeloid cells, and thus serve a significant role in unraveling ontogenetic processes of teleost immunology. Finally, this study associated the first appearance of myeloid cells with an immune response on the molecular level: 2 days post fertilization embryos responded to LPS injection with upregulation of interleukin 1-beta, inducible nitric oxide synthase and serum amyloid A, and down-regulation of complement factor 3 and alpha2-macroglobulin, implying a functional embryonic innate defense system.

Adult worms of the blood-feeding monogenean parasite Neoheterobothrium hirame, which cause anemia in the Japanese flounder Paralichthys olivaceus, attach to the host fish by embedding their posterior part deeply into the host tissue. To investigate the possibility that cellular responses of the host fish can eliminate N. hirame, flounder were experimentally infected with N. hirame larvae and reared in either fed or starved conditions. Mature parasites were identified on the buccal cavity wall of the fish 33 d post-infection (Day 33). Monocytes/macrophages and granulocytes increased rapidly in the blood and infected sites after the appearance of mature parasites. These cells adhered to the tegument of the parasites. In addition, a few cells with large electron-dense granules (DGCs) were observed in the inflammatory foci. On Day 47, the tegument of some parasites collapsed partially and were phagocytosed by the infiltrated host cells. Some infiltrated cells adhered directly to the inner tissues of the parasites. On Day 54, in the fed fish group, the loss of the tegument led to damage of the parasites' inner tissue by a large number of infiltrated cells. In this group, the elimina- tion of the parasites was noted from Day 47 to 54. These observations probably suggest that the cellular response of the host fish destructed the parasite's posterior part embedded in the tissue, thereby eliminating the parasites. On the other hand, a high mortality was observed in the starved group. The starved fish developed much more severe anemia than the fed fish, and the elimination of the parasites was not observed in this group. The results of the present study suggest that flounder can eliminate N. hirame if they are fed sufficiently.

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5–3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

近年ヒラメに頻発している原因不明の貧血症について, 血液学的, 寄生虫学的検査と造血器官の病理組織学的観察を行った。血液学的には, 幼若赤血球の増加と赤血球細胞質の空胞変性および染色性の低下が認められた。病魚の造血器官にヘモジデリンの沈着や組織壊死像はほとんど見られず, 貧血魚にはN.hirameが高率に寄生していた。このことから, 本貧血症は寄生虫による失血性貧血であることが強く示唆された。

The characterization of rainbow trout peripheral blood leucocytes (PBL) was carried out by flow cytometry (FCM) analysis and by transmission electron microscopy. Two major cell populations were identified in FCM analysis. One population showed small values in both forward scatter (FS) and side scatter (SS), and the other population showed large values in both parameters. These major populations were divided into two sub-regions each (A/B and C/D). There was no significant difference in cell populations between sub-regions A and B. However, a difference was observed in the small and large lymphocyte percentages between sub-regions C and D. The majority of regions (A and B) were constituted by neutrophils (56% and 66%, respectively), and monocytes accounted for 9% and 7% of the cells, respectively. Large and small lymphocytes were also present in these regions, accounting for 26% and 6% of the cells in sub-region A and 14% and 7% in sub-region B, respectively. In sub-regions C and D, cells were mainly constituted by large and small lymphocytes (33% and 49% in C, and 7% and 70% in D, respectively) and thrombocytes (14% and 23%).

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.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naive ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4+ and CD8α+ cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α+ cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.

The mucosal surfaces of fish allow for the introduction of foreign substances, including antigens, from the surrounding environment. In this study, uptake of Vibrio anguillarum J-O-3 serotype bacterin by Japanese flounder, and the subsequent immune responses were investigated. Immunohistochemistry revealed that the bacterin was taken up through the epithelial cells of gills. The transcription levels of inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor α were significantly up-regulated in the gills at 3 days following exposure to the bacterin. There was also a corresponding increase in IL-8 receptor, CD4-1, CD4-2 and CD8α transcript levels in the gills. Our findings suggest that the gills play a major role in the uptake of V. anguillarum bacterin and induction of inflammation, which results in an activation of the adaptive immune response in teleost fish.

Hematological characteristics of the anemia prevailing in wild Japanese flounder Paralichthys olivaceus were examined in detail by cell counting and microscopy. The erythrocyte counts, MCH, and MCHC decreased correlatively with the decrease in the hemoglobin concentration. Moreover, the reduction of erythrocyte volume was also observed in many anemic fish. Light microscopy showed erythrocytes with structural abnormalities, such as vacuolated, weakly stained cytoplasm and deformed outlines. In the TEM observations of erythrocytes, an insufficient accumulation of hemoglobin was observed as well. These results indicate that the anemia can be identified as a hypochromic microcytic anemia accompanied with hypoglobulia and a structural abnormality of the erythrocytes.

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8 cm average body length were not susceptible to infection with VHSV at 20 °C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15 °C in the 9.8 cm cohort that had previously been exposed to the virus at 20 °C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8 cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10 cm.

Ontogenetic studies of peripheral blood leucocyte (PBL) subpopulations were conducted in Japanese flounder (Paralichthys olivaceus), from the post-flexion larval stage to the juvenile stage, using three monoclonal antibodies (MAbs) that were specific for membrane components of myeloid cells (JFW1), thrombocytes (JFW10) and immunoglobulin (JFW20). Total leucocyte concentrations decreased between the post-flexion stage (19.1%) and when body length was 29.0 mm (1.4%), remaining unchanged thereafter (1.1-2.8%). The percentage of JFW1(+) cells relative to the other leucocytes was high in the early life stages (6.4-36.6% in larva, 1.1-9.5% in juvenile). No differences in the phagocytic activity of JFW1(+) leucocytes from larval and juvenile flounder were observed, and the ultrastructure of JFW1(+) cells was equivalent to that of granulocytes in adult fish. Conversely, JFW20(+) cells were detected in fish with body lengths of 29.0 mm, and the percentage of the JFW20(+) cells increased with age. No developmental changes were observed in the proportion of the JFW10(+) leucocytes, which had ultrastructural characteristics that differed from the thrombocytes of adult fish. It therefore appears that instead of immature lymphocyte-mediated specific immunity, the non-specific defense mechanism involving myeloid cells plays a major role in host defenses in the early life stages of Japanese flounder.

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.

Objective—To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity.Animals—22 carp.Procedure—Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement.Results—Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of Tlymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum.Conclusion and Clinical Relevance—Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths. (Am J Vet Res2001;62:599–603)