Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms.

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese.

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.

High-level biocidal products effectively eradicate pathogenic γ-proteobacteria biofilms from aquaculture facilities.

The use of effective biocides as disinfectants is essential in aquaculture facilities. However, while most biocides act effectively on free-living planktonic pathogens, they are seldom useful against biofilms. In this study, we evaluate the biocidal efficacy and antimicrobial specific contact time of three disinfectants, Virkon™Aquatic (VirA), peracetic acid (PerA) and hydrogen peroxide (HydP), on Vibrio anguillarum, V. harveyi, V. alginolyticus, and Photobacterium damselae subspecies piscicida against their both life phases. By using the minimum inhibitory, bactericidal, and eradication concentrations of disinfectants acting on the free-living planktonic state (MIC; MBC) and biofilms (MBIC; MBEC), we determined the in vitro susceptibility of each bacterial strain against three different individual concentrations of VirA, PerA, and HydP added at 1, 5, and 10 min intervals. PerA and VirA had the highest bactericidal efficacies against the free-living planktonic state and biofilm of all bacteria. Kinetically, PerA gave a positive result more quickly in both cases regardless of the strain in question, while the weakest HydP required longer than 10 min to act effectively. Moreover, we conducted a short in vivo safety trial by pouring the suggested MIC of each disinfectant into tanks containing juvenile Gilthead seabream (Sparus aurata). A significant mortality after 24 h was observed pointing to the potential risk a mishap of these chemicals might cause to fish. Nevertheless, collectively, our results support the inclusion of biocides within biosecurity protocols in aquaculture facilities and highlight PerA as the most effective disinfectant for fighting against biofilms produced by V. anguillarum, V. harveyi, V. alginolyticus or P. damselae subsp. piscicida.

Phage Therapy as a Focused Management Strategy in Aquaculture.

Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the best-understood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture are mainly restricted to the Myoviridae and Podoviridae, and the Siphoviridae, respectively. However, the current functional use of phages against bacterial pathogens of cultured fish is still in its infancy. Based on the available data, in this review, we summarize the current knowledge about phage, identify gaps, and provide insights into the possible bacterial control strategies they might represent for managing aquaculture-related bacterial diseases.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.

Susceptibility of Malassezia pachydermatis to aminoglycosides.

Previous studies have evaluated the action of gentamicin against Malassezia pachydermatis. The aim of this study was to evaluate in vitro susceptibility of M. pachydermatis to the aminoglycosides- gentamicin, tobramycin, netilmicin and framycetin. The minimum inhibitory concentration (MIC) of gentamicin was determined following methods M27-A3 microdilution and Etest® . The Etest® was used to determine the minimum inhibitory concentration (MIC) of the tobramycin and netilmicin. The Kirby-Bauer test was used to determine the antibiotic susceptibility to the framycetin. The MIC50 and MIC90 were 8.12 µg/mL and 32.5 µg/mL by microdilution method for gentamicin. The MIC50, determined by the Etest® , was 8 µg/mL for gentamicin and netilmicin and 64 µg/mL for tobramycin. The MIC90 was 16 and 32 µg/mL for gentamicin and netilmicin respectively. The MIC90 was outside of the detectable limits for tobramycin. To framycetin, 28 strains (40%) of the 70 M. pachydermatis isolates tested showed a diameter of 22 mm, 22 strains (31.42%) showed a diameter of 20 mm, 16 strains showed a diameter of ≤ 18 mm, and only 5.71% of the isolates showed a diameter of ≥ 22 mm. This study provides evidence of high in vitro activity of the aminoglycosides-gentamicin, tobramycin, netilmicin and framycetin against M. pachydermatis. For gentamicin Etest® showed similar values of MIC50 y MIC90 that the obtained by microdilution method. We considered Etest® method could be a good method for these calculations with aminoglycosides.

Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax).

The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species.

Detection and Localization of IL-8 and CXCR1 in Rainbow Trout Larvae in Response to Pseudomonas aeruginosa Lipopolysaccharide.

The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

Effects of Hypoxia on the Antibacterial Activity of Epidermal Mucus from Chilean Meagre (Cilus gilberti).

Comprehending the immune defense mechanisms of new aquaculture species, such as the Chilean meagre (Cilus gilberti), is essential for sustaining large-scale production. Two bioassays were conducted to assess the impact of acute and intermittent hypoxia on the antibacterial activity of juvenile Chilean meagre epidermal mucus against the potential pathogens Vibrio anguillarum and Vibrio ordalii. Lysozyme and peroxidase activities were also measured. In general, fish exposed to hypoxia showed a 9-30% reduction in mucus antibacterial activity at the end of hypoxic periods and after stimulation with lipopolysaccharide. However, following water reoxygenation, the activity of non-stimulated fish was comparable to that of fish in normoxic conditions, inhibiting bacterial growth by 35-52%. In the case of fish exposed to chronic hypoxia, the response against V. anguillarum increased by an additional 19.8% after 6 days of control inoculation. Lysozyme exhibited a similar pattern, while no modulation of peroxidase activity was detected post-hypoxia. These results highlight the resilience of C. gilberti to dissolved oxygen fluctuations and contribute to understanding the potential of mucus in maintaining the health of cultured fish and the development of future control strategies.

An In-Depth Study on the Inhibition of Quorum Sensing by Bacillus velezensis D-18: Its Significant Impact on Vibrio Biofilm Formation in Aquaculture.

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.

Current Status of Probiotics in European Sea Bass Aquaculture as One Important Mediterranean and Atlantic Commercial Species: A Review.

European sea bass production has increased in recent decades. This increase is associated with an annually rising demand for sea bass, which encourages the aquaculture industries to increase their production to meet that demand. However, this intensification has repercussions on the animals, causing stress that is usually accompanied by dysbiosis, low feed-conversion rates, and immunodepression, among other factors. Therefore, the appearance of pathogenic diseases is common in these industries after immunodepression. Seeking to enhance animal welfare, researchers have focused on alternative approaches such as probiotic application. The use of probiotics in European sea bass production is presented as an ecological, safe, and viable alternative in addition to enhancing different host parameters such as growth performance, feed utilization, immunity, disease resistance, and fish survival against different pathogens through inclusion in fish diets through vectors and/or in water columns. Accordingly, the aim of this review is to present recent research findings on the application of probiotics in European sea bass aquaculture and their effect on growth performance, microbial diversity, enzyme production, immunity, disease resistance, and survival in order to help future research.

Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚß2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

Gill Oxidative Stress Protection through the Use of Phytogenics and Galactomannan Oligosaccharides as Functional Additives in Practical Diets for European Sea Bass (Dicentrarchus labrax) Juveniles.

The aim of the present study is to evaluate the potential of two functional additives as gill endogenous antioxidant capacity boosters in European sea-bass juveniles fed low-FM/FO diets when challenged against physical and biological stressors. For that purpose, two isoenergetic and isonitrogenous diets with low FM (10%) and FO (6%) contents were supplemented with 5000 ppm plant-derived galactomannan-oligosaccharides (GMOS) or 200 ppm of a mixture of garlic and labiate plant essential oils (PHYTO). A control diet was void from supplementation. Fish were fed the experimental diet for nine weeks and subjected to a confinement stress challenge (C challenge) or a confinement stress challenge combined with an exposure to the pathogen Vibrio anguillarum (CI challenge). Both GMOS and PHYTO diets attenuated fish stress response, inducing lower circulating plasma cortisol and down-regulating nfκß2 and gr relative gene-expression levels in the gill. This attenuated stress response was associated with a minor energetic metabolism response in relation to the down-regulation of nd5 and coxi gene expression.

Immune Modulation Ability of Hepcidin from Teleost Fish.

Antimicrobial peptides (AMP) play an essential role in the innate immune system, modulating the defense response. In a previous study, we demonstrated the antimicrobial activity of synthetic hepcidin (hep20) from rainbow trout (Oncorhynchus mykiss), and its protective effect in European sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum. Additionally, we described the uptake and distribution of hep20 in different tissues and leukocyte cells. Interestingly, various AMPs characterized in high vertebrates, called host defense peptides (HDPs), also possess immunomodulation activity. For that reason, the present study explores the immunomodulatory abilities of hep20 through in vitro and in vivo studies. First, a monocyte/macrophage RTS-11 cell line from rainbow trout was used to evaluate hep20 effects on pro- and anti-inflammatory cytokines in fish leukocyte cells. Next, the European sea bass juveniles were used to determine if hep20 can regulate the expression of cytokines in fish immune tissues. The results show that hep20 was uptake inner to RTS-11 cells and was able to induce the expression of IL-10, IL-1ß, and TNFα at transcriptional and protein levels. Then, the European sea bass juveniles were given intraperitoneal injections of the peptide. At 1, 3, 7, 14, and 21 days post-injection (dpi), IL-10, IL -1ß, and TNFα mRNA were quantified in the anterior gut, spleen, and head kidney. The hep20 was able to up-regulate cytokine gene expression in these tissues, mainly in the head kidney. Furthermore, the evaluated cytokines showed a cyclical tendency of higher to lesser expression. Finally, a bioinformatics analysis showed that the structure adopted by hep20 is similar to the γ-core domain described for cysteine-stabilized AMP, defined as immunomodulatory and antimicrobial, which could explain the ability of hep20 to regulate the cytokine expression. This study provides new insights into immunomodulatory function complementary to the previously established antimicrobial activity of hep20, suggesting a role as an HDP in teleost fish. These facts are likely to be associated with molecular functions underpinning the protective effect of fish hepcidin against pathogens.

Dietary Phytogenics and Galactomannan Oligosaccharides in Low Fish Meal and Fish Oil-Based Diets for European Sea Bass (Dicentrarchus labrax) Juveniles: Effects on Gill Structure and Health and Implications on Oxidative Stress Status.

An effective replacement for fish meal (FM) and fish oil (FO) based on plant-based raw materials in the feed of marine fish species is necessary for the sustainability of the aquaculture sector. However, the use of plant-based raw materials to replace FM and FO has been associated with several negative health effects, some of which are related to oxidative stress processes that can induce functional and morphological alterations in mucosal tissues. This study aimed to evaluate the effects of dietary oligosaccharides of plant origin (5,000 ppm; galactomannan oligosaccharides, GMOS) and a phytogenic feed additive (200 ppm; garlic oil and labiatae plant extract mixture, PHYTO) on the oxidative stress status and mucosal health of the gills of juvenile European sea bass (Dicentrarchus labrax). The experimental diets, low FM and FO diets (10%FM/6%FO) were supplemented with GMOS from plant origin and PHYTO for 63 days. GMOS and PHYTO did not significantly affect feed utilization, fish growth, and survival. GMOS and PHYTO downregulated the expression of ß-act, sod, gpx, cat, and gr in the gills of the fish compared with that in fish fed the control diet. The expression of hsp70 and ocln was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of zo-1 was downregulated in the PHYTO group compared with that in the GMOS group. The morphological, histopathological, immunohistochemical, and biochemical parameters of the fish gills were mostly unaffected by GMOS and PHYTO. However, the PHYTO group had lower incidence of lamellar fusion than did the control group after 63 days. Although the tissular distribution of goblet cells was unaffected by GMOS and PHYTO, goblet cell size showed a decreasing trend (-11%) in the GMOS group. GMOS and PHYTO significantly reduced the concentration of PCNA+ in the epithelium of the gills. The above findings indicated that GMOS and PHYTO in low FM/FO-based diets protected the gill epithelia of D. labrax from oxidative stress by modulating the expression of oxidative enzyme-related genes and reducing the density of PCNA+ cells in the gills of the fish.

Organic Selenium (OH-MetSe) Effect on Whole Body Fatty Acids and Mx Gene Expression against Viral Infection in Gilthead Seabream (Sparus aurata) Juveniles.

The supplementation of fish diets with OH-SeMet reduces oxidative stress and modulates immune response against bacterial infection. However, despite the importance of essential polyunsaturated fatty acids in fish nutrition and their high risk of oxidation, the potential protective effect of OH-SeMet on these essential fatty acids has not been studied in detail. Moreover, while viral infection is very relevant in seabream production, no studies have focused the Se effects against viral infection. The aim of the present study was to assess the impact of dietary supplementation with OH-SeMet on gilthead seabream fatty acid profiles, growth performance and response against viral infection. Gilthead seabream juveniles (21.73 ± 0.27 g) were fed for 91 days with three experimental diets, a control diet without supplementation of Se (0.29 mg Se kg diet-1) and two diets supplemented with OH-SeMet (0.52 and 0.79 mg Se kg diet-1). A crowding stress test was performed at week 7 and an anti-viral response challenge were conducted at the end of the feeding trial. Selenium, proximate and fatty acid composition of diets and body tissues were analyzed. Although fish growth was not affected, elevation in dietary Se proportionally raised Se content in body tissues, increased lipid content in the whole body and promoted retention and synthesis of n-3 polyunsaturated fatty acids. Specifically, a net production of DHA was observed in those fish fed diets with a higher Se content. Additionally, both monounsaturated and saturated fatty acids were significantly reduced by the increase in dietary Se. Despite the elevation of dietary Se to 0.79 mg kg-1 not affecting basal cortisol levels, 2 h post-stress plasma cortisol levels were markedly increased. Finally, at 24 h post-stimulation, dietary OH-SeMet supplementation significantly increased the expression of the antiviral response myxovirus protein gene, showing, for the first time in gilthead seabream, the importance of dietary Se levels on antiviral defense.

Isolation and Characterization of a Bacillus velezensis D-18 Strain, as a Potential Probiotic in European Seabass Aquaculture.

Within the food-producing sectors, aquaculture is the one that has developed the greatest growth in recent decades, currently representing almost 50% of the world's edible fish. The diseases can affect the final production in intensive aquaculture; in seabass, aquaculture vibriosis is one of the most important diseases producing huge economical losses in this industry. The usual methodology to solve the problems associated with the bacterial pathology has been the use of antibiotics, with known environmental consequences. This is why probiotic bacteria are proposed as an alternative fight against pathogenic bacteria. The aim of this study was to analyse a strain of Bacillus velezensis D-18 isolated from a wastewater sample collected from a fish farm, for use as probiotics in aquaculture. The strain was evaluated in vitro through various mechanisms of selection, obtaining as results for growth inhibition by co-culture a reduction of 30%; B. velezensis D-18 was able to survive at 1.5-h exposure to 10% seabass bile, and at pH 4, its survival is 5% and reducing by 60% the adhesion capacity of V. anguillarum 507 to the mucus of seabass and in vivo by performing a challenge. Therefore, in conclusion, we consider B. velezensis D-18 isolate from wastewater samples collected from the farms as a good candidate probiotic in the prevention of the infection by Vibrio anguillarum 507 in European seabass after in vitro and biosafety assays.