Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii.

In this study, we established a murine cell line that expresses ginbuna crucian carp (ginbuna) CD4-2 and used it to develop an anti-CD4-2 monoclonal antibody (mAb). An established mAb, named D5, showed good reactivities to BALB/c 3T3 cells expressing CD4-2 and a lymphocyte population in the ginbuna leukocytes. Gene expression analysis showed that D5+ cells express CD4-2 and TCRß genes but not CD4-1 and IgM genes, meanwhile May Grunwald-Giemsa staining of sorted D5+ cells had the typical morphology of lymphocytes. Two-color immunofluorescence analysis with anti-CD4-1 mAb (6D1) and anti-CD4-2 mAb (D5) by flow cytometry revealed that the percentages of CD4-1 single positive (SP) and CD4-2 SP lymphocytes were comparatively higher than CD4-1/CD4-2 double positive (CD4 DP) lymphocytes in all tissues examined in ginbuna. The highest percentage of CD4-2 SP cells (∼40%) was found in the thymus, while the head-kidney exhibited the highest percentages of CD4-1 SP (∼30%) and CD4 DP (∼5%) cells. These findings indicated that ginbuna CD4+ lymphocyte population consists of two major subpopulations (CD4-1 SP and CD4-2 SP) and a minor subset (CD4 DP).

Decreased resistance to bacterial cold-water disease and excessive inflammatory response in ayu (Plecoglossus altivelis) reared at high water temperature.

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

Cell-mediated immune response against mycolic acids of Mycobacteroides salmoniphilum in rainbow trout Oncorhynchus mykiss.

Mycobacteriosis caused by Mycobacterium spp. causes economic damages to the world aquaculture industry. In mammals, mycolic acids contained in the cell wall of Mycobacterium spp. are presented by CD1b molecule as lipid antigens and induce cell-mediated immunity (CMI). Here, we investigated CMI responses against the mycolic acids of Mycobacterioides salmoniphilum in a CD1-lacking teleost fish, rainbow trout. After stimulation of trout leukocytes with mycolic acids, the number and percentage of CD8α+ T cells increased. Fish immunized with mycolic acids showed an up-regulation of IFN-γ. Further, in vitro re-stimulation of leukocytes derived from immunized fish resulted in proliferation of CD8α+ cells. These data suggest that mycolic acids are recognized as lipid antigens resulting in an activation of rainbow trout CD8α+ cells and up-regulation of the Th1 cytokine IFN-γ. The mycolic acids are promising candidates for vaccines to activate CD8α+ T cells against fish mycobacteriosis.

Reactivation of cyprinid herpesvirus 2 (CyHV-2) in asymptomatic surviving goldfish Carassius auratus (L.) under immunosuppression.

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen of goldfish (Carassius auratus) and Prussian carp (Carassius auratus gibelio) causing herpesviral hematopoietic necrosis. Our previous study revealed that CyHV-2 can persistently infect the kidney and spleen of goldfish that recovered from a primary infection. In this study, we tried to identify the cells persistently infected with the virus in surviving fish and investigated virus reactivation in the survivors injected with immunosuppressants, namely dexamethasone (Dex) and cyclosporine A (CsA). Virus DNA was detected from the monocytes that were isolated from the trunk kidney of the asymptomatic survivors, suggesting that monocytes/macrophages are major cells that may be persistently infected with CyHV-2. A significant increase of virus DNA levels was detected in the group injected with Dex at 10 and 21 days post-injection (dpi). In the fish group injected with CsA, the virus DNA level was the same as that in the control group at 10 dpi but increased in some organs at 21 dpi. Compared with Dex-injected fish at 10 dpi, the group injected with both Dex and CsA showed a greater increase in virus DNA levels. The gene expression of phagocytosis-associated genes, major histocompatibility complex (MHC) class II and p47phox, and anti-virus antibody levels increased in the CsA group due to virus reactivation in the infected cells but not in the Dex and Dex & CsA groups, indicating that Dex effectively suppressed monocyte/macrophage function and antibody production. In addition, recombinant interferon γ (IFNγ) supplementation in the kidney leukocyte culture that was isolated from survivors showed a reduction of virus DNA. CsA may inhibit T-helper 1 (Th1) cells and consequently IFNγ production, causing a synergetic effect with Dex on virus reactivation. The results suggest that the activity of monocytes/macrophages stimulated by IFNγ can relate to virus latency and reactivation in asymptomatic virus carriers.

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery.

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20-33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.

Immune responses induced by oil-adjuvanted inactivated vaccine against Flavobacterium psychrophilum in ayu Plecoglossus altivelis.

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV-2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non-permissive temperature for 4 days, initiated at three different time points post-infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post-infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR-positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV-2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.

Granulomatous inflammation in ginbuna crucian carp Carassius auratus langsdorfii against Mycobacterium gordonae.

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.

A Novel Antigen-Sampling Cell in the Teleost Gill Epithelium With the Potential for Direct Antigen Presentation in Mucosal Tissue.

In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1ß, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the "phagosome", "lysosome," and "antigen processing and presentation" pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.

Maturation-associated changes in the non-specific immune response against Flavobacterium psychrophilum in Ayu Plecoglossus altivelis.

In this study, we investigated maturation-associated changes in non-specific immune responses of ayu against Flavobacterium psychrophilum. The gonadosomatic index was minimum on 16 June, began to increase on 17 July, and reached the maximum value during August. The highest phagocytic rate (16.3%) was observed on 16 June, which decreased significantly to 5.6% on 26 August. The number of viable bacteria after the serum treatment was highest during August, suggesting that bactericidal activity of the serum decreased along with the sexual maturation. Gene expression levels of interleukin-8, and tumor necrosis factor-α in the spleen did not change significantly during this period, whereas the level of suppressor of cytokine signaling (SOCS)3 was significantly higher on 26 August than that on 16 July (p < 0.05). These results suggest that phagocytic activity of trunk kidney leukocytes and serum bactericidal activity against F. psychrophilum decreased with sexual maturation, and that SOCS3 may be related to the decrease in non-specific immune activity in ayu.

Complete Genome Sequence of the Lytic Giant Bacteriophage pT24 Infecting Tenacibaculum spp., Isolated from a Shrimp Culture Pond.

The lytic bacteriophage pT24, which infects Tenacibaculum spp., was isolated from the water of a whiteleg shrimp (Litopenaeus vannamei) culture pond in Thailand. This giant bacteriophage with myovirus morphology comprised 234,670 bp with 296 predicted genes.

Expressed sequence tag analyses of three leukocyte subpopulations in ayu Plecoglossus altivelis altivelis, separated by monoclonal antibodies.

Ayu Plecoglossus altivelis altivelis are one of the most economically important fish for freshwater aquaculture in Japan. We conducted expressed sequence tag analyses of three leukocyte subpopulations, thrombocytes, neutrophils, and B lymphocytes in ayu using a next generation sequencer. The sequencing and de novo assembly yielded 22,494, 22,733, and 16,505 contigs from the thrombocyte, neutrophil, and B lymphocyte cDNA libraries, respectively. Pathways involving endocytosis, phagosomes, and lysosomes, were found in all three cDNA libraries using pathway analysis. The thrombocyte cDNA library contained 2894 unique sequences, including CXC chemokine receptor 4 and MHC class II. Cytokine and cytokine receptor genes such as interleukin (IL)-1ß, IL-8, IL-1 receptor (IL-1R), IL-8RA, and IL-8RB were found among the 3056 unique sequences of the neutrophil cDNA library. Typical B lymphocyte related genes such as B cell linker protein, immunoglobulin (Ig) M, IgD and transforming growth factor ß were found in the 1590 unique sequences of the B lymphocyte cDNA library. In summary, a large number of immune-related genes were identified from the three leukocyte cDNA libraries. Our results represent a valuable sequence resource for understanding the immune system function in ayu.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

Protective efficacy and immune responses induced by a DNA vaccine encoding codon-optimized PPA1 against Photobacterium damselae subsp. piscicida in Japanese flounder.

Photobacterium damselae subsp. piscicida (Pdp) kills many cultured marine fish. As it evolves resistance to existing vaccines, new vaccines are needed. PPA1 is a major antigenic protein of Pdp. Here, DNA vaccines encoding wild-type PPA1 (pPPA1(wt)) and codon-optimized PPA1 (pPPA1(opt)) were constructed and tested against Pdp in Japanese flounder. The mRNA levels of the two antigenic genes at the vaccination site were not different, but the protein level was significantly higher in the pPPA1(opt)-vaccinated fish. In addition, after a bacterial challenge, the levels of interleukin (IL)-1ß, IL-6 and IFN-γ mRNA significantly increased in the pPPA1(opt)-vaccinated fish but not in the pPPA1(wt)-vaccinated fish. The relative percent survival (RPS) after the challenge was higher in the pPPA1(opt)-vaccinated fish (90.9) than in the pPPA1(wt)-vaccinated fish (69.2). At the early stage of the infection after the challenge, the number of viable Pdp in the spleen was significantly lower in the pPPA1(opt)-vaccinated fish than in the pPPA1(wt)-vaccinated fish. These data show that codon-optimized DNA vaccine pPPA1(opt) had a strong immunogenicity and conferred protective efficacy against Pdp infection in Japanese flounder.

Protective efficacies and immune responses induced by recombinant HCD, atpD and gdhA against bacterial cold-water disease in ayu (Plecoglossus altivelis).

Protective efficacies of three antigenic proteins (3-hydroxyacyl-CoA dehydrogenase (HCD), ATP synthase beta subunit (atpD), and glutamate dehydrogenase (gdhA)) against Flavobacterium psychrophilum were investigated in ayu (Plecoglossus altivelis). Recombinant proteins of HCD, atpD, and gdhA were expressed in Escherichia coli BL21 cells. Ayu were then vaccinated with inactivated cells via the intraperitoneal route. Compared with the empty BL21- and PBS-injected groups, the vaccinated group had a significantly longer survival time after challenge with F. psychrophilum. The antibody titers against each recombinant protein were significantly higher in serum from vaccinated fish, compared with serum from control fish. Results of indirect immunofluorescence assays using serum indicated that the HCD, atpD, and gdhA proteins are located on the surface of F. psychrophilum. These results suggest that these three surface proteins are protective antigens and are good candidates for development of vaccines against bacterial cold-water disease in ayu.

Antigenic proteins of Flavobacterium psychrophilum recognized by ayu Plecoglossus altivelis antisera.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis altivelis and is responsible for substantial economic losses in ayu culture in Japan. To develop effective vaccines for the disease, we identified antigenic proteins of F. psychrophilum using immunoglobulin from ayu that had recovered from BCWD. The whole protein extracted from the bacterium was separated using 2-dimensional polyacrylamide gel electrophoresis and was transferred to a polyvinylidene fluoride membrane. Subsequently, antigenic proteins of the bacterium were detected using western blotting and ayu antisera against F. psychrophilum. Each protein spot showing antigenicity was subjected to tandem mass spectrometry (MS/MS) analysis using a MALDI-QIT-TOF mass spectrometer. Protein identification based on the MS/MS data was performed using the genome database for F. psychrophilum JIP02/86, and the subcellular localization for each identified protein was predicted with web-based tools (LipoP and PSORTb). In total, 62 antigenic proteins were identified: of these, 46 were putative cytoplasmic proteins (e.g. elongation factor Tu and heat shock protein 90). The remaining 21 proteins were identified as putative membrane-bound or secreted proteins and are potential vaccine candidates. These proteins include OmpA, Omp 121, M13 family metallopeptidase, and M48 family metalloprotease.

Vibrio anguillarum bacterin uptake via the gills of Japanese flounder and subsequent immune responses.

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

CD4 and CD8 homologues in Japanese flounder, Paralichthys olivaceus: Differences in the expressions and localizations of CD4-1, CD4-2, CD8α and CD8ß.

CD4 and CD8 molecules are co-receptors of T cell receptors which interact specifically with MHC class II and I, respectively, during antigen presentation. Here we investigated CD4 and CD8 expression patterns in a fish, Japanese flounder, Paralichthys olivaceus in response to infection and tuberculin injection. The CD4-1 mRNA level was gradually and weakly increased in trunk kidney after infection with Streptococcus iniae, Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV), while the CD4-2 mRNA level was dramatically increased after E. tarda and VHSV infection, but not increased after S. iniae infection. CD4-2 mRNA but not CD4-1mRNA increased in the kidney during tuberculin response which is mediated by memory Th1 cells. The patterns for the change of mRNA level in CD8α and CD8ß were similar to those of the CD4-2 during the infections and tuberculin response. Fluorescent in situ hybridization detected CD4-1 mRNAs on melano-macrophage centers and CD4-2 mRNAs at some cell clusters located near the melano-macrophage centers. CD8α and CD8ß mRNAs were detected at the same cell clusters in the spleen and head kidney. These results suggest that CD4-1 and CD4-2 are expressed in different cells and that CD4-2-positive cells, rather than CD4-1-positive cells, have a main role in Th1-related immune responses collaborating with CD8α- and CD8ß-positive cells in Japanese flounder.