Vibrio Anguillarum Research Articles

A CCL25 chemokine functions as a chemoattractant and an immunomodulator in black rockfish, Sebastes schlegelii

Chemokines are small cytokines that are classified into four groups, one of which is called CC chemokines. In the present study, the full-length cDNA of a CCL25 chemokine was identified from black rockfish, Sebastes schlegelii (named as SsCCL25) by EST (expressed sequence tag) analysis. The cDNA of SsCCL25 consisted of a 5-terminal untranslated region (UTR) of 74 bp, a 3-UTR of 882 bp with a poly (A) tail, and an open reading frame (ORF) of 303 bp encoding a polypeptide of 100 amino acids with the putative molecular mass of 11.5 kDa. There was a SCY domain in the deduced amino acid sequence of SsCCL25. The phylogenetic relationships and syntenic analyses provided evidences for the identities of SsCCL25 with CCL25 group. The mRNA transcripts of SsCCL25 were expressed in all detected tissues and dominantly in liver, muscle and gill. Moreover, after Vibrio anguillarum stimulation, the mRNA expression levels of SsCCL25 were significantly up-regulated at 24 h (p < 0.05) in the liver and during 4–8 h (p < 0.05) in the spleen. Recombinant SsCCL25 protein induced chemotaxis of both control and LPS-stimulated peripheral blood leukocytes (PBL) and enhanced their resistance to bacterial infection in a dose-dependent manner. Furthermore, rSsCCL25 showed significant inhibitory effect on V. anguillarum and Edwardsiella tarda growth. All these results collectively indicated that SsCCL25 might contribute to the defense against microbe infection and function as a chemoattractant in black rockfish.

Recombinant DnaK Orally Administered Protects Axenic European Sea Bass Against Vibriosis.

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (105 CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture.

An in vitro study of the effect of carob (Ceratonia siliqua L.) leaf extracts on gilthead seabream (Sparus aurata L.) leucocyte activities. Antioxidant, cytotoxic and bactericidal properties

Carob leaves, the main residues of the carob tree, were investigated as a renewable and abundant source of bioactive compounds for fish aquaculture. Aqueous and ethanolic extracts obtained from carob leaves were characterized in terms of biochemical composition, antiradical and cytotoxic effects and immunostimulant and antibacterial activities. The ethanolic extract showed higher levels of total phenolics, flavonoids and condensed tannins and higher antioxidant activity than the aqueous extract. No significant immunostimulant effects were observed on gilthead seabream (Sparus aurata) head kidney leucocytes (viability, phagocytosis and respiratory burst activities and peroxidase content) after incubation for 24 h with different extracts. Furthermore, the ethanolic extracts used at 0.5, 0.75 and 1 mg mL−1 and aqueous extracts at 1 g mL−1 had a cytotoxic effect on PLHC-1 cells. When the bactericidal activity was tested against three fish pathogenic bacteria (Vibrio harveyi, Vibrio anguillarum and Photobacterium damselae) notable activity of the different extracts was detected against P. damselae at all three concentrations. A similar effect was demonstrated against V. haryeri when ethanolic extracts were used in the same range of concentrations. This work demonstrates interesting in vitro effects of carob leaf extracts and suggests it could be used as an alternative to chemical compounds with farmed fish. The concentration and nature of the extracts were very important in terms of any positive results.

Purification and Characterization of an Antibacterial Substance from Aerococcus urinaeequi Strain HS36.

A bacterial strain inhibiting the growth of Vibrio anguillarum, the causative agent of vibriosis, was isolated from fish intestines. The isolated strain HS36 was identified as Aerococcus urinaeequi based on the characteristics of the genus according to Bergey's Manual of Systematic Bacteriology and by 16S rRNA sequencing. The growth rate and antibacterial activity of strain HS36 in shaking culture were higher than those in static culture, while the optimal pH and temperature for antibacterial activity were 7.0 and 30°C, respectively. The active antibacterial substance was purified from a culture broth of A. urinaeequi HS36 by Sephadex G-75 gel chromatography, Sephadex G-25 gel chromatography, and reverse-phase high-performance liquid chromatography. Its molecular weight, as estimated by Tricine SDS-polyacrylamide gel electrophoresis, was approximately 1,000 Da. The antibacterial substance produced by strain HS36 was stable after incubation for 1 h at 100°C. Although its antibacterial activity was optimal at pH 6-8, activity was retained at a pH range from 2 to 11. The purified antibacterial substance was inactivated by proteinase K, papain, and β-amylase treatment. The newly purified antibacterial substance, classified as a class II bacteriocin, inhibited the growth of Klebsiella pneumoniae, Salmonella enterica, and Vibrio alginolyticus.

Sequence Analysis of Plasmids in Vibrio anguillarum from Different Fish and Locations.

The genetic diversity of Vibrio anguillarum pJM1-like plasmids was investigated. Plasmids were isolated from 18 V.anguillarum serovar O1 strains collected from different geographic locations and fish species. The plasmids were sequenced and compared with the complete sequence of the published virulence plasmid pJM1. All 18 strains contained pJM1-like plasmids with approximately 65kbp and all plasmids encoded the virulence genes responsible for the anguibactin iron sequestering system. The plasmids were highly conserved but minor differences were observed in some genes. A single nucleotide polymorphisms (SNPs) analysis showed 0-11 nucleotide variations between each of the 18 plasmids and the pJM1 plasmid. Compared with the sequence of pJM1, nonsynonymous SNPs were identified in fatC, angR, angL, pJM1_p19, and angE. In particular, a mutation found in 15 out of 18 sequenced plasmids in angR has previously been linked to hyperproduction of anguibactin and was found in all the European isolates. However, overall the pJM1-like plasmids isolated from V.anguillarum serovar O1 exhibited a high degree of conservation regardless of their geographical origin or fish species.

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.

Identification and characterization of a novel L-type lectin (MjLTL2) from kuruma shrimp (Marsupenaeus japonicus)

L-type lectins (LTLs) belong to the lectin family and are characterized by a conserved structural motif in their carbohydrate recognition domain. LTLs are homologous to leguminous lectins. In this study, we identified and functionally characterized an LTL from kuruma shrimp Marsupenaeus japonicus. We designated this LTL as MjLTL2. MjLTL2 contains a signal peptide, a Lectin_leg domain, a coiled coil, and transmembrane domain. MjLTL2 is distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestine; higher expression levels are seen in hemocytes and the hepatopancreas than in other tissues. MjLTL2 was upregulated following challenge of shrimp with Vibrio anguillarum and white spot syndrome virus (WSSV). MjLTL2 can agglutinate several bacteria without Ca2+. In addition, MjLTL2 could bind to several Gram-positive and -negative bacteria by binding to their lipopolysaccharide and peptidoglycan. However, MjLTL2 could not enhance the clearance of V. anguillarum in vivo. In the presence of WSSV infection, MjLTL2 knockdown by RNA interference resulted in a 7-day lower cumulative mortality of M. japonicus. Moreover, less VP19, VP24, VP26, and VP28 mRNAs were extracted from the hemocytes of MjLTL2 knockdown shrimp than from the control. These results suggest that MjLTL2 is involved in immune responses in shrimp.

Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses.

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.

Antimicrobial activity of mannose binding lectin in grass carp (Ctenopharyngodon idella) in vivo and in vitro

Mannose-binding lectin (MBL) is a crucial pattern recognition receptor in the host innate immune system. Previously, we reported the biological function of Ctenopharyngodon idella MBL (CiMBL) in initiating the lectin pathway of the complement system. In the present study, we further explored its biological function including the agglutinating ability, binding capacity and protective role in vitro and in vivo. After Aeromonas hydrophila infection, western blot analysis revealed that the CiMBL were fluctuated and expressed in the serum and major immune-related tissues. The result of quantitative PCR (qPCR) showed that the recombinant CiMBL (rCiMBL) significantly inhibited the mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in liver, spleen and hepatic cells. Due to rCiMBL bound to d-mannose, d-galactose, d-glucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS), peptidoglycan (PGN) and Agar in the presence of Ca2+, herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca2+-dependent manner. More importantly, rCiMBL enhanced the survival rate of grass carp following bacterial infection. Overall, the results provide an evidence that CiMBL can protect grass carp against A. hydrophila infection in aquaculture.

Phylogenetic Relationship Among Brackishwater Vibrio Species.

Vibriosis is regarded as an important disease of penaeid shrimps affecting larvae in hatcheries. Among the Vibrio species, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio furnissii, Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus, and Vibrio anguillarum are often associated with diseases in finfish and shellfish of brackishwater ecosystem. Accurate species differentiating methods for the organisms present in an ecosystem are required for precise classification of the species and to take steps for their management. Conventional methods like 16s rRNA phylogeny and multilocus sequence typing (MLST) have often failed to correctly identify Vibrio species. This has necessitated a comprehensive investigation on methodologies available to distinguish Vibrio species associated with brackishwater aquaculture system. To achieve this, 35 whole genomes belonging to 7 Vibrio species were subjected to phylogenetic analysis based on 16s rRNA gene, MLST genes, single-copy orthologous genes, and single-nucleotide polymorphisms. In addition, genome-based similarity indices like average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) were computed as confirmatory tests to verify the phylogenetic relations. There were some misclassifications occurred regarding phylogenetic relations based on 16s rRNA genes and MLST genes, while phylogeny with single-copy orthologous genes produced accurate species-level clustering. Study reveals that the species identification based on whole genome-based estimates or genome-wide variants are more precise than the ones done with single or subset of genes.

Vulnerability of glutathione-depleted Crassostrea gigas oysters to Vibrio species

Glutathione (GSH) is a major cellular antioxidant molecule participating in several biological processes, including immune function. In this study, we investigated the importance of GSH to oysters Crassostrea gigas immune response. Oysters were treated with the GSH-synthesis inhibitor buthionine sulfoximine (BSO), and the function of immune cells and mortality were evaluated after a bacterial challenge with different Vibrio species. BSO caused a moderate decrease (20–40%) in GSH levels in the gills, digestive gland, and hemocytes. As expected, lower GSH decreased survival to peroxide exposure. Hemocyte function was preserved after BSO treatment, however, oysters became more susceptible to challenges with Vibrio anguillarum, V. alginolyticus, or V. harveyi, but not with V. parahaemolyticus and V. vulnificus, indicating a species-specific vulnerability. Our study indicates that in natural habitats or in mariculture farms, disturbances in GSH metabolism may pre-dispose oysters to bacterial infection, decreasing survival.

Identification and Expression of Heat Shock Protein 60 (HSP60) from the Ark Shell Scapharca broughtonii

Heat shock protein 60 (HSP60) is evolutionarily highly conserved, and it assists with protein folding in the mitochondria and serves as a molecular chaperone, increasing cell survival under various conditions. Studies of HSP60 in molluscs under stress conditions remain limited. Here, this study first characterize a full-length HSP60 cDNA from the ark shell Scapharca broughtonii (termed SbHSP60) containing a 1,725-bp open reading frame corresponding to a deduced protein of 574 amino acid residues with a predicted molecular mass of approximately 61.4 kDa. The deduced protein has 72.3%–80.3% amino acid identity with homologs from invertebrates and vertebrates. Phylogenetic analysis showed that SbHSP60 grouped with proteins from other molluscs, forming a sister cluster to the HSP60 proteins from vertebrates. The expression pattern of SbHSP60 as examined by quantitative real-time PCR suggested that these mRNA transcripts were distributed across all examined tissues. The highest levels of expression were found in the hepatopancreas and gill, with decreasing levels of expression in the mantle, foot, hemocytes, and adductor muscle. Expression of SbHSP60 after challenge with Vibrio anguillarum showed similar upregulation profiles in the hepatopancreas and gill, indicating that SbHSP60 is involved in the bacterial defense process as an acute-phase protein. Levels of SbHSP60 in the hepatopancreas and gill were higher in cadmium-exposed individuals than in controls. SbHSP60 was significantly upregulated by Cd stress in the gill, but expression was less affected in the hepatopancreas, implying different roles in cellular Cd stress protection. The effects of heat stress on SbHSP60 mRNA expression were duration- and tissue-specific. The induction of SbHSP60 in the heat-stressed group occurred predominantly in the hepatopancreas, suggesting that protein damage and the consequent requirement for the HSP60 protein are increased during the heat shock process.

Effects of dietary arachidonic acid in European sea bass (Dicentrarchus labrax) distal intestine lipid classes and gut health.

The use of low fishmeal/fish oil in marine fish diets affects dietary essential fatty acids (EFAs) composition and concentration and, subsequently, may produce a marginal deficiency of those fatty acids with a direct impact on the fish intestinal physiology. Supplementation of essential fatty acids is necessary to cover the requirements of the different EFAs, including the ones belonging to the n-6 series, such as arachidonic acid (ARA). ARA, besides its structural role in the configuration of the lipid classes of the intestine, plays an important role in the functionality of the gut-associated immune tissue (GALT). The present study aimed to test five levels of dietary ARA (ARA0.5 (0.5%), ARA1 (1%), ARA2 (2%), ARA4 (4%), and ARA6 (6%)) for European sea bass (Dicentrarchus labrax) juveniles in order to determine (a) its effect in selected distal intestine (DI) lipid classes composition and (b) how these changes affected gut bacterial translocation rates and selected GALT-related gene expression pre and post challenge. No differences were found between distal intestines of fish fed with the graded ARA levels in total neutral lipids and total polar lipids. However, DI of fish fed with the ARA6 diet presented a higher (P < 0.05) level of phosphatidylethanolamine (PE) and sphingomyelin (SM) than those DI of fish fed with the ARA0.5 diet. In general terms, fatty acid profiles of DI lipid classes mirrored those of the diet dietary. Nevertheless, selective retention of ARA could be observed in glycerophospholipids when dietary levels are low (diet ARA0.5), as reflected in the higher glycerophospholipids-ARA/dietary-ARA ratio for those animals. Increased ARA dietary supplementation was inversely correlated with eicosapentaenoic acid (EPA) content in lipid classes, when data from fish fed with the diets with the same basal composition (diets ARA1 to ARA6). ARA supplementation did not affect intestinal morphometry, goblet cell number, or fish survival, in terms of gut bacterial translocation, along the challenge test. However, after the experimental infection with Vibrio anguillarum, the relative expression of cox-2 and il-1β were upregulated (P < 0.05) in DI of fish fed with the diets ARA0.5 and ARA2 compared with fish fed with the rest of the experimental diets. Although dietary ARA did not affect fish survival, it altered the fatty acid composition of glycerophospholipids and the expression of pro-inflammatory genes after infection when included at the lowest concentration, which could be compromising the physical and the immune functionality of the DI, denoting the importance of ARA supplementation when low FO diets are used for marine fish.

T and B lymphocytes immune responses in flounder (Paralichthys olivaceus) induced by two forms of outer membrane protein K from Vibrio anguillarum: Subunit vaccine and DNA vaccine

To further elucidate the roles of T and B lymphocytes in fish subunit and DNA candidate vaccines for immunisation, the immune responses of T and B lymphocytes to recombinant protein (rOmpK) and plasmid OmpK (pOmpK) from Vibrio anguillarum plus cyclosporine A (CsA) were investigated in flounder (Paralichthys olivaceus). The results showed that in the rOmpK-immunised groups, the percentages of CD4-1+ and CD4-2+ T (PCD4-1+ and PCD4-2+ T) lymphocytes significantly increased to a peak on days 5 or 7. The percentages of IgM+ B (PIgM+ B) lymphocytes and specific antibodies markedly increased to a peak at weeks 4 or 5. The nine immune-related genes were significantly up-regulated and the expression levels of CD4-1, CD4-2 and MHC II genes were higher than that of CD8α, CD8β and MHC I genes. The CD4+ T lymphocytes, IgM+ B lymphocytes, and specific antibodies were significantly inhibited by CsA. Therefore, the responses of CD4+ T lymphocytes influenced the responses of the B lymphocytes and antibodies. In the pOmpK-immunised groups, the PCD4-1+, PCD4-2+, and PCD8β+ T lymphocytes significantly increased to a peak on days 11 or 14, days 9 or 11, and days 7 or 9, respectively. The PIgM+ B lymphocytes and specific antibodies significantly increased to a peak at weeks 5 or 6. Immune related genes upregulated, and CD4+ and CD8+ T lymphocytes, IgM+ B lymphocytes and specific antibodies all suppressed by CsA, suggesting that the responses of T lymphocytes subpopulations influenced B lymphocytes and antibodies responses. Therefore, the subpopulations of T lymphocytes played an important role in the immune responses induced by subunit and DNA candidate vaccines of OmpK and regulated the immune responses of B lymphocytes in flounder.

Physiological disturbances in juvenile sockeye salmon (Oncorhynchus nerka) exposed to the water-soluble fraction of diluted bitumen

Current and proposed transcontinental pipelines for the transport of diluted bitumen (dilbit) from the Canadian oil sands traverse the coastal watersheds of British Columbia, habitat essential to Pacific salmonids. To determine the potential risks posed to these keystone species, juvenile sockeye (Oncorhynchus nerka; 1+ parr) were acutely (24–96 h) or subchronically (21–42 d) exposed to 4 concentrations of the water-soluble fraction (WSF) of unweathered Cold Lake Blend dilbit (initial total PAC concentrations: 0, 13.7, 34.7 and 124.5 μg/L) in a flow-through system. Dilbit effects on iono-osmoregulation, the physiological stress response, and the immune system were assessed by both biochemical and functional assays. Hydrocarbon bioavailability was evidenced by a significant induction of liver ethoxyresorufin-O-deethylase (EROD) activity in exposed fish. Acute and subchronic exposure significantly reduced gill Na+-K+-ATPase activity and resulted in lower plasma osmolality, Cl−, and Na+ concentrations. Acute exposure to dilbit resulted in a classic physiological stress response, however at 21 d of exposure, plasma cortisol remained elevated while other measured parameters had returned to baseline values. A compromised immune system was demonstrated by a 29.5 % higher mortality in fish challenged with Vibrio (Listonella) anguillarum following dilbit exposure compared to unexposed controls. Exposure of juvenile salmonids to the WSF of dilbit (at TPAC concentrations at the ppb level) resulted in sublethal effects that included a classic physiological stress response, and alterations in iono-osmoregulatory homeostasis and immunological performance.

Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity

Cathepsin L (CTSL) is one of the crucial enzymes in cathepsin family, which has been widely known for its involvement in the innate immunity. However, it still remains poorly understood how CTSL modulates the immune system of teleosts. In this study, we captured three cathepsin L genes (SmCTSL, SmCTSL.1 and SmCTSL1) from turbot (Scophthalmus maximus). The coding sequences of SmCTSL, SmCTSL.1 and SmCTSL1 are 1,026 bp, 1,005 bp and 1,017 bp in length and encode 341, 334 and 338 amino acids, respectively. In details, transcripts of CTSL genes share same domains as other CTSL genes, one signal peptide, one propeptide and one papain family cysteine protease domain. Protein interaction network analysis indicated that turbot CTSL genes may play important roles in apoptotic signaling and involve in innate immune response. Evidence from subcellular localization demonstrated that the three Cathepsin L proteins were ubiquitous in nucleus and cytoplasm. The cathepsin L genes were widely expressed in all the tested tissues with the highest expression level of SmCTSL in spleen, and SmCTSL.1 and SmCTSL1 in intestine. Following Vibrio anguillarum, Edwardsiella tarda and Streptococcus iniae challenge, these cathepsin L genes were significantly regulated in mucosal tissues in all the challenges, especially significant down-regulation occurred rapidly in intestine in all the three challenges. In addition, the three cathepsin L genes showed strong binding ability to all the examined microbial ligands (LPS, PGN and LTA). Further studies should be used to analyze the specific function of these three cathepsin L genes. By then, we can use their function to maintain the integrity of the mucosal barrier, thereby promoting the disease resistance line and family selection in turbot.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48–72 hours.

Molecular and functional characterisation of a mannose-binding lectin-like gene from Japanese sea bass (Lateolabrax japonicus)

Mannose-binding lectin (MBL) plays an important role in host immune responses against pathogens. LjMBL-like-1 was identified from Japanese sea bass (Lateolabrax japonicas), which has selectivity for galactose. Herein, this lectin might be better designated as galactose-binding lectin (LjGalBL-1). LjGalBL-1 transcripts were detected in all tested tissues, with highest expression in liver. Upon Vibrio harveyi infection, LjGalBL-1 mRNA expression was increased in major immune-related tissues, and protein levels in serum were also upregulated. Recombinant LjGalBL-1 (rLjGalBL-1) bound to monosaccharides and polysaccharides, and both rLjGalBL-1 and native LjGalBL-1 (nLjGalBL-1) agglutinated three Gram-positive bacteria (Staphylococcus aureus, Streptococcus iniae and Micrococcus luteus) and four Gram-negative bacteria (Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum and V. harveyi) in a Ca2+-dependent manner in vitro. Moreover, rLjGalBL-1 increased the survival rate of V. harveyi-infected fish and decreased bacterial load in liver, spleen, kidney and blood. Thus, LjGalBL-1 protects L. japonicas against V. harveyi infection.

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.

In Silico Toxicological, Anti-Tubercular Effect Evaluation And In Vitro Marine Pathogenic Bacteria Inhibition of N-[(3-Chloro-4-Nitro-Phenyl)Methyleneamino]Pyridine-4-Carboxamidine

The hydrazone; N-[(3-chloro-4-nitro-phenyl) methyleneamino] pyridine-4-carboxamidine (H) was selected for in silico toxicological and in vitro bactericidal studies. Toxicological investigation was carried out using software program, such as eMolTox and Gusar, for the toxic substructure determination, and acute rat toxicity prediction respectively. In vitro bactericidal effect evaluation was investigated using tow marine pathogenic bacteria; Vibrio anguillarum and Photobacterium damselae. Computational results determinate toxicophores of (H), which are nitro-aromatic part, hydrazine group, and quaternary carbon, were predicted as responsible for Idiosyncratic toxicity metabolic activation, covalent bond with DNA, and hepatotoxicity respectively. In addition, the predicted LD50 of (H) are 1086, 244, 1816, and 823.40 mg/kg in intraperitenial, intravenous, oral and subcutaneous administration respectively. For bactericidal results, H exhibited an excellent effect with inhibition percentages of 98.65 and 98.83% at the concentrations of 1000 and 500 µg/mL against Vibrio anguillarum respectively, the same effect was demonstrated against Photobacterium damselae with inhibition percentages of 97.74 and 97.98 % at the same concentrations. For anti-tubercular effect prediction, results revealed that H has an excellent effect with probability percentage of 84.6%. &#x0D; Keyword: Hydrazone, toxicophore, LD50, Anti-tubercular, Vibrio anguillarum, Photobacterium damselae.