Search

Ichthyophthirius multifiliis is a ciliated protozoan parasite and is known to infect many freshwater teleosts. Characterizing the immune system in epithelial tissues, where the parasites penetrate and settle, is key to understanding host–parasite interactions. This study examined local immune responses in vivo to the infective stage (theront and trophont) of the parasites using intra-fin administration, which has been developed to analyze in vivo immune responses using fish fin. CD8α+ and CD4+ T-cell compositions were increased significantly in the fin cavity injected with theront or trophont antigens. The expression of GATA-3 and T-bet mRNA, which regulate differentiation of helper T-cells, was upregulated significantly in leukocytes from the trophont antigen–injected site. In contrast, the percentages of macrophages and neutrophils, which are innate immunity components, were decreased significantly in the injection sites. These results suggest that I. multifiliis antigens inhibit the migration of macrophages and neutrophils, and T-cells are the first responders to I. multifiliis. Thus, to better understand the interaction of host immunity and I. multifiliis, further studies should focus on exploring the inhibitory factors from I. multifiliis or examining innate functions of teleost T-cells.

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8+ cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8+ CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8+ cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbs) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbs in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbs mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

In this study, we investigated the immune responses against Mycobacterium gordonae in ginbuna crucian carp. Cumulative mortality of ginbuna injected with 2.0 × 107 CFU of M. gordonae was 50% at 170 days post-infection. CD4-1, CD8α, T-bet and IFNγ2 gene expression levels were significantly upregulated in ginbuna injected with 1.9 × 108 CFU of M. gordonae at 21 and 28 days post-infection. The CD4-2 level did not change during the experiment. Granulomatous responses consisted of central macrophage accumulation and surrounding lymphocytes, and Ziehl-Neelsen-positive bacteria were observed in the trunk kidney of the challenged fish. Immunohistochemistry using anti-ginbuna IFNγs and anti-ginbuna CD4-1 polyclonal antibody revealed that the marginal lymphocytes were positive for CD4-1, and the IFNγ-producing cells surrounded the mycobacterial cell-laden phagocytes. These results suggest that CD4-1+ cells and IFNγ2 play important roles in the granulomatous inflammation against Mycobacterial infections in teleosts.

Phytohemagglutinin (PHA) is a well-known mitogen inducing activation and proliferation of lymphocytes, particularly T lymphocytes in vitro. PHA has also been used in vivo for assessing cell-mediated immunity in non-mammalian vertebrates, particularly in birds. However, it has been suggested that local inflammation as a direct result of tissue damage could be responsible for skin swelling after PHA injection, in addition to induction of T lymphocyte mitogenesis. In order to understand the complex nature of this response in fish we investigated the accumulation of cell types chronologically in dorsal fin of ginbuna crucian carp Carassius auratus langsdorfii after PHA injection. Neutrophils appeared first and showed a peak response on day 1, decreasing gradually and followed by macrophages and blast cells while lymphocytes increased later with a peak response on day 5. The number of accumulated cells was significantly higher in PHA-injected fish than controls in most cases. Lymphocytes identified as CD4-1

The development of mammalian megakaryocytes and platelets is regulated by numerous cytokine signals, primarily through the thrombopoietin (TPO)/c-MPL axis. Although non-mammalian vertebrates are known to possess nucleated thrombocytes functionally equivalent to mammalian platelets, the dynamics of the thrombocyte development remains unclear. Here we identified TPO and a splice variant (TPO-v) caused by the intron retention in common carp (Cyprinus carpio). Both the tpo and its variant transcripts were highly expressed in heart and liver. Recombinant carp TPO (rcTPO) was produced and purified in HEK293T cells stably expressing tpo, but TPO-v was shown not to be secreted from the transfectants. rcTPO induced the formation of colony-forming unit-thrombocyte (CFU-T) colonies which were recognized by a monoclonal antibody against carp thrombocytes expressing c-mpl and cd41, in a dose-dependent manner. The combination of rcTPO and recombinant carp Kit ligand A (rcKITLA) exerted a significant synergistic effect on three types of colony formation: thrombocytic colonies, thrombocytic burst colonies and thrombocytic/erythroid colonies. Utilizing this colony assay to examine the distribution of thrombocytic progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in thrombopoiesis, while the spleen does not. Our results indicate that carp possess mechanisms of TPO- and KITLA-dependent thrombopoiesis similar to those in other vertebrates and the sites of thrombopoiesis are restricted to the kidney, the primary hematopoietic organ in the teleost fish.

We have monoclonal antibodies (mAbs) against CD4-1 (6D1) and CD8α (2C3) in ginbuna crucian carp Carassius auratus langsdorfii . In our previous studies we showed that 2C3 mAb positive cells are the primary cell type showing specific cytotoxicity against allogeneic targets, suggesting that CD8α + lymphocytes in ginbuna are equivalent to cytotoxic T lymphocytes (CTLs) in mammals. We further demonstrated the helper T cell function of 6D1 mAb positive cells by studying mixed leukocyte culture (MLC) and hapten/carrier effects. Here, we report that our mAbs cross-react with zebrafish lymphocytes. First, mAbs 6D1 and 2C3 recognized 7–11% of zebrafish lymphocytes that were ZAP-70 positive and had the typical morphology of lymphocytes. Second, to verify the cell types reacting with the 6D1 and 2C3 mAbs we examined the expression profiles of zebrafish lymphocyte surface markers in FACS-sorted lymphocytes from kidney. cd4-1 ( cd8a ) and tcrac but not iglc transcripts were detected in 6D1(2C3) + lymphocytes, whereas cd4-1 ( cd8a ) transcripts were not found in 6D1 (2C3) - lymphocytes. Third, we further confirmed that 6D1 reacted with zebrafish CD4-1 but not CD4-2, and 2C3 recognized zebrafish CD8α expressed on HEK293T cells. Collectively, these findings suggest that 6D1 + and 2C3 + lymphocytes in zebrafish are equivalent to CD4 + and CD8α + T lymphocytes in mammals, respectively. Furthermore, we found the cross-reactivity of our 6D1 and 2C3 mAbs with other cyprinid species including goldfish, common carp and grass carp.

Immunocompetence is an important parameter that reflects disease resistance in fish. Very few methods to examine immunocompetence in vivo have been developed, even for mammals. In the present study, we present a unique method for analyzing local immune responses using fish fin. We first demonstrated the migration of granulocytes to the site of zymosan injection in fin in a dose-dependent manner and that this could be easily observed macroscopically due to the fin membrane transparency. We also demonstrated phagocytic activity of accumulated leukocytes after zymosan administration and that almost all phagocytes were granulocytes. In addition, we succeeded to detect respiratory burst activity of granulocytes in vivo without any in vitro treatment of cells, indicating that our present method is suitable for the analysis of granulocyte phagocytic function in vivo. The method provides a unique tool applicable for fishes that possess transparent fins and may lead to better understanding of the mechanisms of local immune responses in fish.

Intracellular bacterial and viral diseases are widespread in the aquaculture industry and cause serious economic losses. Development of effective vaccines and adjuvants that can induce cell-mediated immunity is urgently needed for prevention of these diseases. Here we report the immunostimulatory effects of probiotic bacteria ‘‘ E. faecalis ’’ in ginbuna crucian carp Carassius auratus langsdorfii . Intraperitoneal injection of heat-killed E. faecalis induced an increase in CD4-1 + lymphocytes, CD8α + lymphocytes and macrophages in vivo . Expression of Th1 cytokine genes was enhanced by exposure to the bacteria in vitro . We identified the leukocyte subsets that expressed specific Th1 cytokine genes: granulocytes and macrophages produced IL12 and IFNγrel2, respectively, while lymphocytes produced IFNγs including IFNγ1 and IFNγ2. Finally, expression of Th1 cytokines was also enhanced by intraperitoneal injection of heat-killed E. faecalis in vivo , while expression of Th2 cytokine was unchanged. Together, these findings suggest that heat-killed E. faecalis can induce cell-mediated immunity in fish.

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.

Granzymes are serine proteases involved in the induction of cell death against non-self cells. The enzymes differ in their primary substrate specificity and have one of four hydrolysis activities: tryptase, Asp-ase, Met-ase and chymase. Although granzyme genes have been isolated from several fishes, evidence for their involvement in cytotoxicity has not yet been reported. In the present study, we attempted to purify and characterize a fish granzyme involved in cytotoxicity using ginbuna crucian carp. The cytotoxicity of leukocytes was significantly inhibited by the serine protease inhibitor ''3, 4-dichloroisocoumarin''. In addition, we found that granzymeA-like activity (hydrolysis of Z-GPR-MCA) was inhibited by the same inhibitor and significantly enhanced by allo-antigen stimulation in vivo. Proteins from leukocyte extracts were subjected to two steps of chromatographic purification using benzamidine-Sepharose and SP-Sepharose. The molecular weight of the purified enzyme was estimated to be 26,900 Da by SDS-PAGE analysis. The purified enzyme displayed a Km of 220 μM, a Kcat of 21.7 sec(-1) and a Kcat/Km of 98,796 sec(-1) M(-1) with an optimal pH of 9.5 for the Z-GPR-MCA substrate. The protease was totally inhibited by serine protease inhibitors and showed granzymeA-like substrate specificity. Therefore, we conclude that the purified enzyme belongs to the mammalian granzymeA (EC 3.4.21.78) and appears to be involved in cytotoxicity in fish.

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8

The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, β-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.

Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a "per-gill infection method", which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)-infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8(+) cells in kidney leukocytes increased after the secondary infection, whereas IgM(+) cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination.

Herpesviral hematopoietic necrosis caused by goldfish hematopoietic necrosis virus (now identified as cyprinid herpesvirus 2, CyHV-2) has contributed to economic losses in goldfish Carassius auratus culture and is becoming a major obstacle in Prussian carp C. gibelio aquaculture in China. Several reports have described difficulties in culturing the virus, with the total loss of infectivity within several passages in cell culture. We succeeded in propagating CyHV-2 with a high infectious titer in a RyuF-2 cell line newly derived from the fin of the Ryukin goldfish variety using culture medium supplemented with 0.2% healthy goldfish kidney extract. The addition of kidney extract to the medium enabled rapid virus growth, resulting in the completion of cytopathic effect (CPE) within 4 to 6 d at 25°C. The extract also enabled reproducible virus culture with a titer of 105-6 TCID50 ml-1. The virus cultured using this protocol showed pathogenicity in goldfish after intraperitoneal injection. The virus grew in RyuF-2 cells at 15, 20, 25, 30, and 32°C but not at 34°C or higher. Higher incubation temperatures allowed earlier development of CPE, but culture at 30 and 32°C yielded a lower virus titer than that obtained at other temperatures because of heat inactivation of the propagated virus during cultivation. Cell lines derived from goldfish and ginbuna C. langsdorfii showed high susceptibility to the virus; cell lines from carp were susceptible to the virus using a medium containing goldfish kidney extract, but EPC, FHM, and BF-2 cell lines did not produce any CPE, even in the presence of the extract.

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.

Mycobacteriosis and nocardiosis in cultured fish caused by infections with acid-fast bacteria, are responsible for large economic losses globally. In this study, we suggest a novel adjuvant using glycolipids that activates host immune systems. The immune response to glycolipids stimulation was investigated using ginbuna crucian carp. Ginbuna vaccinated with FKC (formalin-killed cells) + glycolipids isolated from Mycobacterium sp., upregulated inflammatory- and Th1-related cytokines, and a DTH (delayed-type hypersensitivity) response was confirmed only in ginbuna vaccinated with FKC + glycolipids. These observations suggest that glycolipids activated host innate and cell-mediated immunity. Subsequently, we evaluated the adjuvant effect of glycolipids against amberjack nocardiosis. In a challenge test, a higher survival rate was observed in amberjack vaccinated with FKC + glycolipids emulsified with conventional oil adjuvant than in fish vaccinated with FKC + oil adjuvant without glycolipids. Therefore, glycolipids potentially could be used as a practical, economical and safe adjuvant for aquaculture fish.

Teleosts and elasmobranchs are the lowest vertebrates that possess adaptive immunity akin to mammalian ones. However, the fish immune system is different from that of mammals, that is, a lack of bone marrow, lymph nodes and germinal center. Their immune system is also characterized by the fact that their surface is covered by live cells with mucus in direct contact with environmental water and the fact that the immune response is greatly affected by temperature.

Granzymes (Gzms) are serine proteases released from cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Gzms induce apoptosis within virus-infected and transformed cells. In fish as well as mammals, Gzms appear to play a major role in inducing target cell death. However, information on the function of fish Gzms is limited, although Gzm-like genes have been reported in several species. We identified and characterized a fish Gzm (termed gcGzm) in ginbuna crucian carp, Carassius auratus langsdorfii. The primary structure of gcGzm resembled mammalian GzmB, and gcGzm clustered with mammalian GzmB by phylogenetic tree analysis. gcGzm was secreted from HEK293T cells transfected with gcgzm cDNA and was predominantly expressed in CD8(+) T cells, as in mammals. Expression of gcgzm mRNA was greatly enhanced by allo-sensitization and infection with the intracellular pathogen Edwardsiella tarda, indicating that gcGzm is involved in cell-mediated immunity. However, its enzymatic activity was different from mammalian Gzms because gcGzm did not cleave the known substrates for mammalian Gzms. Thus we conclude that the newly discovered gcGzm is a novel secretory serine protease involved in cell-mediated immunity in fish, with similar structure to human GzmB but different substrate specificity.

Cytotoxic T lymphocytes (CTL) recognize and kill cells infected with viruses, intracellular bacteria and tumors with MHC restriction and antigen specificity. In addition to these activities, recent studies in mammals have suggested that CTL can exhibit direct microbicidal activity. In our previous study we documented direct antibacterial activity of CD4(+) T cells and sIgM(+) cells as well as CD8α(+) T cells from immunized fish. However, we also found weak non-specific killing activity of lymphocytes against bacteria. In the present study we further analyzed the weak killing activity of lymphocytes, increasing the effector cell to target bacteria ratio from 10:1 to 10(3):1. Sensitized and non-sensitized effector lymphocytes (CD8α(+), CD4(+) and sIgM(+)) separated by MACS were incubated with target bacteria. CD8α(+) T cells from Edwardsiella tarda-immunized ginbuna crucian carp killed 98%, 100% and 70% of E. tarda, Streptococcus iniae and Escherichia coli, respectively. CD8α(+) T cells from non-immunized fish showed similar but slightly lower killing activity than sensitized cells. CD4(+) and sIgM(+) lymphocytes also showed high killing activity against E. tarda and S. iniae as found for CD8α(+) T cells, although the activity was lower against E. coli. Supernatants from all three types of lymphocytes showed microbicidal activity, although the activity was lower than that evoked by effector lymphocytes. Furthermore, the presence of a membrane between effectors and targets did not affect the killing activity. The present results suggest that both sensitized and non-sensitized lymphocytes non-specifically killed target bacteria without the need of contact. The major difference between the present and previous experiments is the E:T ratio. We suspect that there are two different mechanisms in the direct bacterial killing by lymphocytes in ginbuna.

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

The development of rapid and simple gene amplification tests is required for detection of pathogens to prevent transmission of infectious diseases between animals or from animals to humans. An easy-to-use rapid gene amplification method that can directly detect RNA and DNA viruses in clinical samples was developed. This method is based on combining loop-mediated isothermal amplification (LAMP) or reverse transcription-LAMP (RT-LAMP) and RNA GEM Tissue, a thermophilic enzyme that extracts nucleic acid by quickly digesting proteins and ribonucleases. The authors named these methods GEM LAMP and GEM RT-LAMP. These methods were able to detect viral DNA and RNA within 70 min in a single tube using only a water bath. The detection capacities were 10-100-fold more sensitive than those of previously established LAMP and RT-LAMP methods. The GEM LAMP and GEM RT-LAMP methods were used to detect macroscopically the presence of DNA and RNA viruses in sera or fecal samples from cattle, pigs, horses, dolphins, penguins, and sea lions using SYBR green I. The GEM LAMP and GEM RT-LAMP methods thus have considerable versatility as tools for detecting pathogens and are applicable to basic human and veterinary medicine, environmental hygiene, and point-of-care-testing.

Previous studies have suggested that anti-viral cytotoxic effector cells induced by infection with a sublethal dose of crucian carp hematopoietic necrosis virus (CHNV) correspond to mammalian cytotoxic T-lymphocytes (CTLs), because the mRNA expression patterns of the effector cells are similar to those of mammalian CTLs. To further characterize the effector population in cell-mediated cytotoxic (CMC) activity, we isolated the effector cells using an anti-CD8α monoclonal antibody, a density gradient and plastic adherence. As expected, the purified CD8α-positive cells killed CHNV-infected cells, indicating that the fish CTLs are one of anti-viral effector cells similar those to in mammals. However, it appeared that cytotoxic cells other than CTLs were the dominant effectors, because CTL-depleted peripheral blood leukocytes (PBL) exhibited significant cytotoxic activity against CHNV-infected cells. In addition, the adoptive transfer of CTL-depleted PBL provided as efficient protection against CHNV-infection as the transfer of PBL containing CTLs. Further analyses showed that sIg/CD8α-negative cells and monocyte-enriched effectors possessed activities that were comparable to or were higher than that of CD8α-positive cells, suggesting that natural killer (NK)-like cells and monocytes are among the dominant effector cells. CMC inhibition assays with concanamycin A suggested that CTLs and CD8α-negative lymphocytes lysed virus-infected cells by a perforin-based cytotoxic pathway. These results indicate that CMC induced by viral-infection is executed by not only CTLs but monocytes and CD8/IgM-negative lymphocytes.

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.

Haptoglobin (Hp) is a positive acute-phase protein and a valuable marker of inflammation in both human and veterinary medicine. The aim of this study was to validate the molecular characterization of Hp in dolphins and to validate commercially available Hp measurement methods such as Hp-ELISA (originally designed for pigs) and Hp–hemoglobin (Hb) binding assay. The dolphin Hp (dHp) amino acid sequence appeared most similar to pig Hp by sequence homology and phylogenetic clustering. Amino acid sequence analysis revealed that dHp comprises the Hp1 form of α1 and β chains. The anti-pig Hp antibody cross-reacted with both recombinant dHp, expressed by Escherichia coli, and dHp from serum. The intra- and inter-assay levels of imprecision of pig Hp-ELISA and the Hp–Hb binding assay were found to be tolerable for the determination of Hp in dolphin, and there was no significant discrepancy between the two determination methods. The ability of the assay to differentiate between healthy and inflammation groups was investigated, and a significant increase in Hp concentration was detected in inflammatory conditions. Thus, Hp is a useful inflammation marker for dolphin, and the Hp concentration in dolphin serum samples can be reliably measured using commercially available pig Hp-ELISA and Hp–Hb binding assay.

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α(+) T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α(+) T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1-2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α(+) T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α(+) T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4(+) T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM(+) cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α(+), CD4(+) T-cells and surface IgM(+) cells in fish.

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.

Nocardiosis, one of the most systemic and devastating diseases, is currently emerging as an important disease of cultured marine and freshwater fishes. The causative agent of this disease is Nocardia seriolae, a Gram-positive acid-fast bacterium. An effective vaccine/vaccination strategy against this pathogen is necessary to control the significant loss in aquaculture practices. In this chapter, we present the vaccination/immunization protocol in fish against both live (sublethal) and inactivated form of N. seriolae using ginbuna crucian carp (Carassius auratus langsdorfii) as a model. N. seriolae either in live (sublethal) form or inactivated antigenic form is found to elevate immunity in ginbuna and also can induce protective immunity upon challenge. In order to develop live vaccine, determination of sublethal dose is critical and needs to be established in the host fish species through pathogenicity and persistence studies. Herein for ginbuna, a sublethal dose of 10(6) CFU/mL was determined by pathogenicity study through a series of challenge doses followed by pathogen persistence study by microbiological and molecular techniques. On the other hand, for inactivated antigenic form, the concentration of the N. seriolae was approximately 10(8) CFU/mL. Although this study showed significant potential of both the forms of N. seriolae as candidate for vaccination, factors such as dose, duration and form need to be optimized in individual fish species.

In vertebrates, the rejection of allografts is primarily accomplished by cell-mediated immunity. We recently identified four IFNγ isoforms with antiviral activity in ginbuna crucian carp, Carassius auratus langsdorfii. However, involvement of the IFNγ isoforms in cell-mediated immunity, especially in T cell function remains unknown. Here we investigate expression of the IFNγ isoforms and effects of administration of recombinant IFNγ (rgIFNγ) isoforms in ginbuna scale allograft rejection. All four IFNγ isoforms showed significantly higher expression with the progression of graft rejection. Administration of rgIFNγrel 1 but not rgIFNγrel 2, rgIFNγ1 nor rgIFNγ2 enhanced allograft rejection. The number of CD4(+) and CD8α(+) cells increased in early stages of rejection, while sIgM(+) cells were higher than controls at day 0 and 5 in the rgIFNγrel 1 administrated group. Expression of IFNγ1 and IFNγ2 mRNA was significantly up-regulated by rgIFNγrel 1 administration, while that of IFNγrel 1 and IFNγrel 2 was not. These results suggest different contributions of the four IFNγ isoforms toward the immune responses comprising allograft rejection.

Aeromonas veronii is a Gram-negative opportunistic pathogen in fish. We have previously investigated the intestinal microbiota of carp Cyprinus carpio and revealed that most bacterial isolates with high mucus-binding capability were A. veronii. With regard to infection, mucosal adhesion of bacteria is regarded as a prerequisite for colonization and invasion. In the present study, we selected three A. veronii strains with different mucus-binding capability and pathogenicity, and compared the colonization capability and localization of these strains in the intestinal tract of carp, using an in vivo imaging system. We showed that only a pathogenic strain was mainly visible around the intestinal bulb and rectum of the intestinal tract at 48 h after administration. Frozen sections with fluorescence signals revealed that the administered bacteria were localized on the mucosal surfaces and in the lamina propria of the intestinal bulb, whereas they were mainly detected in the remaining intestinal contents in the rectum. These results suggest that the intestinal bulb is the primary region for A. veronii to invade and proliferate in healthy carp.

The present study examined the effects of high-concentration ascorbic acid (AsA) supplementation over a long period on growth, plasma components, nonspecific immune responses, and thermal tolerance in rainbow trout Oncorhynchus mykiss. Four commercial diets supplemented with 0, 100, 1000, and 5000 mg AsA per kg of diet (designated AsA0, AsA100, AsA1000, and AsA5000, respectively) were fed to rainbow trout (initial weight 1.85 g) for 100 days. AsA contents in liver increased with increasing dietary AsA levels. Feeding period, growth performance, and plasma components did not differ significantly between the AsA groups. On the other hand, the phagocytic assay [nitro-blue tetrazolium (NBT) assay] was significantly higher in fish fed AsA1000 and AsA5000 than those fed a diet without AsA (AsA0) at the end of feeding trial. Superoxide dismutase (SOD) activity in the intestines of fish fed AsA5000 was also significantly higher than that of fish fed AsA0. In the thermal tolerance test, fish fed AsA5000 only showed significantly lower cumulative mortality compared with the AsA0 group. In conclusion, high-concentration AsA supplementation such as AsA5000 over a long period does not induce any adverse effects and can enhance disease and stress resistance in rainbow trout.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.

Rainbow trout and Atlantic salmon interleukin-4/13A (IL-4/13A) genes were identified. They were found expressed at high level in thymus, gill, and skin, in concert with the transcription factor gene GATA-3. High expression levels of IL-4, IL-13, and GATA-3 were also detected in murine thymus, suggesting similar importance of the fish and mammalian homologues for early T cell development. In mammals, combined high expression of IL-4/13 and GATA-3 in tissues other than thymus is mostly indicative of Th2 responses. Th2-skewage may protect fish skin and gill from parasites and from damage by inflammatory Th1 and Th17 responses. The immune milieus of fish gill and skin are relevant to aquaculture, because these tissues are preferred sites for vaccine administration. The similarities between the immune milieus of fish gill and thymus may reflect an evolutionary relationship, since these tissues map close together lining the gill cavity. Expression patterns of IL-4/13A and interferon gamma (IFN-γ) in isolated trout gill cells and pronephrocytes were consistent with Th2 identity of IL-4/13A.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes. Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8β. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4. Moreover, 5'-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vβ sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8- αβT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αβT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cβ, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8β and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.

T cell-mediated cytotoxicity occurs via pathways based on perforin or Fas mechanisms. Perforin is a protein present in the cytoplasmic granules of CD8 + cytotoxic T lymphocytes and is secreted to form pores on target cell membranes. In fish, although the involvement of perforin in cytotoxicity have been suggested for several species, perforin-mediated cytotoxicity of CD8α + lymphocyte in conjunction with expression of the perforin gene has not been reported. In order to investigate the killing mechanism of CD8α + lymphocytes by perforin-mediated pathway in fish, we measured apoptosis of target cells triggered by CD8α + lymphocytes, performed cytotoxic assays in the presence or absence of perforin inhibitor; concanamycin A and EGTA, and analysed the expression of perforin1 , perforin2 and perforin3 isotypic genes in ginbuna crucian carp. In the present study, we found that CTLs attached with target cells. CTL should have direct contact with target cells to kill them. Approximately 50% of target cells were positive for annexin V after co-cultured with CD8α + lymphocytes, indicating the induction of apoptotic cell death. Concanamycin A, which induces depolymerization of perforin resulting in lytic function, suppressed the cytotoxicity of CD8α + cells in a dose-dependent manner. In addition, cytotoxicity mediated by CD8α + lymphocytes were significantly suppressed by the addition of the Ca 2+ -chelating agents EGTA or EGTA–Mg 2+ , and the addition of Ca 2+ restored the killing mechanism of target cells. We further found enhanced expression of perforin1 but not perforin2 or perforin3 in CTLs from allo-sensitized fish. The present study has demonstrated that ginbuna CTLs kill target cells through perforin-mediated pathway, suggesting that perforin-mediated pathway is conserved throughout vertebrate.

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.

Single hematopoietic cells from carp (Cyprinus carpio) kidney were seeded to each well of 96-well plates and cultured in the presence of a supporting cell layer and conditioned media (CM). The CM were obtained from bulk-cultured carp hematopoietic cells, in which T and macrophage-lineage cells rapidly proliferated as previously reported. After 2-3 weeks, colony formation was found in 0-4 wells of each plate. Three different morphological types of colonies were observed: "type I colonies", "type II colonies" and "mixed-type colonies". Type I colony cells were interpreted as composed by macrophage-lineage cells, since they expressed a specific macrophage marker, M-CSFR/csf1r gene, and most of them phagocytosed latex particles. Type II colony cells were interpreted as composed by T lineage cells, since they expressed several T cell marker genes including gata3, lck and TCRbeta, but did not engulf latex particles. Mixed-type colonies were interpreted as composed by both macrophages and T lineage cells. They expressed not only the M-CSFR gene but also a T cell marker gene, gata3, but not other T cell markers, such as lck and TCRbeta. These results indicated that the mixed-type colonies were developed from immature common progenitors of macrophage and T cell. In contrast, type I and type II colonies were developed from more mature and mono-potent progenitors of macrophage and T cell, respectively.