Inactivated Vibrio Anguillarum Research Articles

Genome-wide identification, phylogenetic relationships and expression patterns of the NOD-like receptor (NLR) gene family in flounder (Paralichthys olivaceus)

NOD-like receptors (NLRs) are one of the pattern recognition receptors which have been widely known for identifying pathogens and regulating innate immunity in mammals, but the functions of the NLR gene family in teleost fish remain poorly understood. In this study, we conducted a comprehensive identification and analysis of the flounder (Paralichthys olivaceus) NLR gene family, including bioinformatics information, evolutionary relationships, gene structures, conserved motifs, domain composition, expression patterns and protein-protein interaction (PPI). We identified 22 NLRs in flounder (flNLRs) which were clustered into three subfamilies according to their domain organizations and phylogenetic features, i.e., NLR-A (6 members) resembling mammalian NODs, NLR-B (1 member) resembling mammalian NLRPs, and NLR-C (15 members) unique to teleost fish. All flNLRs shared a conserved NACHT domain including an N-terminal nucleotide-binding domain, a middle helical domain 1, and a winged helix domain. Gene structure analysis displayed that flNLRs were significantly different, with exon numbers from 1 to 52. Conserved domain analysis showed that the N-terminus of flNLRs possessed different characteristics of the domains including CARD domain, PYRIN domain, RING domain, and fish-specific FISNA domain, and the C-terminus of seven NLR-C members contained an extra B30.2 domain, named NLRC-B30.2 group. Notably, flNLRs were expressed in all nine tested tissues, showing higher expressions in the systemic and mucosal immune tissues (e.g., kidney, spleen, hindgut, gills, skin, liver) in healthy flounder, and significant responses to intraperitoneal injection and immersion immunization of inactivated Vibrio anguillarum in mucosal tissues, especially the NLR-C members. In addition, PPI analysis demonstrated that some flNLRs of NLR-A and NLR-C shared the same interacting proteins such as RIPK2, TRAF6, MAVS, CASP, ASC, and ATG5, suggesting they might play crucial roles in host defense, antiviral innate immunity, inflammation, apoptosis and autophagy. This study for the first time characterized the NLR gene family of flounder at the genome-wide level, and the results provided a better understanding of the evolution of the NLR gene family and their immune functions in innate immunity in fish.

Identification and functional analysis of histone 1.2-like in red sea bream (Pagrus major)

Histone H1 acts as an essential chromatin component and participates in the formation of higher chromatin structures together with core histones. In addition, H1 also has important functions in physiological processes such as gene expression regulation, DNA repair, and the immune response. In this study, the histone homologous protein Pm-H1.2-like was identified from the transcriptome database of Pagrus major we studied previously. Conservatism of evolution was investigated by sequence alignment and phylogenetic analysis. Transcripts of Pm-H1.2-like were detected in P. major tissues. The highest expression level was found in gill and skin tissues. Consistent with the data from the transcriptome database, we observed that the expression of Pm-H1.2-like was rapidly induced in nonspecific cytotoxic cells (NCCs) infected with inactivated Vibrio anguillarum. Gene silencing of Pm-H1.2-like by RNAi significantly suppressed the expression of NK-lysin and GZMB in NCCs at 12 h after pathogen stimulation, but had no significant effect on IFN-γ expression. Next, we obtained the fusion proteins rPm-H1.2-like and rPm-H1.2-like (36–80) through prokaryotic expression. ELISA showed that rPm-H1.2-like bound to oligonucleotide (ODN) in a concentration-dependent manner, while no binding activity of rPm-H1.2-like (36–80) with ODN was observed. This study confirmed that Pm-H1.2-like actively participates in the immune response of NCCs to bacterial infection, deepening the understanding of the immune features of histone H1 in fish.

Transepithelial Secretion of Mucosal IgM Mediated by Polymeric Immunoglobulin Receptor of Flounder (Paralichthys olivaceus): In-Vivo and In-Vitro Evidence.

Secretory immunoglobulin (SIg) is crucial for mucosal surface defenses, but the transepithelial secretion of SIg mediated by polymeric immunoglobulin receptor (pIgR) is not clarified in fish. We previously found that flounder (Paralichthys olivaceus) pIgR (fpIgR) and secretory IgM (SIgM) increased in gut mucus post-vaccination. Here, the fpIgR-positive signal was mainly observed in the intestinal epithelium, whereas the IgM-positive signal was mainly distributed in the lamina propria, before immunization. IgM signals increased in the lamina propria and then in the epithelium after immunization with inactivated Vibrio anguillarum, and co-localization between IgM and fpIgR in the epithelium was determined, while the presence of EdU+IgM+ cells in the lamina propria identified the proliferative B cells, revealing that the secretion and transepithelial transport of SIgM locally occurred in the gut of flounder. Subsequently, we established an in-vitro model of transfected MDCK cells that stably expressed the fpIgR. After a recombinant eukaryotic expression plasmid (pCIneoEGFP-fpIgR) was constructed and transfected into MDCK cells, stable expression of the fpIgR in transfected MDCK-fpIgR cells was confirmed, and the tightness and integrity of the polarized cell monolayers grown on Transwells were evaluated. Afterward, the serum IgM of flounder was purified as a binding ligand and placed in the lower compartment of Transwells. An ~800-kDa protein band in the upper compartment was shown to be IgM- and fpIgR-positive, and IgM-positive fluorescence was seen in MDCK-fpIgR cells but not in MDCK-mock cells. Hence, the fpIgR helped polymeric IgM to pass across MDCK-fpIgR cells via transcytosis in a basolateral-to-apical fashion. These new findings provide a better understanding of the pathways shaping mucosal IgM responses and the local mucosal immune mechanisms in teleosts.

Upregulation of polymeric immunoglobulin receptor expression in flounder (Paralichthys olivaceus) gill cells by cytokine tumor necrosis factor-α via activating PI3K and NF-κB signaling pathways

The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11−7082, and the mixtures of TNF-α and wortmannin / Bay11−7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11−7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11−7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11−7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11−7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.

A CpG-riched plasmid as vaccine adjuvant reduce antigen dose of an inactivated Vibrio anguillarum vaccine in turbot (Scophthalmus maximus L.)

Inactivated vaccines are often applied with adjuvants in commercial fish farming. Although some mineral or non-mineral oil adjuvants show efficient improvement with inactivated vaccines, but sometimes bring side effects such as tissue adhesion and granulomatous lesion at the injection site. CpG ODN is a novel type of soluble adjuvant which has been proved to possess excellent advantages in fish vaccine development. In this study, we designed a tandem sequence of CpG ODN synthesized in plasmid pcDNA 3.1, and an inactivated Vibrio anguillarum vaccine developed in our previous work was chosen for determining the efficiency of the CpG-riched plasmids (pCpG) as an adjuvant. Results showed that pCpG we designed can offer higher immunoprotection with the vaccine. Interestingly, even below the minimum immune dosage of the vaccine, a high RPS of 84% was observed once the vaccine was administrated with the pCpG. Serum specific antibody titer, superoxide dismutase and total protein were enhanced and some immune genes related to both innate and adaptive immune response were upregulated, implying an effective auxiliary function of the pCpG. Totally, our study suggested that the pCpG is a potential and available adjuvant for turbot vaccine development.

Polymeric immunoglobulin receptor and mucosal IgM responses elicited by immersion and injection vaccination with inactivated Vibrio anguillarum in flounder (Paralichthys olivaceus)

Polymeric immunoglobulins (Igs) and polymeric immunoglobulin receptor (pIgR) play crucial roles in teleost mucosal surface defenses, but their mucosal immune responses, especially under different routes of vaccine administration, needs to be better clarified. In the present study, the flounder (Paralichthys olivaceus) was immunized with inactivated Vibrio anguillarum by intraperitoneal injection and immersion, and pIgR and IgM responses were analyzed. Real-time PCR showed that pIgR and IgM mRNA expression were up-regulated with a similar variation trend, but pIgR mRNA responded earlier than IgM; the pIgR mRNA levels were higher in spleen, gills, skin and hindgut in both immunized groups, while IgM mRNA response was higher in skin, gills, hindgut and liver in immersion group, or head kidney, spleen, hindgut and liver in injection group. Immunohistochemical results revealed that pIgR and IgM were localized in the epithelium of skin, gills and hindgut, and biliary epithelium of liver. Western blot demonstrated that a ~800 kDa protein band in skin, gill and gut mucus and bile reacted with anti-pIgR and anti-IgM antibody, indicating that secretory component (SC) of pIgR associated with polymeric IgM as a complex in these secretions. ELISA assay revealed that the vaccine-specific IgM and SC of pIgR levels were up-regulated in skin, gill and gut mucus and bile, reaching a higher peak level at earlier time in skin and gill mucus in immersion than injection group, and a higher peak level in bile and gut mucus in injection group. These results illustrated that immersion and injection vaccination stimulated pIgR and secretory IgM production in mucus and bile, and a disparity in pIgR and IgM response was observed under different administration routes, which provided better understanding for fish mucosal immunity and in turn helped to develop vaccination strategies aimed at maximizing mucosal and consequently fish health.

Molecular cloning of polymeric immunoglobulin receptor-like (pIgRL) in flounder (Paralichthys olivaceus) and its expression in response to immunization with inactivated Vibrio anguillarum

In the present work, the polymeric immunoglobulin receptor-like (pIgRL) from flounder (Paralichthys olivaceus) was firstly cloned and identified. The full length cDNA of flounder pIgRL was of 1393 bp including an open reading frame of 1053 bp, and the deduced pIgRL sequence encoded 350 amino acids, with a predicted molecular mass of 39 kDa. There were two immunoglobulin-like domains in flounder pIgRL. In healthy flounder, the transcriptional level of pIgRL was detected in different tissues by real-time PCR, showing the highest level in the skin and gills, and higher levels in the spleen and hindgut. After flounders were vaccinated with inactivated Vibrio anguillarum via intraperitoneal injection and immersion, the pIgRL mRNA level increased firstly and then declined in all tested tissues during 48 h, and the maximum expression levels in the gills, skin, spleen and hindgut in immersion group, or in the spleen, head kidney, skin and gills in injection group, were higher than in other tested tissues. In addition, recombinant protein of the extracellular region of flounder pIgRL was expressed in Escherichia coli BL21 (DE3), and rabbit anti-pIgRL polyclonal antibodies were prepared, which specifically reacted with the recombinant pIgRL, and a 39 kDa protein confirmed as natural pIgRL by liquid chromatography-mass spectrometry in skin mucus of flounder. Co-immunoprecipitation assay and western-blotting demonstrated that the pIgRL, together with IgM, could be immunoprecipitated by anti-pIgRL antibody in gut, skin and gill mucus of flounder, suggesting the existence of pIgRL-IgM complexes. These results indicated that the flounder pIgRL was probably involved in the mucosal IgM transportation and played important roles in mucosal immunity.

Modulatory in vitro effect of stress hormones on the cytokine response of rainbow trout and gilthead sea bream head kidney stimulated with Vibrio anguillarum bacterin

In fish, the stress response and their consequences in the immune system have been widely described. Recently, a differential cytokine regulation between rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) was reported after treatment with stress hormones together with their receptor antagonists. Nevertheless, there is no evidence of whether antagonists for stress hormone receptors may influence the interaction between hormones and cytokines after bacterial administration. Thus, the aim of our study was to evaluate the cytokine expression in the presence of stress hormones (cortisol, ACTH, adrenaline), hormone receptor antagonists and inactivated Vibrio anguillarum bacterin in rainbow trout and gilthead sea bream head kidney primary cell culture (HKPCC). Mifepristone, spironolactone, propranolol and phentolamine were used to block GR, MR, MC2R, and β-/α-adrenoreceptors.Our results showed an expected increase of the pro-inflammatory and anti-inflammatory response after inactivated V. anguillarum bacterin treatment in both species. Cortisol, ACTH and adrenaline did not modulate the expression of immune-related genes in rainbow trout, while in sea bream cortisol was able to reduce the stimulated gene expression of all cytokines. This effect was only restored to basal expression level in IL-1β and TNF-α by mifepristone. ACTH reduced both pro-inflammatory and anti-inflammatory cytokine expression, excluding IL-1β, only in sea bream. Adrenaline enhanced the expression of IL-1β and TGF-β1 stimulated by inactivated V. anguillarum in sea bream, and the effect was diminished by propranolol. In sum, our results confirm that the immunoendocrine differences reported at gene expression profile between two teleost species are also observed after exposure to inactivated V. anguillarum bacterin, suggesting that stress hormones would differentially modulate the immune response against pathogens in teleost species.

Influence of moderate hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus (Nile tilapia).

Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55±5% DO) or normoxia (85±5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V.anguillarum were significantly higher in normoxic fish. Serum killing of V.anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 β, TCR-β, MHC-II β), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.

Deep transcriptome sequencing of Pecten maximus hemocytes: A genomic resource for bivalve immunology

Pecten maximus, the king scallop, is a bivalve species with important commercial value for both fisheries and aquaculture, traditionally consumed in several European countries. Major problems in larval rearing, however, still limit hatchery-based seed production. High mortalities during early larval stages, likely related to bacterial pathogens, represent the most relevant bottleneck. To address this issue, understanding host defense mechanisms against microbes is extremely important.In this study next-generation RNA-sequencing was carried on scallop hemocytes. To enrich for immune-related transcripts, cDNA libraries from hemocytes challenged in vivo with inactivated-Vibrio anguillarum and in vitro with pathogen-associated molecular patterns, as well as unchallenged controls, were sequenced yielding 216,444,674 sequence reads. De novo assembly of the scallop hemocyte transcriptome consisted of 73,732 contigs (31% annotated). A total of 934 contigs encoded proteins with a known immune function, grouped into several functional categories. Particular attention was reserved to Toll-like receptors (TLRs), a family of pattern recognition receptors (PRRs) involved in non-self recognition. Through mining the scallop hemocyte transcriptome, at least four TLRs could be identified. The organization of canonical TLR domains demonstrated that single cysteine cluster and multiple cysteine cluster TLRs co-exist in this species. In addition, preliminary data concerning their mRNA level following bacterial challenge suggested that different members of this family could exhibit opposite responses to pathogenic stimuli. Finally, a global analysis of differential expression comparing gene-expression levels in in vitro and in vivo stimulated hemocytes against controls provided evidence on a large set of transcripts involved in the great scallop immune response.

Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149bp and contained a 168bp 5′ UTR, a 580bp 3′ UTR and a 1401bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P<0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.

Analysis of Immune Response in Yellowtail upon Vaccination with a Bivalent Vaccine of Vibrio anguillarum and Lactococcus garvieae

We inoculated yellowtail Seriola quinqueradiata with a commercial bivalent vaccine containing inactivated Vibrio anguillarum and Lactococcus garvieae to better understand the immunological responses to the vaccination. Serum samples were collected from vaccinated and control (PBS-injected) fish at 1, 3, 5, 7, 10, and 14 days post-vaccination (dpv). Compared to the controls, the serum from the vaccinated fish inhibited the proliferation of V. anguillarum from 7 dpv. However, vaccination had no effect on L. garvieae proliferation. The antibody titer against V. anguillarum obviously increased from 7 dpv, but no such increase was detected for L. garvieae. We confirmed that the protection conferred by the V. anguillarum vaccine was a result of the antibodies against the pathogen. However, humoral factors related to protection were not observed for L. garvieae. The expression of immune-related genes in the head kidney was profiled by microarray analysis. Twenty-two immune-related genes were up-regulated during the experimental period. Up-regulation of cellular immune-related genes, such as tumor necrosis factor, CC chemokine, and macrophage colony-stimulating factor receptor, was mostly observed from 1 to 3 dpv, whereas that of humoral immune-related genes was observed from 3 to 14 dpv. Notably, immunoglobulin-related genes showed continuous up-regulation during the 2-week experimental period.

MRNA expression patterns of the BPI/LBP molecule in the Atlantic cod ( Gadus morhua L.)

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are important components of the mammalian innate defence system against Gram-negative infections. cDNA encoding a protein related to mammalian BPI and LBP have been cloned from several teleosts including the Atlantic cod ( Gadus morhua L.). Using real-time PCR an increase in cod BPI/LBP expression in whole blood and peritoneal cells was demonstrated one, two and four days after intraperitoneal injection of inactivated Vibrio anguillarum. Although constitutively produced in the head kidney, a moderate rise of BPI/LBP mRNA production was seen on day two in this organ. After seven days the BPI/LBP mRNA levels at the three locations examined were almost back to normal. In situ hybridisation demonstrated a leucocytic localisation and morphology of the BPI/LBP expressing cells in various tissues. A combination of in situ hybridisation and peroxidase staining of head kidney leucocytes showed that the BPI/LBP producing cells are peroxidase positive and possible neutrophil like cells. The results suggest that the cod BPI/LBP is important in the first-line defence against bacterial infections and has a function more related to the mammalian BPI molecule than the LBP counterpart.

Immunological suppression in gilthead sea bream Sparus aurata of the North-West Mediterranean at low temperatures

Non-specific immunological indicators were determined in gilthead sea bream Sparus aurata affected by winter syndrome. Affected fish showed a severe immunosuppression involving significant decrease of serum complement activity, decrease of plasma lysozyme activity, and reduction of circulating lymphocytes. When these fish were subjected to an injection of inactivated Vibrio anguillarum, a transient increase of circulating lymphocytes and respiratory burst activity was detected after 4 weeks of injection compared to uninjected fish. However, no significant signs of improved resistance were observed.

The measurement of disease susceptibility in dab, Limanda limanda L., following long-term exposure to contaminated sediments: Preliminary studies

Exploratory studies into the immunocompetence of fish held on contaminated and uncontaminated bottom sediments have been conducted at the Fish Diseases Laboratory, Weymouth. The approach has been to expose dab, Limanda limanda L. (Pleuronectidae) long-term (> 3 months) on highly contaminated harbour sludge and less-contaminated sediment, and to compare the immune response and uptakes of contaminants between the two groups. Following exposure to the sediments, mortalities and clinical signs of disease were recorded. The dab were then challenged individually by intraperitoneal injection with 0–1 ml of a 1% suspension of inactivated Vibrio anguillarum. The fish were examined for clinical signs of disease. Blood and samples of organs, including brain, were taken for histological examination. The remainder of these fish (and all fish dying during the experiments) were frozen for contaminant uptake analysis. Clinical and histopathological changes were observed in both groups of fish with skin ulcerations and gill hyperplasia, showing a slightly higher prevalence in the fish exposed to contaminated harbour sludge. Quantification of the immune response was by agglutination assay and an enzyme-linked immunosorbent assay (ELISA). Fish maintained on the contaminated harbour sludge had a lower mean antibody titre than those on the reference sediment and this difference in mean titres was statistically significant. However, because the fish on the contaminated sediment were still able to produce a marked response to the antigen, in biological terms there may have been no difference in the susceptibility to disease between the two groups. Further work will be needed to clarify this point.