Immune Responses In Japanese Flounder Research Articles

Enhancing immune responses in Japanese flounder (Paralichthys olivaceus) to Keyhole Limpet Hemocyanin by intramuscularly conjugating a new type of chemokine, JfCXCL8_L1b

CXCL8 is a high-profile chemokine in vertebrates. In our previous study, CXCL8_L1b was first identified in Japanese flounder and triggered varying immune responses in infected fish compared with Japanese flounder CXCL8_L1a (JfCXCL8_L1a). To further understand the immune functions of Japanese flounder CXCL8_L1b (JfCXCL8_L1b), in vitro and in vivo experiments were performed using the recombinant protein rCXCL8_L1b. Our results showed that rCXCL8_L1b significantly induced all the detected immune genes except IgM and IgD in peripheral blood leukocytes (PBLs) of Japanese flounder. Intramuscular injection of Keyhole Limpet Hemocyanin (KLH) did not cause inflammation responses in fish muscle. In the spleen of injected fish, IgM was the only detected gene expression. In contrast, all detected genes in the KLH group were significantly up-regulated in the head kidney, indicating the primary immune organ response to KLH was the head kidney instead of the spleen. At the injection site, we observed significantly induced receptor gene CXCR1 in KLH + rCXCL8_L1a, KLH + rCXCL8_L1b, and KLH + rCXCL8_L1a + rCXCL8_L1b groups on day 1, while significantly induced receptor gene CXCR2 was only observed in the KLH + rCXCL8_L1b group on day 3. Conversely, gene expression of JfCXCL8_L1a and CXCR2 were down-regulated in the head kidney of the KLH + rCXCL8_L1b group compared with the KLH + rCXCL8_L1a group. Intramuscular injection of KLH significantly induced an antibody titer response to KLH at weeks 2 and 4. Compared with rCXCL8_L1a, rCXCL8_L1b, as the immunologic adjuvant of KLH, significantly enhanced the antibody titer response to KLH at weeks 2 and 4. Collectively, this study reveals that JfCXCL8_L1a and JfCXCL8_L1b have different immune pathways, and JfCXCL8_L1b plays a significant role in enhancing the adaptive immunity of T cell-dependent antigen.

Characterization of Japanese flounder (Paralichthys olivaceus) STAT members: An immune-related gene family involved in Edwardsiella tarda and temperature stress.

The signal transducer and activator of transcription (STAT) family members are not only the transcriptional activators, but also play important roles in regulating inflammatory response. Some members have been reported to be involved in innate bacterial and antiviral immunity in aquatic organisms. However, no systematic research on STATs has been found in teleost. In this present study, we characterized six STAT genes in Japanese flounder based on bioinformatics methods, namely PoSTAT1, PoSTAT2, PoSTAT3, PoSTAT4, PoSTAT5 and PoSTAT6. The phylogenetic analysis of STATs in fish indicated that STATs were highly conserved and revealed an absence of STAT5 in a few species. Further analysis of gene structures and motifs showed STAT proteins shared a similar structure and probably had similar functionality in Japanese flounder. The expression profiles of different development stages and tissues demonstrated that PoSTATs exhibited specificity in temporality and spatiality as well as PoSTAT4 was highly expressed in gill. The transcriptome data analysis of E. tarda and temperature stress showed that PoSTAT1 and PoSTAT2 were more respective to these two kinds of stress. In addition, the results also demonstrated that these PoSTATs might regulate immune response in different ways, manifested by up-regulation in E. tarda infection and down-regulation in temperature stress. In a word, this systematic analysis of PoSTATs would provide valuable information about the phylogenetic relationship of STATs in fish species and help understand the role of STAT genes in the immune response of Japanese flounder.

Genome-wide identification of TNF receptor-associated factors in Japanese flounder (Paralichthys olivaceus) and functional analysis of resistance to temperature and Edwardsiella tarda stress.

Tumor necrosis factor receptor-associated factors (TRAFs), as the signaling mediators of the tumor necrosis factor (TNFR) superfamily, toll-like receptors (TLR) and interleukin-1 receptor (IL-1R) superfamily, can activate downstream signal transduction pathways and play an important role in the body's immune process. In this study, six TRAF genes, namely PoTRAF2a, PoTRAF2b, PoTRAF3, PoTRAF4, PoTRAF6 and PoTRAF7, were identified and annotated in Japanese flounder by using bioinformatics methods. Phylogenetic analysis confirmed that TRAF genes can be divided into seven groups. Analysis of motif composition and gene structure demonstrated that all PoTRAF members were evolutionarily conserved. The expression patterns of PoTRAF genes were then further investigated in six different developmental stages and eleven tissues of healthy fish, and it was found that there were spatial and tissue specificities among the members. To investigate the immune response of Japanese flounder to abiotic and biotic stresses, we further analyzed the expression profile of PoTRAFs after temperature stress and pathogen challenge. The result showed that PoTRAF3 and PoTRAF4 were observably differentially expressed under temperature stress, indicating that they were involved in the immune response after temperature stress. The expression of PoTRAF2a, PoTRAF2b and PoTRAF4 was significantly different after E. tarda infection, suggesting that they might have antibacterial effects. These results would help to clarify the molecular roles of PoTRAF genes in the regulation of immune and inflammatory responses in Japanese flounder.

Edwardsiella tarda triggers the pyroptosis of the macrophage of Japanese flounder (Paralichthys olivaceus)

Pyroptosis is a caspase-1-dependent form of programmed cell death and is characterized by cell lysis and inflammatory cytokine release, which can remove replication niches by releasing intracellular pathogens and facilitating the direct killing of intracellular pathogens by neutrophils or other cells. A method for separating and identifying macrophages of Japanese flounder was described in this study. Upon the stimulation with Edwardsiella tarda, significant swelling with characteristic large bubbles from the membrane was observed in macrophages, which was also positive for Annexin-V and propidium iodide (PI) stain. DNA fragmentation of the macrophage was detected by TUNEL and DNA ladder assays. LDH release assay showed that E. tarda infection impaired cell membrane integrity. The expression levels of pyroptosis-related genes and inflammatory cytokines were substantially up-regulated after E. tarda infection by qRT-PCR, strongly indicating that E. tarda triggered the pyroptosis in the macrophages which played a vital role in the immune response of Japanese flounder after E. tarda invasion. Additionally, treatment of VX-765, a small-molecule inhibitor of caspase-1, significantly reduced macrophages pyroptosis caused by E. tarda, implying that pyroptosis was associated with caspase-1. This study could improve our understanding of the immune mechanism underlying the resistance of teleosts to pathogen invasion.

Gene network analysis reveals a core set of genes involved in the immune response of Japanese flounder (Paralichthys olivaceus) against Vibrio anguillarum infection

Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine fish cultured in north Asia. Vibrio anguillarum is a severe bacterial pathogen to Japanese flounder and many other aquaculture species. In order to understand the immune response of flounder during bacterial infection, we systematically examined the transcriptome profiles of flounder spleen at three time points after V. anguillarum challenge. More than one billion high quality reads were obtained, approximately 80.70% of which were successfully mapped to the reference genome of flounder. A total of 6060, 4688 and 4235 differentially expressed genes (DEGs) were captured at 6, 12 and 24-h post-infection, respectively. The DEGs exhibited dynamic changes in expression and were assigned into four different profiles based on expression trend. GO and KEGG analysis showed that the DEGs were enriched in various immune-related terms, including response to stimulation, immune system and pathways of cytokine-cytokine receptor interaction, Jak-STAT signaling and Toll-like receptor signaling. Furthermore, a network of highly interactive DEGs involved in 11 immune-related pathways was detected by utilizing the weighted co-expressing network analysis (WGCNA). Accordingly, 26 hub genes were discovered that constituted an elaborate immune regulatory network and functioned mainly in pathogen recognition, antigen processing, and molecular signaling. The results of this study provided the first systematical transcriptome profile of flounder in association with V. anguillarum infection and can serve as a valuable resource of target genes for future studies on the molecular mechanisms underlying the immune defense of flounder against bacterial infection.

Identification and characterization of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) involved in regulating extracellular ATP-mediated innate immune responses in Japanese flounder (Paralichthys olivaceus)

Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.

Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda

We evaluated the effects of Freund's adjuvants (FCA/FIA) on protection and immune response of Japanese flounder Paralichthys olivaceus immunized by the formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA did not confer protection against the challenge, while they significantly induced higher antibody titers than that of FKC only. The suppression of FKC-dependent induction of interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after FKC+FCA or +FIA. These results suggest that the mineral oil in Freund's adjuvants might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.

Transcriptome profiling provides gene resources for understanding gill immune responses in Japanese flounder (Paralichthys olivaceus) challenged with Edwardsiella tarda

Marine organisms are commonly under threats from various pathogens. Edwardsiella tarda is one of the fish pathogens that seriously infect cultured and wild fish species. Bacteremia caused by E. tarda can be a fatal disease in humans. Fish gill is a mucosa-associated lymphoid tissue that directly contacted with sea water. Generating gill transcriptomic resources that challenged by E. tarda is crucial for understanding the molecular mechanisms underlying gill immune responses. In this study, we performed transcriptome profiling of gene expression in Japanese flounder gills (Paralichthys olivaceus) challenged by E. tarda with different stress duration. An average of 40 million clean reads per library were obtained, of which approximately 83.2% were successfully mapped to the reference genome. 456 and 1037 differential expressed genes (DEGs) were identified at 8 h and 48 h post-injection, respectively. Gene annotation analysis and protein–protein interaction networks were conducted to obtain the key interaction relationships of immune-related DEGs during pathogens infection. 24 hub genes with multiple protein-protein interaction relationships or involved in multiple KEGG signaling pathways were discovered and validated by qRT-PCR. These hub genes mainly participated in Leukocyte transendothelial migration signaling pathway, B cell receptor signaling pathway, Wnt signaling pathway and Apoptosis signaling pathway. This study represents the first gill transcriptomic analysis based on protein–protein interaction networks in fish and provides valuable gene resources for understanding the fish gill immunity, which can pave the way to understand the molecular mechanisms of immune responses with E. tarda infection.

Proteome profiling reveals immune responses in Japanese flounder (Paralichthys olivaceus) infected with Edwardsiella tarda by iTRAQ analysis

The liver is an important organ for bacterial pathogen attack in fish. The differential proteomic response of the Japanese flounder liver to Edwardsiella tarda infection was examined using isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 3290 proteins were identified and classified into categories related to biological process (51.4%), molecular function (63.6%), and cellular component (57.7%). KEGG enrichment analysis indicated the complement and coagulation cascade pathways and the mineral absorption pathway were significantly enriched. Among the differentially expressed proteins, those involved in mediating complement cascade (e.g. complement component C7, C8, C9, complement factor H, complement factor Bf/C2) and mineral absorption (e.g. ferritin, STEAP-4) were most significantly upregulated during infection. Subsequently, five significantly upregulated (C4, C8beta, ferritin middle subunit, PRDX4-like and KRT18) and one significantly downregulated (transferrin) candidate immune proteins were validated by multiple reaction monitoring (MRM) assays. Furthermore, changes in expression of 15 proteins in the complement cascade and mineral absorption pathways were validated at the transcriptional level using quantitative real-time PCR (qPCR). The transcriptional levels of four transcription factors (p21Ras, Rab-31-like, NF-κB, STAT3) were also investigated by qPCR following infection with E. tarda. This study contributes to understanding the defense mechanisms of the liver in fish.

The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder (Paralichthys olivaceus).

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (109, 108, 107, 106cfuml-1) for 30, 60 and 90min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 109-30, 109-60, 108-60, 108-90 and 108-90 groups than other groups (P<0.05), especially the 108-60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡα, TcRα, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1β, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 107-90, 108 and 109cfuml-1 groups than other groups (P<0.05), especially the 108-60 group displayed the highest up-regulation. These results demonstrated that immersion with formalin-inactivated E.tarda, especially under 108-60min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time.

Poly(I:C) induces antiviral immune responses in Japanese flounder (Paralichthys olivaceus) that require TLR3 and MDA5 and is negatively regulated by Myd88.

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

Adjuvant and immunostimulatory effects of LPS and β-glucan on immune response in Japanese flounder, Paralichthys olivaceus

In order to investigate the adjuvant and immunostimulatory effects of LPS and β-glucan, a highly effective vaccine candidate for Edwardsiella tarda known as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was recombinantly expressed in Escherichia coli, which was intramuscularly injected into Japanese flounder alone or containing LPS or β-glucan. ELISA analysis of sera showed that the fish immunized with rGAPDH containing LPS or β-glucan had significant higher antibody titers than rGAPDH immunization group at day 14 and 30 (P<0.05). Flow cytometric analysis showed that the surface immunoglobulin positive (sIg+) lymphocytes percentages in head kidney of the immunized fish exhibited significant higher than the fish of control group at day 14 and 30, and immunization with rGAPDH containing LPS or β-glucan could induce significant higher sIg+ percentages than immunization with rGAPDH alone. Simultaneously, the expressions of genes coding for immunoregulatory molecules involved in innate and acquired responses, including IFN-γ, IL-1β, IL-6, TNF-α, TCRα, IgM, C-type lysozyme and MHC-IIα were analyzed by quantitative PCR. The transcription levels of all selected genes except TNF-α displayed up-regulation after immunization, and immunization with rGAPDH containing LPS or β-glucan could induce significant higher levels than that induced only by rGAPDH. Moreover, to investigate the protection against infection, challenge with E. tarda was performed at day 30 after immunization. Fish immunized with rGAPDH containing β-glucan exhibited the highest relative percentage survival (RPS) of 63% compared to the other groups. These results indicated that LPS and β-glucan employed as co-immunostimulatory molecules could significantly trigger the innate and acquired immune responses in Japanese flounder.

Mycobacterium bovis BCG vaccine induces non-specific immune responses in Japanese flounder against Nocardia seriolae

Nocardiosis caused by Nocardia seriolae has been causing severe loss of fish production, so that an effective vaccine is urgently needed. Mycobacterium bovis BCG (BCG) is a live attenuated vaccine for tuberculosis, which is effective against various infectious diseases including nocardiosis in mammals. In this study, the protective efficacy of BCG against N. seriolae was evaluated in Japanese flounder Paralichthys olivaceus and antigen-specific immune responses induced in BCG vaccinated fish were investigated. Cumulative mortality of BCG-vaccinated fish was 21.4% whereas that of PBS-injected fish was 56.7% in N. seriolae challenge. However, gene expression level of IFN-γ was only slightly up-regulated in BCG-vaccinated fish after injection of N. seriolae antigen. In order to reveal non-specific immune responses induced by BCG vaccination, transcriptome of the kidney after BCG vaccination was investigated using oligo DNA microarray. Gene expression levels of antimicrobial peptides such as C-type and G-type lysozyme were significantly up-regulated after BCG vaccination. Consistently, BCG vaccination appeared to increase the bacteriolysis activity of the serum against Micrococcus luteus and N. seriolae. These results suggest that BCG-vaccinated Japanese flounder fight N. seriolae infection mainly by non-specific immune responses such as by the production of bacteriolytic lysozymes.

A novel immune-related gene, microtubule aggregate protein homologue, is up-regulated during IFN-γ-related immune responses in Japanese flounder, Paralichthys olivaceus

Delayed-type hypersensitivity (DTH) response mediated by antigen-specific Th1 cells is used as a test to detect exposure to tuberculosis in humans. Japanese flounder ( Paralichthys olivaceus) microtubule aggregate protein homologue (PoMTAP) was identified as a gene strongly induced during fish DTH response. In this study, PoMTAP gene was cloned and its expression profile was analyzed. The PoMTAP gene has a transcriptional regulatory region that includes two interferon-stimulated response elements and two IFN-γ activated sites. Expressions of PoMTAP and IFN-γ genes were up-regulated at the same time points during the DTH response, Edwardsiella tarda infection and VHSV infection. Furthermore, PoMTAP gene expressing cells also expressed CD3ε, confirming that PoMTAP is expressed by T lymphocytes. These results suggest that PoMTAP is a novel immune-related gene expressed by T lymphocytes that is preferentially induced by IFN-γ and has a role in Th1-mediated immune responses in Japanese flounder.

Effect of temperature on immune response of Japanese flounder ( Paralichthys olivaceus) to inactivated lymphocystis disease virus (LCDV)

Using flow cytometric analysis, the dynamics of surface immunoglobulin positive (sIg+) cells in lymphoid organs of Japanese flounder ( Paralichthys olivaceus) reared at 9, 15, 21 and 26 °C, was investigated following intraperitoneal injection with inactivated lymphocystis disease virus (LCDV). The results showed that the percentages of sIg+ cells were suppressed in peripheral blood leucocytes (PBL), spleen leucocytes (SL) and head kidney leucocytes (HKL) from 9 °C to 15 °C immunized groups, and arrived at their peaks (9 °C: 26.12% in PBL, 18.84% in SL, 17.53% in HKL; 15 °C: 38.82% in PBL, 25.38% in SL, 23.95% in HKL) at 9th and 7th week after immunization, respectively. While the proportions of sIg+ cells in PBL, SL and HKL increased most prominent in the 21 °C group and reached the peaks (54.16% in PBL, 30.32% in SL, 30.23% in HKL) at 5th week. The responses of sIg+ cells from 26 °C group were similar to that from 21 °C group and reached the peaks (35.3% in PBL, 26.24% in SL, 21.83% in HKL) at 5th week. Simultaneously, the kinetics of the specific antibody titer against LCDV in sera was determined. It was shown that the antibody response in the 21 °C group was most prominent and reached the peak earliest. These results indicated inactivated LCDV elicited the most powerful immune response when Japanese flounder maintained at the optimal temperature (21 °C) and obtained the most effective immunization, while the response were suppressed at 9 °C, 15 °C or 26 °C.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5.

Effect of Total Genomic DNA from Probiotic Bacteria on Non-specific Immune Response of Japanese Flounder, Paralichthys olivaceus

Effect of Total Genomic DNA from Probiotic Bacteria on Non-specific Immune Response of Japanese Flounder, Paralichthys olivaceus

Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis

A viral hemorrhagic septicemia virus (VHSV), recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg) were injected in Japanese flounder ( Paralichthys olivaceus). Each fish was injected with 10 μg vaccine dissolved in 50 μl phosphate buffer saline (PBS). One month after the vaccination, the fish were challenged intraperitoneally with either 1 × 10 2 or 1 × 10 3 TCID 50 of virus. Fish that received the VHSg DNA vaccine were highly protected against virus infection over a 21-day observation period, with cumulative mortalities ranging from 4 to 10%. However, the recombinant protein vaccine group appeared to have low survival rates. Using microarray analysis, humoral defense-related genes such as complement component C3, complement regulatory plasma proteins, IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were observed to be up-regulated by the VHSg recombinant protein vaccine at 1 or 21 days post vaccination. On the other hand, cellular defense-related genes such as CD8 alpha chain, T-cell immune regulator, MIP1-α and apoptosis-associated protein were not detected. Specific antibodies against VHSVg protein were detected in both vaccinated groups at a titer of 1:40 at 28 days post vaccination.

Detection of Antibody against Lymphocystis Disease Virus in Japanese Flounder by Enzyme Linked Immunosorbent Assay.

Enzyme linked immunosorbent assay (ELISA) was applied to detect the antibody of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus (LCDV). Purified Japanese flounder LCDV was used as a capture antigen of ELISA. Monitoring the immune response of Japanese flounder to LCDV was done using the sera obtained from apparently healthy, LCD-diseased and LCD-recovered fish. ELISA absorbances of these three groups were clearly different. Using the serum with high ELISA absorbance, the optimum ELISA condition was set up. Apparently healthy fish were injected with inactivated LCDV. One to three months after the injection, the antibody against LCDV was detected by ELISA established for LCDV and their ELISA antibody titer increased during that period.

Acquired Protection against Neobenedenia girellae in Japanese Flounder.

The immune response of Japanese flounder (Paralichthys olivaceus) against Neobenedenia girellae (Hargis, 1955) (Monogenea : Capsalidae) was investigated. An acquired protection of fish against secondary infection with the parasite was demonstrated by a reduction in the number and body size of parasites on primed fish which had previously been infected with N. girellae and treated by freshwater bath. In the same experiment, however, no raise in the level of antibody in the sera of primed fish was detected by the enzyme-linked immunosorbent assay (ELISA). In another experiment, although antibody was detected by ELISA in the sera of fish immunized by injection with sonicated parasite antigen, there was no significant difference in the parasite counts between antigen-injected and PBS-injected control fish when challenged with oncomiracidia of N. girellae. These results indicate that the protection induced by the previous infection was not associated with the humoral antibody.