Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii.

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.

Adjuvant effect in aquaculture fish of cell-wall glycolipids isolated from acid-fast bacteria.

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.

Adjuvant effect of recombinant interleukin-12 in the Nocardiosis formalin-killed vaccine of the amberjack Seriola dumerili.

Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2- related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection.

Naringenin suppresses Edwardsiella tarda infection in GAKS cells by NanA sialidase inhibition.

Edwardsiella tarda (E. tarda) is a gram-negative bacterium, which causes Edwardsiellosis in aquaculture. Previous studies indicate that E. tarda NanA sialidase plays crucial roles in infection through the desialylation of glycoproteins in fish cells. On the other hand, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, classic sialidase inhibitor, negatively regulates E. tarda infection of goldfish scale GAKS cells. Here, to development the suppression model of E. tarda infection for aquaculture application, the possibility of NanA inhibitory activities in citrus phytochemicals was evaluated as citrus extracts have widely been used as a supplement in fish diets for the improvement of meat quality. Some flavanones such as naringenin, hesperetin, hesperidin and naringin showed sialidase inhibitory activity toward recombinant NanA in vitro. Among them, naringenin showed the most potent inhibitory activity and its inhibitory pattern was non-competitive. Naringenin significantly suppressed E. tarda infection in GAKS cells at 200 and 400 µM without bactericidal effect on E. tarda. On the other hand, naringin, glycosylation form of naringenin, showed slight suppression of E. tarda infection toward GAKS cells, suggesting the glycosides on flavanone could be important for NanA inhibition. Fluorescence microscopy analysis verified that number of invading E. tarda in GAKS cells was declined by naringenin treatment. The present study exhibited the possibility of naringenin as an effective ingredient in fish diet for the inhibition of E. tarda infection.

Identification and functional characterization of multiple interleukin 12 in amberjack (Seriola dumerili).

Interleukin (IL) -12 is a heterodimeric cytokine mainly produced by monocytes, macrophages, and dendritic cells in mammals. IL-12p70 composed of IL-12p35 and IL-12p40, is known to play a crucial role in promoting cell-mediated immunity (CMI) through Th1 differentiation and IFN-γ production. Although two types of IL-12p35 (p35a, p35b) and three types of IL-12p40 (p40a, p40b and p40c) have been identified in several fish species, the knowledge on functional characteristics of teleost IL-12 is still limited. In the present study, we cloned two types of IL-12p35 and three types of IL-12p40 genes in amberjack and yellowtail, and analyzed their expressions in response to stimulation with Nocardia seriolae in amberjack. As a result, four types of IL-12 (IL-12p35a, p35b, p40a and p40b) and IFN-γ mRNA were increased by live-N. seriolae stimulation but not by formalin-killed N. seriolae, suggesting that four types of IL-12 (p35, p35b, p40a and p40c) participate in promoting CMI. Subsequently, we produced six types of recombinant IL-12p70 (rIL12p70) protein in insect cells. Head kidney leukocytes were cultured with formalin-killed N. seriolae and six types of rIL-12p70 to elucidate the role of amberjack IL-12p70 in induction of CMI. After stimulation, IFN-γ expression was elevated whereas IL-10 expression was suppressed in Head kidney leukocytes stimulated with four types of rIL-12 (p40a/p35a, p40c/p35a, p40a/p35b, p40a/p35b). On the other hand, two types of rIL-12 (p40b/p35a, p40b/p35b) only elicited down regulation of IL-10 expression. These results indicate that all amberjack IL-12p70 isoforms are involved in Th1 -differentiation and promotion of CMI with different manners. Fish IL-12 has a potential for the promising vaccine adjuvant.

Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate α2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection.

Comparative analysis of adaptive immune response after vaccine trials using live attenuated and formalin-killed cells of Edwardsiella tarda in ginbuna crucian carp (Carassius auratus langsdorfii).

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.

Transcription analysis of two Eomesodermin genes in lymphocyte subsets of two teleost species.

Eomesodermin (Eomes), a T-box transcription factor, is a key molecule associated with function and differentiation of CD8(+) T cells and NK cells. Previously, two teleost Eomes genes (Eomes-a and -b), which are located on different chromosomes, were identified and shown to be expressed in zebrafish lymphocytes. For the present study, we identified these genes in rainbow trout and ginbuna crucian carp. Deduced Eomes-a and -b amino acid sequences in both fish species contain a highly conserved T-box DNA binding domain. In RT-PCR, both Eomes transcripts were readily detectable in a variety of tissues in rainbow trout and ginbuna. The high expression of Eomes-a and -b in brain and ovary suggests involvement in neurogenesis and oogenesis, respectively, while their expression in lymphoid tissues presumably is associated with immune functions. Investigation of separated lymphocyte populations from pronephros indicated that both Eomes-a and -b transcripts were few or absent in IgM(+) lymphocytes, while relatively abundant in IgM(-)/CD8α(+) and IgM(-)/CD8α(-) populations. Moreover, we sorted trout CD8α(+) lymphocytes from mucosal and non-mucosal lymphoid tissues and compared the expression profiles of Eomes-a and -b with those of other T cell-related transcription factor genes (GATA-3, T-bet and Runx3), a Th1 cytokine gene (IFN-γ) and a Th2 cytokine gene (IL-4/13A). Interestingly, the tissue distribution of Eomes-a/b, T-bet, and Runx3 versus IFN-γ transcripts did not reveal simple correlations, suggesting tissue-specific properties of CD8α(+) lymphocytes and/or multiple modes that drive IFN-γ expressions.

Role of CD4(+) and CD8α(+) T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp, Carassius auratus langsdorfii.

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 naïve 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.

Peculiar monomeric interferon gammas, IFNγrel 1 and IFNγrel 2, in ginbuna crucian carp.

The existence of fish-specific isoforms of interferon (IFN)γ, known as IFNγ-related (IFNγrel), has been reported in several fish species. However, comparisons with deduced amino acid sequences of known IFNγrels among several fish species have indicated significant differences at the C-terminus basic amino acid continuous sequences, which indicate the existence of multiple IFNγrel isoforms. Two distinct cDNAs, encoding two IFNγrels, ifngrel 1 and ifngrel 2, were cloned from ginbuna crucian carp (Carassius auratus langsdorfii). Recombinant IFNγrel 1 and IFNγrel 2 have shown high antiviral activities against the lethal crucian carp hematopoietic necrosis virus. Both ligands exhibit biological activity as monomers despite the fact that the functional conformation of IFNγ is a homodimer. Both interferons have a high degree of sequence similarity, but differ in the C-terminus region. In this region, IFNγrel 1 contains a functional nuclear localization sequence which induces the translocation of green fluorescent protein from the cytoplasm to the nucleus. IFNγrel 2 lacks this sequence. These results indicate that IFNγrel 1 and IFNγrel 2 are functional antiviral cytokines. These structurally related ligands play distinct antiviral roles through different intracellular translocation mechanisms. Thus, IFNγrels form a novel, distinct subtype included in type II IFNs. The cyprinid fish IFNγ subtype currently consists of four members, including two IFNγ isoforms and two distinct additional IFNγrel isoforms specific to the fish.

Serum GlcNAc-binding IgM of fugu (Takifugu rubripes) suppresses the growth of fish pathogenic bacteria: a novel function of teleost antibody.

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.

Adaptive immune response to Edwardsiella tarda infection in ginbuna crucian carp, Carassius auratus langsdorfii.

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.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors.

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.

A unique epidermal mucus lectin identified from catfish (Silurus asotus): first evidence of intelectin in fish skin slime.

The present study reports a new type of skin mucus lectin found in catfish Silurus asotus. The lectin exhibited calcium-dependent mannose-binding activity. When mannose eluate from chromatography with mannose-conjugated agarose was analysed by SDS-PAGE, the lectin appeared as a single 35-kDa band. Gel filtration showed that the lectin forms monomers and dimers. A 1216-bp cDNA sequence obtained by RACE-PCR from the skin encoded a 308 amino acid secretory protein with homology to mammalian and fish intelectins. RT-PCR demonstrated that the lectin gene was expressed in the gill, kidney and skin. Subsequent sequencing revealed the presence of an isoform in the gills. Antiserum detected the intelectin protein in club cells in the skin and gill, renal tubules and blood plasma. Although intelectin gene expression was not induced by in vivo bacterial stimulation, the intelectin showed agglutination activity against the pathogenic bacterium Aeromonas salmonicida, suggesting that the lectin plays an important role in self-defence against bacteria in the skin surface of the catfish. These findings represent one of the few examples of characterization and functional analysis of a fish intelectin protein.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish.

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.

Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in ginbuna crucian carp, Carassius auratus langsdorfii.

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.

A novel C1q family member with fucose-binding activity from surfperch, Neoditrema ransonnetii (Perciformes, Embiotocidae).

The C1q family is a growing group of proteins with a globular C1q domain in the C-terminal region. We purified a new member of this family with L-fucose-binding activity from the plasma of surfperch, Neoditrema ransonnetii through L-fucose-affinity chromatography and anion-exchange chromatography. N-terminal amino acid sequencing followed by cDNA sequencing revealed that the protein was composed of 212 amino acids including a signal peptide of 20 amino acids. The gene expression analysis by RT-PCR showed that the gene was transcribed in the liver, stomach and intestine. The hepatic gene expression was up-regulated within 3 h of an intraperitoneal injection of formalin-killed Edwardsiella tarda. A phylogenetic analysis of gC1q domains placed the 23 kDa protein in the same cluster as other fish non-complement C1q-like proteins including a precerebellin-like protein of rainbow trout and ovary-specific protein of crucian carp. Interestingly, sialic acid-binding lectins of mollusca were located on the neighboring branch. Though the lectin activity has yet to be ascribed to the gC1q domain, these findings, together with former findings on lectin activity of lamprey and human C1q, indicate that sugar-binding activity is relatively common among the C1q family.

Co-culture of carp (Cyprinus carpio) kidney haematopoietic cells with feeder cells resulting in long-term proliferation of T-cell lineages.

To characterise fish haematopoietic stem/progenitor cells, it is necessary to develop a culture system that supports proliferation and differentiation of these cells. In the present study, we established cell lines from various tissues of carp (Cyprinus carpio) and ginbuna (Carassius auratus langsdorfii). By using these cell lines, we developed a culture system in which carp haematopoietic cells proliferated and were successively passaged. Cell lines from carp thymus (KoT), carp fin (KoF1) and ginbuna thymus (GTS6 and GTS9) were newly established. In addition to these cell lines, ginbuna fin (CFS) cell lines were also used as feeder layers. Kidney haematopoietic cells co-cultured with these feeder layers proliferated rapidly and were passaged over 20 times for more than 60 days. To characterise the proliferating cells, expression of marker genes for blood cell development were analysed. In the primary culture, marker genes for myeloid/erythroid progenitors (gata1), haematopoietic stem cells (gata2), neutrophils (mpx/mpo), B-cells (IgH) and T-cells (lck, TCRbeta and gata3) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Expression of most of the genes disappeared after the third passage, only T-cell marker genes were highly expressed after passages. These results indicate that multiple blood cells developed in the primary culture and then T-cell lineages dominantly proliferated after several passages.

Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection.

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.

GATA3 mRNA in ginbuna crucian carp (Carassius auratus langsdorfii): cDNA cloning, splice variants and expression analysis.

GATA3, a transcriptional activator, plays a critical role in the development of T-cells and differentiation to T helper type 2 cells. To date, no information is available on the role of GATA3 in the teleost immune system. We identified full-length cDNA and alternatively spliced variants of ginbuna crucian carp GATA3 (gbGATA3). The gbGATA3 gene is transcribed into multiple splice variants lacking either one or both zinc finger domains, although the sequences of both domains are fully conserved between ginbuna and other vertebrates. We found that alternative splice site and stop codon in gbGATA3 intron 3, located between exons that separately encode the two zinc finger domains, are conserved among teleosts, suggesting that teleost GATA3 gene can be translated into multiple isoforms. RT-PCR analysis revealed that the gbGATA3 is strongly expressed in the brain, thymus and gill of unstimulated fish. Moreover, gbGATA3 expression was detected in surface-IgM-negative lymphocytes among kidney cells sorted by FACS. Real-time PCR demonstrated that expression levels of full-length gbGATA3 and the splice variants differed with tissue type, but full length was always the predominantly expressed form. These results suggest that gbGATA3, including its splice variants, is involved in teleost T-cell function.