Vibrio Anguillarum Research Articles

Extracts of the green algae Ulva prolifera possess antioxidant and antibacterial activities in vitro

Macroalgae are currently being explored as novel and sustainable sources of bioactive compounds. Ulva prolifera is a major member of the green algae, as it includes various bioactive compounds, including carotenoids, fucoidans, and phlorotannins, with various biological activities in pharmaceutical, nutraceutical, cosmeceutical, and functional foods industries. In the current work, the rbcL gene products were utilized in identifying U. prolifera molecularly. The antibacterial and antioxidant activities of the hexane, ethyl acetate, and methanol extracts of U. prolifera were carried out In vitro. The total antioxidant capacity method was utilized in assessing the antioxidant activity. Estimation of nutritional value of U. prolifera showed 5.4±0.04%, 1.7±0.04% and 43±0.5% dry weight for total proteins, lipids, and carbohydrates, respectively. The total antioxidant capacity of algal hexane, ethyl acetate, and methanol extract was 0.97±0.09, 1.23±0.04 and 1.63±0.09 mg equivalent ascorbic acid/gdw, respectively. Investigation of the antibacterial activity of U. prolifera against Staphylococcus aureus and Aeromonas hydrophila showed that the recorded diameter of the inhibition zones for the methanolic extract was 1.1cm. The antibacterial effect of hexane extract against Edwardsiella tarda was high which had a diameter of 1.5cm. Of interest, the ethyl acetate extract of U. prolifera showed potent antibacterial activity against the three bacteria, including Vibrio anguillarum, Aeromonas hydrophila, and Edwardsiella tarda. These results indicate that macroalgae U. prolifera contain bioactive substances with potential antioxidant and antibacterial effects.

Coadministration of citric acid allows oxytetracycline dose reduction in yellowtail Seriola quinqueradiata infected with Vibrio anguillarum.

Oxytetracycline (OTC) has been commonly used as an effective antibiotic against various fish bacterial diseases, including vibriosis. In this study, the absorption-enhancing effect of citric acid on oral OTC pharmacokinetics and treatment of artificial Vibrio anguillarum infection was evaluated in juvenile yellowtail Seriola quinqueradiata followed by serum OTC concentration analysis. When 25 mg kg-1 body weight (BW) OTC was administered in combination with 1250 mg kg-1 BW citric acid, the serum OTC concentration reached almost the same concentration as that of the group treated with 50 mg kg-1 BW OTC. This coadministration successfully suppressed mortality due to vibriosis similar to the group treated with 50 mg kg-1 BW OTC. Conversely, poor efficacy was observed when only 25 mg kg-1 BW OTC was administered. These results suggest that coadministration of citric acid can be beneficial in reducing the dose of OTC needed for effective treatment, and thus contributes to the goal of reduced use of this antibiotic in aquaculture.

Micro-Transcriptome Analysis Reveals Immune-Related MicroRNA Regulatory Networks of Paralichthys olivaceus Induced by Vibrio anguillarum Infection.

MicroRNAs (miRNAs) are non-coding regulatory RNAs that play a vital part in the host immune response to pathogen infection. Japanese flounder (Paralichthys olivaceus) is an important aquaculture fish species that has suffered from bacterial diseases, including that caused by Vibrio anguillarum infection. In a previous study, we examined the messenger RNA (mRNA) expression profiles of flounder during V. anguillarum infection and identified 26 hub genes in the flounder immune response. In this study, we performed the micro-transcriptome analysis of flounder spleen in response to V. anguillarum infection at 3 different time points. Approximately 277 million reads were obtained, from which 1218 miRNAs were identified, including 740 known miRNAs and 478 novel miRNAs. Among the miRNAs, 206 were differentially expressed miRNAs (DEmiRs), and 104 of the 206 DEmiRs are novel miRNAs identified for the first time. Most of the DEmiRs were strongly time-dependent. A total of 1355 putative target genes of the DEmiRs (named DETGs) were identified based on integrated analysis of miRNA-mRNA expressions. The DETGs were enriched in multiple functional categories associated with immunity. Thirteen key DEmiRs and 66 immune DETGs formed an intricate regulatory network constituting 106 pairs of miRNAs and DETGs that span five immune pathways. Furthermore, seven of the previously identified hub genes were found to be targeted by 73 DEmiRs, and together they formed interlinking regulatory networks. These results indicate that V. anguillarum infection induces complicated miRNA response with extensive influences on immune gene expression in Japanese flounder.

Antibacterial functions of a novel fish-egg lectin from spotted knifejaw (Oplegnathus punctatus) during host defense immune responses

Fish-egg lectins (FELs) have been identified in several teleost species and have been proved to play important roles in innate immune system against pathogen infection. In this study a novel fish-egg lectin (OppFEL) was identified from spotted knifejaw (Oplegnathus punctatus), and the expression patterns against bacterial infection was characterized. The amino acid sequence is highly homologous to other teleost FELs, containing five repeats of the conserved TECPR domain. Expression of OppFEL was widely observed in examined tissues, with the most abundant transcripts observed in gill, showing a pattern of tissue specific expression. The OppFEL expression was significantly up-regulated following a Gram-negative bacterium (Vibrio anguillarum) challenge in vivo, suggesting participation in host antibacterial immune responses. Recombinant OppFEL protein (rOppFEL) possessed calcium dependent binding capacities and agglutination to four Gram-negative bacterium and two Gram-positive bacterium. Sugar binding assay revealed that rOppFEL specifically bound to insoluble lipopolysaccharide and peptidoglycan. In addition, rOppFEL was also proved to have hemagglutinating activity against erythrocytes from Mus musculus, O. punctatus, Sebastes schlegelii and Paralichthys olivaceus. Dual-luciferase analysis showed that overexpression of OppFEL could suppress the activity of NF-κB in a dose dependent manner. Taken together, these results suggest that OppFEL is a unique fish-egg lectin that possesses apparent immunomodulating property and is involved in host defense against pathogens invasion.

Characterization of lamprey (Lampetra japonica) tnfr10-like gene: A potential granulocyte marker molecule and its immune functions

Tumor necrosis factor receptor superfamily (TNFRSF) is an ancient protein superfamily. By binding to tumor necrosis factor (TNF), it can participate in inflammatory response, apoptosis, lymphocyte homeostasis and tissue development. Seven TNFR members have previously been identified in lampreys but detailed functions of TNFR members are not yet to be resolved. Here, we demonstrate some of the distinguishing features of TNFR10-like member which belongs to TNFRSF. The immunohistochemical results indicate that the TNFR10-like protein is abundant in vascular epithelial cells of the lamprey typhlosole and gills. The expression of tnfr10-like gene has a significantly increased at transcription level after Vibrio anguillarum, Staphylococcus aureus and Poly (I:C) stimulation. Notably, TNFR10-like is specifically expressed in the granulocytes of lamprey peripheral blood and supraneural body. Besides, overexpression tnfr10-like gene in HEK-293 T cells cause a decrease in cell activity and able to activate nuclear transcription factor-κB (NF-κB). Together, these results imply that L-TNFR10-like may play a vital role as a potential marker in lamprey granulocytes and may also be involved in regulation of immune response mediated by itself.

Involvement of a novel Ca2+-independent C-type lectin from Sinonovacula constricta in food recognition and innate immunity

Bivalve lectins perform a crucial function in recognition of foreign particles, such as microalgae and pathogenic bacteria. In this study, a novel C-type lectin form Sinonovacula constricta (ScCL) was characterized. The full-length cDNA of ScCL was 1645 bp, encoding a predicted polypeptide of 273 amino acids with one typical carbohydrate-recognition domain. ScCL has the highest similarity and closest phylogenetic relationship with the C-type lectin from Solen grandis. Real-time PCR analysis showed that ScCL was expressed in all tested tissues, with the highest expression in the foot and the lowest expression in hemocytes. Agglutination activity of ScCL was Ca2+-independent. ScCL showed the strongest agglutination on Chlorella vulgaris, the modest agglutination on Platymonas subcordiformis, Nannochloropsis sp., and Thalassiosira pseudonana, the weakest agglutination on Chaetoceros sp., and no agglutination on Isochrysis zhanjiangensis. Meanwhile, agglutination tests and western blot analysis revealed that the recombinant ScCL protein could agglutinate Staphylococcus aureus and Vibrio harveyi, but could not agglutinate Vibrio anguillarum, Bacillus cereus, or Vibrio parahaemolyticus. Furthermore, ScCL had a high binding activity with LPS and mannose, a low binding activity with LTA, and no binding activity with PGN. The expression of ScCL in the gill of S. constricta fed with C. vulgaris and T. pseudonana was significantly increased at 1 and/or 3 h. After injection with S. aureus, the expression of ScCL in the gill was significantly increased at 3, 6, and 24 h. These results indicated that ScCL was involved in food particle recognition and immunity of S. constricta.

Cloning, expression profile of the complement component C9 gene and influence of the recombinant C9 protein on peripheral mononuclear leukocytes transcriptome in half-smooth tongue sole (Cynoglossus semilaevis)

The ninth complement component (C9) is a terminal complement component (TCC) that is involved in creating the membrane attack complex (MAC) on the target cell surface. In this study, the CsC9 (C9 of Cynoglossus semilaevis) cDNA sequence was cloned and characterized. The full-length CsC9 cDNA measured 2,150 bp, containing an open reading frame (ORF) of 1,803 bp, a 5′-untranslated region (UTR) of 24 bp and a 3′-UTR of 323 bp. A domain search revealed that the CsC9 protein contains five domains, including two TSP1s, an LDLRA, an EGF, and a MACPF. Quantitative real-time PCR analysis showed that CsC9 at the mRNA level was expressed in all the tested tissues, with the highest expression being observed in the liver. CsC9 expression is significantly upregulated in the tested tissues after challenge with Vibrio anguillarum. To further characterize the role of CsC9, peripheral blood mononuclear cells of C. semilaevis were used for transcriptome analysis after incubation with recombinant CsC9 (rCsC9) protein. A total of 3,775 significant differentially expressed genes (DEGs) were identified between the control and the rCsC9-treated group, including 2,063 upregulated genes and 1,712 downregulated genes. KEGG analyses revealed that the DEGs were enriched in cell adhesion molecules, cytokine-cytokine receptor interactions, T cell receptor signaling pathways, B cell receptor signaling pathways and Toll-like receptor signaling pathways. The results of this study indicate that in addition to participating in MAC formation, CsC9 might play multiple roles in the innate and adaptive immunity of C. semilaevis.

Dietary citrulline improves survival of rainbow trout Oncorhynchus mykiss juveniles challenged with Vibrio anguillarum

Arginine is a versatile amino acid that plays important roles in various physiological functions. However, supplementation with arginine can also increase urea excretion of the fish. Thus, given than ornithine and citrulline can potentially be used to increase arginine availability or improve fish immunity, this study aimed to compare the effects of dietary supplementation of arginine, ornithine, and citrulline on the survival of rainbow trout Oncorhynchus mykiss against Vibrio anguillarum. Dietary treatment consisted of a control diet (CTRL) and three treatment diets comprising CTRL supplemented with 2% L-arginine (+ARG), L-ornithine (+ORN), or L-citrulline (+CIT). Protein and lipid content of the diets were confirmed at 48.9 and 18.9%, 53.1 and 18.7%, 51.7 and 18.1% and 52.9 and 18.9% for the CTRL, +ARG, +ORN & + CIT diets respectively. Postprandial plasma amino acid levels were measured at 6, 15, and 30 h after feeding. A feeding trial was conducted for 30 days, and at the end of the trial, the rainbow trout were challenged with V. anguillarum. At 24 h post-injection, plasma amino acid and renal gene expression levels were examined. Fish exhibited better weight gain in +ARG treatment compared to +CIT treatment. Similar postprandial levels of plasma arginine were observed in +ARG and + CIT, and the levels in both treatments were significantly higher than those in CTRL. A survival analysis revealed that fish in +CIT (69.2%) were more resistant than those in CTRL (25.0%). There was significantly higher inducible nitric oxide synthase (iNOS) expression in the kidneys of fish in +CIT than those in CTRL. A significant difference in interleukin-1-beta expression was found between +ORN and CTRL treatments, while there was no significant difference in arginase II expression among the treatments. These result shows that citrulline supplementation can be used to increase arginine availability and better at enhancing the immune performance of rainbow trout compared to arginine supplementation when challenged with V. anguillarum.

Antibacterial activities and mechanisms of action of a defensin from manila clam Ruditapes philippinarum

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum.

Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-γ, IL-4/13A, and IL-17A Expression in Atlantic Salmon.

The overexpression of GATA-3, T-bet and TGF-ß may theoretically induce IL-4/A, IFN-γ and IL-17A expression, respectively. Whether this also applies to fish is not yet known. The plasmid vectors encoding reporter gene (RFP)-tagged T-bet, GATA-3 and TGF-ß were used as overexpression tools, transfected into cells or injected intramuscularly to monitor the expression of IFN-γ, IL-4/13A and IL-17A. In addition, the fish were either experimentally challenged with Vibrio anguillarum (VA group) or Piscirickettsia salmonis (PS group). The reporter gene (RFP) inserted upstream of the GATA-3, T-bet and TGF-ß genes, was observed in muscle cell nuclei and in inflammatory cells after intramuscular (i.m.) injection. PS group: following the injection of GATA-3 and T-bet-encoding plasmids, the expression of GATA-3 and T-bet was high at the injection site. The spleen expression of IFN-γ, following the injection of a T-bet-encoding plasmid, was significantly higher on day 2. VA group: The T-bet and GATA-3-overexpressing fish expressed high T-bet and GATA-3 mRNA levels in the muscles and on day 4 post-challenge. The expression of TGF-ß in the muscles of fish injected with TGF-ß-encoding plasmids was significantly higher on days 7 (8 days pre-challenge) and 19 (4 days after challenge). The protective effects of the overexpression of T-bet, GATA-3 and TGF-ß on both bacterial infections were negligible.

Identification and characterization of outer membrane vesicles from the fish pathogen Vibrio ordalii.

Vibriosis outbreaks due to Vibrio ordalii occur globally, but Chilean salmon aquaculture, in particular, has suffered significant monetary losses in the last 15years. Little is known about the virulence mechanisms employed by V.ordalii. However, most Vibrio pathogens (e.g., Vibrio anguillarum, a very close taxonomic species) present outer membrane vesicles (OMVs) that are released extracellularly and implicated in the delivery of virulence factors to host cells. This study provides the first reported evidence of the fish pathogen V.ordalii producing and releasing OMVs under normal growth conditions. Analyses were conducted with the V.ordalii strain Vo-LM-18 and the type strain ATCC 33509T . For comparative purposes, the reference strain V.anguillarum ATCC 43307 was employed. The average size for the three Vibrio strains was 0.215±0.6µm (via scanning electron microscopy) or between 0.19 and 1.8µm (via dynamic light scattering), with each bacterium presenting a wide range. SDS-PAGE revealed similarities in OMV patterns, but neither total nor external proteins were identical. Comparing V.ordalii ATCC 33509T and Vo-LM-18, bands were most evident in the total proteins, and the greatest degree of similarity in OMV profiles was between 37 and 50kDa. The purified OMVs demonstrated haemolytic enzyme activity, which could play a role during V.ordalii infection. These data represent an initial step towards gaining new insights into this virulence factor, of which a lot is known in other pathogenic microorganisms.

Dietary inclusion effect of yacon, ginger, and blueberry on growth, body composition, and disease resistance of juvenile black rockfish (Sebastes schlegeli) against Vibrio anguillarum

BackgroundTo minimize the use of antibiotics and to obtain a more sustainable fish culture and aquaculture industry, development of alternative natural source of immunostimulant to replace antibiotic in aquafeed is highly needed.ObjectiveDietary inclusion effect of yacon (YC), ginger (GG), and blueberry (BB) on growth, body composition, and disease resistance of black rockfish against Vibrio anguillarum was compared to ethoxyquin (EQ).MethodsFour hundred eighty juvenile (an initial weight of 4.2 g) fish were randomly distributed into 12 of 50 L flow-through tanks (forty fish per tank). Four experimental diets were prepared; the control (Con) diet with 0.01% EQ inclusion, and YC, GG, and BB diets at 1% each additive inclusion. Each additive was included into the experimental diets at the expense of wheat flour. Each diet was assigned to triplicate tanks of fish and hand-fed to satiation twice daily for 8 weeks. At the end of 8-week feeding trial, 20 fish from each tank fish were artificially infected by intraperitoneal injection with 0.1 mL of culture suspension of pathogenic V. anguillarum containing 3.3 × 106 cfu/mL respectively. Fish were monitored for the following 8 days after V. anguillarum infection and dead fish were removed every 6 h for the first 4 days and 12 h for the rest of the study.ResultsWeight gain, specific growth rate (SGR), and feed efficiency ratio (FER) of fish fed the YC diet was higher than those of fish fed all other diets. However, feed consumption, protein efficiency ratio, and protein retention was not affected by dietary additive. Moisture, crude protein, and crude lipid content of the whole body of fish were affected by dietary additive. Analysis of the Kaplan-Meier survival curves showed that survival of fish fed the YC, BB, and GG diets was higher than the Con diet.ConclusionOral administration of YC can improve not only weight gain, SGR, and FER of black rockfish, but lower mortality of rockfish at occurrence of V. anguillarum.

High cell densities favor lysogeny: induction of an H20 prophage is repressed by quorum sensing and enhances biofilm formation in Vibrio anguillarum.

Temperate ϕH20-like phages are repeatedly identified at geographically distinct areas as free phage particles or as prophages of the fish pathogen Vibrio anguillarum. We studied mutants of a lysogenic isolate of V. anguillarum locked in the quorum-sensing regulatory modes of low (ΔvanT) and high (ΔvanO) cell densities by in-frame deletion of key regulators of the quorum-sensing pathway. Remarkably, we find that induction of the H20-like prophage is controlled by the quorum-sensing state of the host, with an eightfold increase in phage particles per cell in high-cell-density cultures of the quorum-sensing-deficient ΔvanT mutant. Comparative studies with prophage-free strains show that biofilm formation is promoted at low cell density and that the H20-like prophage stimulates this behavior. In contrast, the high-cell-density state is associated with reduced prophage induction, increased proteolytic activity, and repression of biofilm. The proteolytic activity may dually function to disperse the biofilm and as a quorum-sensing-mediated antiphage strategy. We demonstrate an intertwined regulation of phage-host interactions and biofilm formation, which is orchestrated by host quorum-sensing signaling, suggesting that increased lysogeny at high cell density is not solely a strategy for phages to piggy-back the successful bacterial hosts but is also a host strategy evolved to take control of the lysis-lysogeny switch to promote host fitness.

Characterization of a novel lncRNA (SETD3-OT) in turbot (Scophthalmus maximus L.)

LncRNAs have been demonstrated to play pivotal roles in virous biological processes, especially the gene expression regulation, including transcriptional regulation, posttranscriptional control and epigenetic processes. However, most of the current studies of lncRNAs are still limited in mammalian species, the investigations of functional roles of lncRNAs in teleost species are still lacking. In current study, we identified a novel lncRNA (SETD3-OT) in turbot, with 2,504 bp full-length obtained by 5′ and 3′ RACE, located in turbot chromosome 17, ranged from 20,933,835 to 20,936,302 bp. In addition, 8 neighboring genes of SETD3-OT were identified within 100 kbp in genome location. From the annotation of the neighboring adjacent genes, SETD3-OT might involve in regulation of cell apoptosis and cycle, the immune cell development, and the immune response against infection, and its expression pattern is similar to majority of the neighboring genes following Aeromonas salmonicida challenge. Intriguingly, SETD3-OT showed significant high expression levels in mucosal surfaces (intestine, gill and skin), and was dramatically down-regulated in these mucosal tissues following Vibrio anguillarum challenge, especially in gill and skin. In addition, SETD3-OT was distributed in nucleus, it might regulate the neighboring genes in cis or in trans. Taken together, our results provide insights for lncRNA in fish innate immunity, further studies should be conduct to explore the detailed molecular mechanism of the gene regulation between SETD3-OT and its neighboring genes.

The efficacy and side-effects of oil-based adjuvants emulsified Vibrio anguillarum bivalent inactivated vaccine in turbot (Scophthalmus maximus) under production mode

Turbot (Scophthalmus maximus) is an important commercial fish in China, and Vibrio anguillarum is a common pathogen of turbot. Oil emulsified vaccines have been widely used to protect fish against V. anguillarum with long-term immune responses in several fish species. Side-effects, such as adhesions and growth reduction caused by oil-based adjuvants, however, limit the application of oil emulsions in turbot. In this study, turbot V. anguillarum serotype O1 and O2 bivalent inactivated vaccines were emulsified as water-in-oil (W/O) emulsions with four kinds of oil-based adjuvants: Freund's incomplete adjuvant (FIA), MOBIL™ Marcol 52 (Marcol 52), SEPPIC Montanide™ ISA 763 A VG (ISA 763) and ISA 780 (ISA 780). Vaccine protection efficiency, fish adhesions, and growth were tested under production mode. The relative percent of survival (RPS) of W/O vaccines was all higher than that of the water-based (WB) vaccine at 7 days post vaccination. At 30 days post vaccination, the RPS of all vaccinated groups was over 90%, and most of the specific antibody titers of W/O vaccine-vaccinated fish were significantly higher than those of WB vaccines. Fish immunized with FIA, ISA 763, and ISA 780 adjuvants W/O vaccine showed serious tissue adhesions after vaccination, and their weight was significantly lower than control fish at 30 days post vaccination. Only slight or minor adhesions were observed in WB and Marcol 52 adjuvant vaccinated fish, which did not show growth reduction. These results indicate that Marcol 52 is a well-tolerated oil-based adjuvant for turbot, and could be used in vaccine development in further study.

Influence of incubation time on antimicrobial susceptibility testing of pathogenic Vibrio anguillarum and Vibrio vulnificus isolated from fish

A multi-laboratory study was performed to investigate the most suitable incubation time for susceptibility tests of fish pathogens Vibrio anguillarum and Vibrio vulnificus performed at 28 °C. An isolate set consisting of 30 V. anguillarum and 26 V. vulnificus was used by four participating laboratories in Denmark, France, Sweden, and the Netherlands. Inhibition zone diameters were determined by agar disc diffusion for eight agents and Minimum Inhibitory Concentration (MIC) values were determined for seven agents using the standard CLSI testing protocols for non-fastidious organisms that specify 24-28 h incubation. In this work an additional set of readings was made after 48 h incubation. In total, 1120 paired zone sizes and 399 paired MIC observations were made at the two incubation times. Examination of the data demonstrated that incubation time had a small but statistically significant effect on the numerical values of susceptibility measures. However, the effects of incubation time on the precision of the data sets and the categorisation of isolates based on the application of epidemiological cut-off values were slight and statistically non significant. These analyses suggest that the susceptibility of these Vibrio species could be established using protocols that specify either 24-28 h or 44-48 h incubation.This study does not provide evidence that prolonged incubation to 48 h improves the quality of data generated by the tests. Therefore, it is recommended that the existing standard CLSI protocols with 24-28 h at 28 °C should be adopted for susceptibility testing of V. anguillarum and V. vulnificus.

Chemical composition and antibacterial activity of Algerian propolis against fish pathogenic bacteria

Five different varieties of propolis from four sites from Sétif region (East of Algeria) (Babor, Setif; Ain-Abbassa and El-Hamma), and one site from the center of Algeria (Tizi-Ouzou) were chemically analysed by gas chromatography-mass spectrometry. One hundred and two compounds were identified including aromatic acids, linear hydrocarbons and their acids, terpenes and alcaloïdes. Furthermore, the in vitro bacteriostatic and bactericidal activities of the aqueous extracts were evaluated against one Gram positive (Bacillus subtilis, used as probiotics in aquaculture) and two Gram negative (Vibrio anguillarum and Vibrio harveyi, pathogenic for fish) bacteria. The obtained results showed that all aqueous extracts of propolis inhibit the growth of B. Subtilis while the growth inhibition of fish pathogens was achieved when using higher propolis concentrations. These antibacterial properties would warrant further studies on the clinical applications of propolis in aquaculture field.
 Keywords: Bactericidal activity; Chemical characterization; Propolis; Vibrio.

Shewanella algae and Vibrio spp. strains isolated in Italian aquaculture farms are reservoirs of antibiotic resistant genes that might constitute a risk for human health

The aquatic environment can represent a reservoir of antimicrobial resistance genes. In the present study, phenotypical, biochemical and molecular techniques were used to screen a collection of marine strains isolated in Italian aquaculture farms to investigate their beta-lactam resistance profiles. The genome of 12 carbapenemase and/or beta-lactamase producing strains was sequenced and a phylogenetic analysis of the beta-lactamases found in their chromosomes was performed. Gene annotation and prediction revealed the presence of blaAmpC and blaOXA-55-like in all the Shewanella algae isolates whereas in Vibrio anguillarum and Vibrio parahaemolyticus strains, blaAmpC and blaCARB-19 were found, respectively. Multiple alignments of OXA-55-like and AmpC protein sequences showed different point mutations. Finally, comparisons between enzyme phylogeny and strain clusterization based on sampling sites and dates indicate the diffusion of specific Multi Drug Resistant (MDR) Shewanella algae clones along the Italian Adriatic coast.

Prebiotics and phytogenics functional additives in low fish meal and fish oil based diets for European sea bass (Dicentrarchus labrax): Effects on stress and immune responses

The use of terrestrial raw materials to replace fish meal (FM) and fish oil (FO) in marine fish diets may affect fish growth performance and health. In the last years functional additives have been profiled as good candidates to reduce the effects on health and disease resistance derived from this replacement, via reinforcement of the fish immune system. In the present study, three isoenergetic and isonitrogenous diets with low FM and FO (10% and 6% respectively) were tested based on supplementation either with 0.5% galactomannanoligosaccharides (GMOS diet) or 0.02% of a mixture of essential oils (PHYTO diet), a non-supplemented diet was defined as a control diet. Fish were fed the experimental diets in triplicate for 9 weeks and then they were subjected to a stress by confinement as a single challenge (C treatment) or combined with an experimental intestinal infection with Vibrio anguillarum (CI treatment). Along the challenge test, selected stress and immunological parameters were evaluated at 2, 24 and 168h after C or CI challenges. As stress indicators, circulating plasma cortisol and glucose concentrations were analyzed as well as the relative gene expression of cyp11b hydroxylase, hypoxia inducible factor, steroidogenic acute regulatory protein, heat shock protein 70 and heat shock protein 90 (cyp11b, hif-1α, StAR, hsp70 and hsp90). As immune markers, serum and skin mucus lysozyme, bactericidal and peroxidase activities were measured, as well as gene expression of Caspase-3 (casp-3) and interleukin 1β (il-1ß).The use of functional additives induced a significant (p < 0.05) reduction of circulating plasma cortisol concentration when confinement was the unique challenge test applied. Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1β gene expression 2h after stress test, whereas StAR expression was significantly (p < 0.05) up-regulated. However, when combination of confinement stress and infection was applied (CI treatment), the use of PHYTO significantly (p < 0.05) down-regulated StAR and casp-3 gene expression 2h after challenge test, denoting that PHYTO diet reinforced fish capacity of stress response via protection of head kidney leucocytes from stress-related apoptotic processes, with lower caspase-3 gene expression and a higher il-1β gene expression when an infection occurs. Additionally, dietary supplementation with GMOS and PHYTO compounds increased fish serum lysozyme after infection. Both functional additives entailed a better capability of the animals to cope with infection in European sea bass when fed low FM and FO diets.

Stain capacity of three fungi on two fast-growing wood

We investigated the stain of fast-growing wood (Cunninghamia lanceolate, CL; Paulownia, PT) inoculated with three fungi (Arthrinium phaeospermum, AP; Vibrio anguillarum, VA; Aspergillacea, AS) to explore the new wood dyeing ways and the better combination of wood and fungi for dyeing. Only AP could dye on CL and PT. Especially for CL, its percentage of internal spalting, percentage of external spalting and dyeing depth were the highest (48%, 15% and 5.06 mm, respectively). Surprisingly, the bigger weight loss occurs on PT. The results showed that the dyeing effect of AP dyeing CL was the best, and the wood color change was obviously (Orange to dark red). AP could produce more pigments than the other two fungi (VA; AS), CL was more suitable for fungus staining than PT, indicating that AP could offered a new potential market and a chance for areas to earning higher income for CL. This research paves the way for improving color change was obviously (Orange to dark red). AP could produce more pigments than the other two fungi (VA; AS), CL was more suitable for fungus staining than PT, indicating that AP could offer a new potential market and a chance for areas to earn higher income for CL.