Effect of ß-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells.

ß-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of ß-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of ß-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. ß-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, ß-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, ß-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of ß-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a ß-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of ß-glucans on fish adaptive responses.

Yersinia ruckeri infection activates local skin and gill B cell responses in rainbow trout.

Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.

Mucosal and systemic immune effects of Bacillus subtilis in rainbow trout (Oncorhynchus mykiss).

Bacillus spp. are well known for their probiotic properties. Hence, the long-term feeding of Bacillus spp. strains to different fish species has been proved to confer beneficial effects regarding growth or pathogen resistance, among others. However, whether these strains could function as mucosal adjuvants, up-regulating immune responses after a single administration, has not yet been investigated in fish. Thus, in the current work, we have performed a series of experiments in rainbow trout (Oncorhynchus mykiss) aimed at establishing the potential of two Bacillus subtilis spore-forming strains, designated as ABP1 and ABP2, as oral adjuvants/immunostimulants. As an initial step, we evaluated their transcriptional effects on the rainbow trout intestinal epithelial cell line RTgutGC, and in gut tissue explants incubated ex vivo with the two strains. Their capacity to adhere to RTgutGC cells was also evaluated by flow cytometry. Although both strains had the capacity to modulate the transcription of several genes related to innate and adaptive immune responses, it was the ABP1 strain that led to stronger transcriptional effects, also exerting a higher binding capacity to intestinal epithelial cells. Consequently, we selected this strain to establish its effects on splenic B cells upon in vitro exposure as well as to determine the transcriptional effects exerted in the spleen, kidney, and gut after a single oral administration of the bacteria. Our results showed that B. subtilis ABP1 had the capacity to modulate the proliferation, IgM secreting capacity and MHC II surface expression of splenic B cells. Finally, we confirmed that this strain also induced the transcription of genes involved in inflammation, antimicrobial genes, and genes involved in T cell responses upon a single oral administration. Our results provide valuable information regarding how B. subtilis modulates the immune response of rainbow trout, pointing to the usefulness of the ABP1 strain to design novel oral vaccination strategies for aquaculture.

Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Evaluation of the Potential of Marine Algae Extracts as a Source of Functional Ingredients Using Zebrafish as Animal Model for Aquaculture.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.

Zebrafish Nk-lysins: First insights about their cellular and functional diversification.

Nk-lysins are antimicrobial proteins produced by cytotoxic T lymphocytes and natural killer cells with a broad antimicrobial spectrum (including bacteria, fungi and parasites). Nevertheless, the implication of these proteins in the protection against viral infections is still poorly understood. In this work, four different Nk-lysin genes (nkla, nklb, nklc and nkld) were identified in the zebrafish genome. That means that zebrafish is the species with the higher repertoire of Nk-lysin genes described so far. The differential expression pattern of the Nk-lysins in several tissues, during ontogeny, among the different kidney cell populations, as well as between Rag1(-/-) and Rag1(+/+) individuals, could suggest a certain specialization of different cell types in the production of different Nk-lysin. Moreover, only two of these genes (nkla and nkld) were significantly up-regulated after viral infection, and this observation could be also a consequence of a functional diversification of the zebrafish Nk-lysins.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction.

Type I interferons (IFNs) are considered the main cytokines directing the antiviral immune response in vertebrates. These molecules are able to induce the transcription of interferon-stimulated genes (ISGs) which, using different blocking mechanisms, reduce the viral proliferation in the host. In addition, a contradictory role of these IFNs in the protection against bacterial challenges using murine models has been observed, increasing the survival or having a detrimental effect depending on the bacteria species. In teleosts, a variable number of type I IFNs has been described with different expression patterns, protective capabilities or gene induction profiles even for the different IFNs belonging to the same species. In this work, two type I IFNs (ifn1 and ifn2) have been characterized for the first time in turbot (Scophthalmus maximus), showing different properties. Whereas Ifn1 reflected a clear antiviral activity (over-expression of ISGs and protection against viral haemorrhagic septicaemia virus), Ifn2 was not able to induce this response, although both transcripts were up-regulated after viral challenge. On the other hand, turbot IFNs did not show any protective effect against the bacteria Aeromonas salmonicida, although they were induced after bacterial challenge. Both IFNs induced the expression of several immune genes, but the effect of Ifn2 was mainly limited to the site of administration (intramuscular injection). Interestingly, Ifn2 but not Ifn1 induced an increase in the expression level of interleukin-1 beta (il1b). Therefore, the role of Ifn2 could be more related with the immune regulation, being involved mainly in the inflammation process.

Microarray-based identification of differentially expressed genes in families of turbot (Scophthalmus maximus) after infection with viral haemorrhagic septicaemia virus (VHSV).

Viral haemorrhagic septicaemia virus (VHSV) is one of the major threats to the development of the aquaculture industry worldwide. The present study was aimed to identify genes differentially expressed in several turbot (Scophthalmus maximus) families showing different mortality rates after VHSV. The expression analysis was conducted through genome-wide expression profiling with an oligo-microarray in the head kidney. A significant proportion of the variation in the gene expression profiles seemed to be explained by the genetic background, indicating that the mechanisms by which particular species and/or populations can resist a pathogen(s) are complex and multifactorial. Before the experimental infections, fish from resistant families (low mortality rates after VHSV infection) showed high expression of different antimicrobial peptides, suggesting that their pre-immune state may be stronger than fish of susceptible families (high mortality rates after VHSV infection). After infection, fish from both high- and low-mortality families showed an up-modulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Low levels of several molecules secreted in the mucus were observed in high-mortality families, but different genes involved in viral entrance into target cells were down-regulated in low-mortality families. Moreover, these families also showed a strong down-modulation of marker genes related to VHSV target organs, including biochemical markers of renal dysfunction and myocardial injury. In general, the expression of different genes involved in the metabolism of sugars, lipids and proteins were decreased in both low- and high-mortality families after infection. The present study serves as an initial screen for genes of interest and provides an extensive overview of the genetic basis underlying the differences between families that are resistant or susceptible to VHSV infection.

Rainbow trout interleukin-2: cloning, expression and bioactivity analysis.

In this study the rainbow trout (Oncorhynchus mykiss) interleukin-2 (IL-2) cDNA has been cloned, and its expression and bioactivity analysed in head kidney leucocytes. The IL-2 precursor encoded an open reading frame of 429 bp, that translates into a predicted protein of 142 aa, with a 20 aa signal peptide. The trout IL-2 had moderate protein homology (30.9% identity/48.3% similarity) with Fugu IL-2, the only IL-2 homologue identified in fish to date, with lower homology to avian (17.8% identity/23.2% similarity) and mammalian (34.2 identity/46.5% similarity) IL-2s. IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. The Escherichia coli produced recombinant IL-2 was shown to increase the expression of two transcription factors, STAT5 and Blimp-1, known to be involved in IL-2 signalling in mammals, as well as IFN-gamma, gIP and IL-2 itself. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines are discussed.

Effect of dietary administration of Porphyridium cruentum on the respiratory burst activity of sole, Solea senegalensis (Kaup), phagocytes.

The stimulatory effect of the red microalga Porphyridium cruentum on respiratory burst activity of sole phagocytes was evaluated in vivo. Oral administration of a diet supplemented with lyophilized P. cruentum cells (10 g kg(-1)) stimulated respiratory burst activity after 4 weeks feeding in sole vaccinated with Photobacterium damselae subsp. piscicida bacterin.

Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi.

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (approximately 93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi. Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of approximately 93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.

Superoxide dismutase and catalase activities in Photobacterium damselae ssp. piscicida.

The ability of a set of Photobacterium damselae ssp. piscicida strains isolated from different fish species to produce different superoxide dismutase (SOD) and catalase enzymes was determined. Unlike other bacterial pathogens, P. damselae ssp. piscicida is not able to produce different isoforms of SOD or catalase containing different metal cofactors when cultured under oxidative stress induced by hydrogen peroxide or methyl viologen, or under iron depleted conditions. However, iron content of the growth medium influenced the levels of SOD and catalase activity in cells, these levels decreasing with iron availability of the medium. Comparison of virulent and non-virulent strains of P. damselae ssp. piscicida showed similar contents of SOD, but higher levels of catalase were detected in cells of the virulent strain. Incubation of bacteria with sole, Solea senegalensis (Kaup), phagocytes has shown that survival rates range from 19% to 62%, these rates being higher for the virulent strain. The increased levels of catalase activity detected in the virulent strain indicates a possible role for this enzyme in bacterial survival.

Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup).

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.

Effectiveness of a divalent vaccine for sole, Solea senegalensis (Kaup), against Vibrio harveyi and Photobacterium damselae subsp. piscicida.

The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.