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Hematopoiesis in teleost fish is maintained in the kidney. We previously reported that Hoechst dye efflux activity of hematopoietic stem cells (HSCs) is highly conserved in vertebrates, and that Hoechst can be used to purify HSCs from teleost kidneys. Regulatory molecules that are strongly associated with HSC activity may also be conserved in vertebrates. In this study, we identified evolutionarily conserved molecular components in HSCs by comparing the gene expression profiles of zebrafish, murine, and human HSCs. Microarray data of zebrafish kidney side population cells (zSPs) showed that genes involved in cell junction and signal transduction tended to be up-regulated in zSPs, whereas genes involved in DNA replication tended to be down-regulated. These properties of zSPs were similar to those of mammalian HSCs. Overlapping gene expression analysis showed that 40 genes were commonly up-regulated in these 3 HSCs. Some of these genes, such as egr1, gata2, and id1, have been previously implicated in the regulation of HSCs. In situ hybridization in zebrafish kidney revealed that expression domains of egr1, gata2, and id1 overlapped with that of abcg2a, a marker for zSPs. These results suggest that the overlapping genes identified in this study are regulated in HSCs and play important roles in their functions.

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho(-)) cells with that of SP cells by ginbuna transplantation experiments. Rho(-) cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho(-) cells was lower than that of SP cells, while Rho(-) cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho(-) cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.

Interleukins 4 and 13 (IL-4 and IL-13) are related cytokines important for Th2 immune responses and encoded by adjacent genes on human chromosome 5. Efforts were made previously to detect these genes in fish, but research was hampered by a lack of sequence conservation. A Tetraodon nigrovirides (green spotted pufferfish) gene was annotated as IL-4 by Li et al. (Mol Immunol, 44:2078-2086, 2007), but this annotation was not well substantiated. However, the present study concludes that the reported pufferfish gene belongs to the IL-4/13 lineage indeed, while also describing an additional IL-4/13 copy in a paralogous genomic region. Our analyses of IL-4/13 loci in fish describe (1) genomic region history, (2) characteristic intron-exon organization, (3) deduced IL-4/13 molecules for several teleost fish species, (4) IL-4/13 lineage-specific protein motifs including a cysteine pair (pair 1), and (5) computer software predictions of a type I cytokine fold. Teleost IL-4/13 molecules have an additional cysteine pair (pair 2) or remnants thereof, which is absent in mammalian IL-4 and IL-13. We were unable to determine if the teleost IL-4/13 genes are orthologous to either IL-4 or IL-13, or if these mammalian genes separated later in evolution.

The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56(lck) binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCR beta mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.

We previously demonstrated that some fish have unique response in the form of reactive oxygen species (ROS) production. In the present study, we cloned and sequenced the full-length cDNAs for carp (Cyprinus carpio) phagocyte NADPH oxidase components: gp91phox, p22phox, p47phox, p67phox and p40phox. These amino acid sequences were compared with other teleost and mammalian homologues, to elucidate the features of ROS production of fish neutrophils. The phylogeny analysis clearly demonstrates that the radiation of phagocyte oxidase components took place in the common ancestor of teleosts and mammals. Thereafter, the overall structure and expression pattern of phagocyte oxidase have been highly conserved in two different strains. However the amino acid identity of p67phox and p47phox was relatively lower than the amino acid identities of other components. Moreover, a synteny analysis supports the hypothesis that there was strong selective pressure in the p67phox and p47phox genes. Thus, it is likely that the higher divergence of p67phox and p47phox are responsible for the difference of ROS responses between different species of teleosts.

Macrophage migration inhibitory factor (MIF) was discovered as the first cytokine that inhibited the random migration of macrophages. Recently, MIF has been reported to be involved in embryonic development in higher vertebrates. In fish, however, nothing is known about the function of MIF at early life stages, although immunological functions of MIF have been reported in adult fish. To elucidate the function of MIF during embryonic development in fish, we examined expression patterns and function of the zebrafish MIF gene using antisense morpholino-mediated knockdown (morpholino oligonucleotide-MO). In whole-mount in situ hybridization analysis, zebrafish MIF mRNA was detected in developing eyes, tectum, branchial arches, pectoral fin buds, liver and gut. The onset of MIF mRNA expression coincided with the beginning of tissue differentiation during embryogenesis. MIF-MO-injected embryos (morphants) displayed malformed eyes, abnormal swelling in the tectum and fourth ventricle region, and undeveloped jaw cartilage and pectoral fins. An increased number of apoptotic cells in the eye and neural tissues were observed in MIF morphants by histological analysis and acridine orange staining. Moreover, proliferating cell nuclear antigen (PCNA)-positive cells were reduced in morphant eyes. These results suggest that MIF is essential for normal embryonic development even at the level of teleosts and that it functions as a growth factor for the proliferation and differentiation of embryonic tissues.

In the adaptive immune system of mammals, naive helper T (Th) cells differentiate into Th1 or Th2 cells. The T-box expressed in T cells (T-bet) is a member of a family of T-box transcription factors that regulates the expression of IFN-gamma and plays a crucial role in Th1 cell differentiation and cell-mediated immunity. We cloned and sequenced T-bet cDNA for the first time from non-mammalian species, ginbuna crucian carp. Ginbuna T-bet was composed of 608 predicted amino acids and showed 41.5% identity with human T-bet (Tbx21), and human and ginbuna T-bet share 77.3% identity in their T-box regions. Comparative genomic analysis showed conserved synteny in these regions between zebrafish, fugu, medaka and human T-bet. Phylogenetic analysis indicated that ginbuna T-bet is closely related to that of mouse and human. In unstimulated fish, ginbuna T-bet mRNA was strongly expressed in peripheral blood leukocytes (PBL), head kidney (HK) and spleen. RT-PCR analysis in kidney cells sorted by FACS revealed that T-bet was strongly expressed in surface-IgM-negative lymphocytes in comparison to IgM-positive lymphocytes. These results suggest that ginbuna T-bet is involved in the immune system, especially in T-cell function, and is an important tool to analyze teleost cell-mediated immunity.

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.

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus-2 (CyHV-2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV-2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV-2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non-permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV-2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV-2 infection and immunity.

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.

Lck is a member of the Src-related protein tyrosine kinase family. In mammals, it is predominantly expressed in T cells, and plays crucial role in T-cell maturation and activation. In this report, the cloning, characterization, and expression analysis of lck genes from the S3n strain of ginbuna crucian carp Carassius auratus langsdorfii is described. Three distinct lck homologs, designated as gblck-1, gblck-2, and gblck-3 were isolated from ginbuna thymus. The deduced amino acid sequences of gblck-1 and gblck-2 cDNAs exhibit conserved lck essential motif and overall structures, but gblck-3 lacks an essential region related to regulation of Src kinase activity. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated that gblck-1 was expressed in peripheral blood leukocytes (PBLs), lymphoid organs, and mucosal tissues, as has been reported for T-cell marker genes, i.e. CD3, TCR, and lck in other fish species. In contrast, the expression of gblck-2 and gblck-3 were observed only in PBLs and lymphoid organs. In the sorted lymphocytes from kidney leukocytes using antiginbuna IgM monoclonal antibody, only slg−lymphocytes expressed lck genes as well as the TCR\ gene. Collectively, these results suggest that lck is a useful marker for the identification of T cells.

Eomesodermin (Eomes) is a T-box transcription factor that is involved in mesoderm formation in most vertebrates. Eomes is also expressed in CD8+ T cells and NK cells. No information is available on the role of Eomes in the immune system of lower vertebrates to date, although developmental studies on Eomes (Eomes1) have been performed in zebrafish. Here we report the identification of a second Eomes (Eomes2) in zebrafish and compare expression of the two Eomes genes in the immune system. Zebrafish Eomes1 and Eomes2, composed of 661 and 534 amino acids, respectively, share 49.3% amino acid identity in their coding regions and 88.7% amino acid identity in their T-box regions. Conserved synteny between regions of the human and zebrafish genomes, gene organization and phylogenetic analysis all indicate that the zebrafish Eomes2 gene is a homologue of mammalian Eomes, as previously found for zebrafish Eomes1. Eomes1 mRNA was found to be expressed in the gonad, body kidney, spleen and gill, while Eomes2 mRNA was not detected in any of these tissues. However, strong expression of both Eomes mRNAs was detected in the leukocytes from the spleen, followed by those from body kidney and peripheral blood, with expression of Eomes1 always stronger than that of Eomes2. RT-PCR analysis of body kidney cells sorted by FACS revealed that Eomes1 was expressed strongly in lymphocytes, weakly in blast cells, and was not expressed in granulocytes, while Eomes2 was expressed weakly in lymphocytes. These results suggest that both Eomes genes are involved in the zebrafish immune response, particularly in lymphocyte function as has been found in mammals.

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.

To elucidate the degradation process of the posterior silk gland (PSG) during metamorphosis of the silkworm Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP), and after cocoon formation (CO) were used to analyze macroautophagy, chaperon-mediated autophagy (CMA), and the adenosine triphosphate (ATP)-dependent ubiquitin-proteasome. Immediately after entering the metamorphosis stage at PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form-II at all stages. The levels of ubiquitinated-proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the PSG during metamorphosis.

Viral and intracellular bacterial diseases are big problem in aquaculture. Defense against these diseases is largely dependent on cell-mediated immunity in which cytotoxic T lymphocytes and NK cell...

In mammals the rejection of allografts is primarily accomplished by cell-mediated immunity including T cells. Recently, considerable studies reveal the existence of helper and cytotoxic T cell subsets in fish. Here we investigate the kinetics of CD4(+) and CD8α(+) T cells along with sIgM(+) cells and phagocytic cells in an allogeneic scale graft model using ginbuna crucian carp for understanding the mechanisms of cell-mediated immune response. The results showed that CD4(+) T cells first infiltrated into allogeneic scales followed by CD8α(+) and sIgM(+) cells, and finally phagocytic cells appeared in the graft. Furthermore, most of the CD8α(+) T cells appeared on the border of the allografted scales at the time of rejection. These results suggest that T cells play crucial roles and work together with other cell types for completion of allograft rejection.

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8alpha and TCRbeta genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8alpha and TCRbeta mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8alpha and TCRbeta mRNAs. In addition, mRNA expressions of CD8alpha and TCRbeta in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8+ T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (Tc) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.

A model system of ginbuna carp (clonal triploid fish, Carassius auratus langsdorfii) and ginbuna-goldfish hybrids (tetraploid fish, having three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish) was employed to demonstrate the presence of hematopoietic stem cells (HSCs) in kidney hematopoietic tissues. Kidney hematopoietic cells were obtained from ginbuna carp (S3n, donor) and injected into a ginbuna-gold fish hybrid (S4n, recipient) via the caudal sinus. The percentage of S3n cells in the S4n blood was measured by flow cytometry by the difference of DNA content from 3 to 18 months after the injection. Donor-derived major types of blood cells including erythrocytes, thrombocytes, neutrophils, basophils, monocytes, T and B lymphocytes were detected in the recipient blood over a long period (9-18 month posttransplantation). Moreover, recipient lympho-hematopoietic organs such as head kidney, body kidney, spleen, and thymus were reconstituted by donor cells. These results indicate that long-term multipotent repopulating HSCs reside in the donor kidney.

Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

p,p#-DDE, the main metabolite of DDT, is still detected in aquatic environments throughout the world. Here, the effects and mechanisms by which p,p#-DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha) was studied. Isolated salmon splenic and pronephric leukocytes were incubated with different concentrations of p,p#-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p#-DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p#-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leukocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p#-DDE. The effect of p,p#-DDE on leukocytes appeared to vary between developmental stages or seasonal differences. The mitogenic response of leukocytes of chinook salmon exposed to p,p#-DDE in vivo exhibited a biphasic doseeresponse relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p#-DDE had a significantly lower percentage of IgC blasting cells than controls, although the response was biphasic. These results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression. 2004 Elsevier Ltd. All rights reserved.

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and cytosolic proteins, p40phox, p47phox and p67phox. In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91phox has three potential N-linked glycosylation sites. gp91phox and p22phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47phox and p67phox have two potential Src homology 3 domains and p40phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91phox, p47phox and p67phox expression were high only in the blood and kidney, while p22phox and p40phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

We have isolated and characterized cDNAs and genes for pufferfish, Fugu rubripes, (Fugu) orthologues of mammalian interleukin (IL)-12 subunits (IL-12 p35 and IL-12 p40). The deduced amino acid sequences of the Fugu IL-12 subunits showed homology with mammalian IL-12 subunits (p35: 50.4-58.0% similarity; p40: 51.2-55.4% similarity). Phylogenetic analysis confirmed that Fugu IL-12 p35 and p40 genes cluster with their mammalian counterpart lineages. The genomic organization of each of the Fugu IL-12 subunit genes is similar to that of the corresponding mouse IL-12 subunit genes, although the Fugu genes are very compact due to small intron size. Comparative genomic analysis showed conserved syntenies within the IL-12 p35 and p40 regions between Fugu and human, indicating that the Fugu IL-12 p35 and p40 genes are orthologues for mammalian IL-12 p35 and p40 encoding genes, respectively. Expression of IL-12 p35 mRNA was observed in lymphoid tissues and several non-lymphoid tissues, while expression of IL-12 p40 mRNA was constitutive and nearly ubiquitous. In the spleen and head kidney, expression of IL-12 p35 was induced by polyriboinosinic polyribocytidylic acid [poly(I:C)] and not by lipopolysaccharide (LPS), while expression of IL-12 p40 was constitutive and unresponsive to both poly(I:C) and LPS. These results indicate that IL-12 levels are regulated by production of IL-12 p35 mRNA and suggest that IL-12 in fish may be involved in antiviral defense. This is the first report of the identification and characterization of IL-12 subunit cDNAs and genes in a non-mammalian vertebrate.

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu ( Plecoglossus altivelis ) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout ( Oncorhynchus mykiss ), carp ( Cyprinus carpio ), eel ( Anguilla japonica ), and pond smelt ( Hypomesus nipponensis ). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3–7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2–19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.

Bacterial coldwater disease is considered to be one of the most serious diseases in ayu Plecoglossus altivelis in Japan. We evaluated the efficacy of three highly water-soluble adjuvants (Montanidae IMS-1311, IMS-1312 and IMS-2212) combined with formalin-killed bacterin of Flavobacterium psychrophilum (FKB) in ayu, in comparison with a minimally water-soluble adjuvant (Montanidae ISA-763A). The retention period and toxicity of these adjuvants in ayu were also examined. Experimental challenge 4 wk after vaccination showed that FKB adjuvanted with IMS-1311 and IMS-1312 had higher potency than FKB alone and significantly (p

The objective of the study was to investigate the dynamics of changes in number of granulocytes in bacterial infections of carp ( Cyprinus carpio ). Carp were inoculated with non-pathogenic or pathogenic bacteria and changes in type I (neutrophils) and type II granulocyte (basophils/eosinophils) counts in kidney, circulating blood and peritoneal cavity were assessed. After the injection of non-pathogenic bacteria ( Escherichia coli ), the number of type I and II cells in blood increased after 6–12 h, but retuned to the control level after 24–48 h. In contrast, after the injection of pathogenic bacteria ( Aeromonas hydrophila ), the number of type I cells initially increased followed by an increase in the number of type II cells. The peak counts of type I and II cells were at 12 and 24 h after the injection, respectively. When the fish were given serial-injections of formalin-killed bacteria at 12-h intervals, the type II cells also predominantly increased and remained at high levels, following the peak count of type I cells.

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA , for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A–F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the α3 and transmembrane domains is unprecedented.

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.

A symposium entitled “Current status of fish vaccine development in Japan” was held by the Japanese Society of Fish Pathology in Tokyo on 29th March, 2001. This was aimed to have an overview of the status quo of fish vaccine developments in Japan, especially those for marine fishes. In Japan, a vibrio vaccine to ayu Plecoglossus altivelis was first developed in 1988 and five vaccines have been totally developed until now. In this symposium, ten subjects were included in five categories : (1) Vaccine-use system, utilization and practical problems, (2) Vaccines developed or being developed, (3) Genetic engineering techniques and adjuvants for vaccines (4) General control methods to prevent fish diseases, and (5) Problems confronting the development of fish vaccines in Japan. Vaccines will contribute to make safe products in aquaculture, which can give “peace of mind” to consumers. Vaccines developed in Japan have been effective to protect fish from the target diseases and it was emphasized that fish condition should be kept healthy to get a good result by vaccination.

We investigated the potency of two oil adjuvants, Montanidae ISA 763A (763A) and a squalene emulsion (Squalene), to enhance the response of ayu Plecoglossus altivelis to a formalin-killed bacterin (FKB) made from the coldwater disease etiologic agent, Flavobacterium psychrophilum. Ayu were challenged 4 wk after vaccination by an intramuscular injection with live pathogenic F. psychrophilum. Mortalities of fish injected with FKB of F. psychrophilum combined with either adjuvant showed that the adjuvanted vaccines had significantly higher (Pl0.05) potencies than the FKB vaccine alone or sterile distilled water. Moreover, both adjuvanted vaccines produced significantly higher antibody titers than the FKB vaccine without adjuvant. From these investigations it is concluded that the use of Squalene and 763A adjuvants provides enhanced protection against coldwater disease in ayu compared to the use of FKB alone.

To elucidate the process of the uptake of suspended particles into the skin, fins and gills of rainbow troutOncorhynchus mykiss, 100 fingerlings were immersed in a suspension of 1μm diameter fluorescent microspheres and rinsed with water. The fate of microspheres adhering to body surface was traced for elapsed times ranging from 1 min to 24 h. Although most microspheres were removed in a 2 h-rinse with water, a few were observed by light microscopy to be located in both epidermal and dermal tissues which had microscopic injuries. As observed by electron microscopy, microspheres were taken up by migrating epithelial cells, or embedded in the dermis covered with these cells. Some of those microspheres embedded in dermis were endocytosed by macrophages. Such features were confirmed in artificial wound experiments. We conclude that microscopic injuries to trout could be an important route for the uptake of antigen administered by immersion, 2 to 24 h after fish suffer from injuries.

This review describes the fish immune system, focusing on specific cell-mediated immunity. Specific in vivo cell-mediated immune responses have been shown by allograft rejection, graft-versus-host reaction (GVHR) and delayed hypersensitivity reaction (DTH). Recent in vitro studies also showed specific cell-mediated cytotoxicity against allogeneic target cells. These in vivo and in vitro experiments strongly suggest the presence of cytotoxic T cells in fishes. Also described are current studies on shark and trout MHC class I polymorphism and function that demonstrate strong similarities between fish and mammals.

To determine if a gynogenetic teleost might have a sexually bipotential brain, we tested whether implantation of 11-ketotestosterone (KT) induces male-type sexual behavior and gonadotropin (GTH) secretion in adult gynogenetic crucian carp, "ginbuna," Carassius auratus langsdorfii. KT-implanted female ginbuna were tested for male spawning behavior by pairing them with a stimulus female in which sexual receptivity and attractivity were induced by prostaglandin F(2alpha) (PG) injection. When KT-implanted female ginbuna were paired with a PG-injected stimulus female ginbuna, all the KT-implanted fish tested showed male spawning behavior in response to the PG-injected females. KT-implanted fish also showed female spawning behavior when they were injected with PG. When the KT-implanted female ginbuna were exposed to waterborne 17alpha,20beta-dihydroxy-4-pregnen-3-one (a female sex pheromone that stimulates male-typical GTH secretion in goldfish), all the KT-implanted fish showed an elevation of plasma GTH levels in response to the pheromone. These results demonstrate that gynogenetically evolved ginbuna, like goldfish, is sexually plastic and can be behaviorally and endocrinologically masculinized by androgen treatment without behavioral defeminization. These results support our hypothesis that adult teleosts retain a sexually bipotential brain regardless of reproductive strategy, i.e., hermaphroditism, gonochorism, or gynogenesis.

The effect of temperature (15, 20 and 25 deg C) on the suppression of in vitro cell-mediated cytotoxicity was investigated in clonal, triploid ginbuna crucian carp (S3n) from Lake Suwa, Nagano Prefecture, Japan sensitized by scale grafts from tetraploid hybrids (S4n) bred by insemination of S3n with goldfish sperm. Sensitized S3n effector cells were separated from blood on Percoll gradients (1.085 g/ml); 82% of separated cells were lymphocytes. Haemoglobin release was detected by tetramethylbenzidine. Injection (i.p.) of sensitized effector PBL from S3n to S4n recipients resulted in graft versus host disease (GVHD) characterized by pale gills, scale protrusion, ascites, haemorrhage and necrosis of the ventral skin, muscle layers and peritoneum. PM findings included peritonitis and intestinal and liver necrosis. Death rate was highest in fish kept at 25 deg C and lowest at 15 deg C. Further work needed to explain these results is discussed

A model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids was employed to demonstrate the presence of graft-versus-host reaction (GVHR) in a teleost fish. Tetraploid scale grafts on triploid clone members evoked an acute rejection, whereas the reverse transplants were accepted. When sensitized triploid cells were injected into tetraploid recipients, a typical GVHR was induced, leading to death of the recipients within one month. The onset of illness appeared about one week after cell injection as a loss of appetite and constipation, followed by a scale protrusion, severe haemorrhage, local destruction of the ventral skin and prominent splenomegaly. GVHR was most effectively induced by head-kidney cells and peripheral blood leukocytes (PBL), followed by spleen and thymus cells. Donors had to be sensitized at least twice by scale grafting to induce the reaction. A considerable number of recipients injected with cells from donors which had been sensitized by allogenetically different tetraploids died, suggesting a limited polymorphism or heavy cross-reactions between the alleles of the histocompatibility antigens. Ploidy analyses revealed that donor cells greatly increased in the host liver and spleen, constituting approximately 30% of total cells after 2 3 weeks. Most of these features of acute GVHR observed in this fish system are quite similar to those found in mammals and birds. thereby suggesting the presence of allo-reactive cytotoxic T lymphocytes in teleosts.

In order to clarify the effect of a prolonged exposure period on vaccine effectiveness, juvenile rainbow trout were immersed in 100, 1, 000 and 10, 000 times dilutions of a commercial vibrio bacterin for 3 min (brief immersion, BI) and for 24 h (prolonged immersion, PI). Two weeks and 2, 4 and 6 mo after the vaccination, fish were challenged by waterborne exposure to Vibrio ordalii. Prolonged exposure to dilute bacterin resulted in better protection than the standard BI method. Two weeks after vaccination, the cumulative mortality was 0% in the group vaccinated by PI with the 100, 1, 000 and 10, 000 times diluted vaccine. In contrast, mortality was 58 and 77% in the groups vaccinated by BI with the 100 and 10, 000 times diluted vaccine respectively, and was 100% in the unimmunized control group. Furthermore, 6 mo after vaccination, the mortality was 20% (100 times dilution) to 77% (10, 000 times dilution) in the PI group, while it was 64% (100 times dilution) to 93% (10, 000 times dilution) in the BI group. Mortality in the corresponding control group was 97%. These results suggest that prolonged exposure period improves vaccine effectiveness.

This study examined the uptake of BSA-conjugated 1 μ m fluorescent latex microspheres from bath suspensions by 1·5–3·0 g rainbow trout, Oncorhynchus mykiss (Walbaum). Uptake was quantified using a novel method based on dissolving fish tissues (excluding the gastrointestinal tract) in an alkaline solution and capturing microspheres on a filter for microscopic counting. In suspensions of 1·0×10 7 particles ml −1 , the rate of uptake was approximately linear over 48 h of continuous immersion, with a 24 h mean±SE of 98 000±12 000 microspheres fish −1 . Uptake was logarithmically proportional to particle concentration in suspensions of 1·0×10 5 , 1·0×10 6 and 1·0×10 7 particles ml −1 . Prolonged immersion in a dilute suspension resulted in greater uptake than brief immersion in concentrated suspensions. Quantification of microspheres in separate tissues indicated that both skin and gill participate in uptake, with the skin playing a major role. Particles remained in skin and gill tissues for at least 24 days following exposure, while a minority were transported to the spleen and kidney. This observation supports an important role for local immunity in the protective responses generated during immersion immunisation. Histologically, microspheres were observed primarily within epithelial cells and underlying phagocytes of both skin and gill. Collectively these findings describe a new method for quantifying antigen uptake, which was used to demonstrate the effectiveness of prolonged exposure to dilute antigen suspensions and the importance of the skin as a site of antigen uptake during immersion immunisation.

Life span and half-life of different populations of circulating peripheral blood cells from ginbuna crucian carp (Carassius auratus langsdorfii) were investigated. Erythrocytes or peripheral blood leukocytes (PBL) were stainedex vivowith a fluorescent cell linker kit, then reinjected into the same fish. Blood cells were reisolated from peripheral blood and the decrease in levels of labelled cells was plotted against time. The decrease of stained cells in peripheral blood was exponential, from which the half-life of the cells was calculated. Circulating erythrocytes showed a half-life of approximately 51 days. However, a few erythrocytes survived in the peripheral blood for more than 270 days. The life span of circulating thrombocytes was estimated to be approximately 70 days, with a half-life of less than 5 days. The half-life of circulating monocytes and granulocytes was between 2 and 3 days, respectively, with their life span varying from between 2 and 27 days, while the life-span of circulating lymphocytes ranged from between 10 and 23 days. However, some stained lymphocytes persisted in the peripheral blood for more than 145 days and these may represent memory cells.

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