Broodstock nutritional programming differentially affects the hepatic transcriptome and genome-wide DNA methylome of farmed gilthead sea bream (Sparus aurata) depending on genetic background.

BACKGROUND: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program. RESULTS: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2). CONCLUSIONS: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism.

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 1ß (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-1ß 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-1ß 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.

The immune status against tetanus and diphtheria in healthcare workers in Catalonia.

The objective was to investigate the immune status against tetanus and diphtheria of healthcare workers in Catalonia. A cross-sectional multicentre study was conducted in seven health centres. Blood samples were obtained, and demographic and clinical variables collected. 509 health workers were included. The prevalence of protective antibodies against tetanus was 94.7% (95% CI: 92.3-96.4) overall and 85.1% (95% CI: 74.5-92.0) in workers aged ≥55 years. The prevalence of protective antibodies against diphtheria was 68.6% (95% CI: 64.3-72.5%) overall and 29.7% (95% CI: 19.9-41.6) in workers aged ≥55 years. Protection against tetanus in healthcare workers is high, but should be improved in workers aged ≥55 years. Protection against diphtheria has improved in healthcare workers over the past decade (68.6% vs 46.5%) but should be improved in all ages, especially in workers aged ≥55 years.

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-1ß 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.

Feeding European sea bass (Dicentrarchus labrax) juveniles with a functional synbiotic additive (mannan oligosaccharides and Pediococcus acidilactici): An effective tool to reduce low fishmeal and fish oil gut health effects?

The aim of this study was to assess the effects of dietary mannan oligosaccharides (MOS), Pediococcus acidilactici or their conjunction as a synbiotic in low fish meal (FM) and fish oil (FO) based diets on European sea bass (Dicentrarchus labrax) disease resistance and gut health. For that purpose, sea bass juveniles were fed one of 6 diets containing different combinations of MOS (Biomos® and Actigen©; Alltech, Inc., Kentucky, USA) and Pediococcus acidilactici (BAC, Bactocell®; Lallemand Inc., Cardiff, UK) replacing standard carbohydrates as follows (MOS (%)/BAC (commercial recommendation): high prebiotic level (HP) = 0.6/0, low prebiotic level (LP) = 0.3/0, only probiotic (B) = 0/+, high prebiotic level plus probiotic (HPB) = 0.6/+, low prebiotic level plus probiotic (LPB) = 0.3/+, control (C) = 0/0 for 90 days. After 60 and 90 days of feeding trial, fish were subjected to an experimental infection against Vibrio anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity and major histocompatibility complex class II (MHCII), transforming growth factor ß (TGF-ß), regulatory T-cell subset (CD4+T lymphocytes) and effector T cell (CD8α+T lymphocytes) gene expression patterns in gut by in situ hybridization were evaluated after 90 days of feeding. The effects of both additives on posterior gut through Gut Associated Lymphoid Tissue (GALT) gene expression was also studied. Fish fed the prebiotic and its combination with P. acidilactici presented increased weight regardless of the dose supplemented after 90 days of feeding, however no effect was detected on somatic indexes. For posterior gut, morphometric patterns and goblet cells density was not affected by MOS, P. acidilactici or its combination. Anti-iNOS and anti-TNFα gut immunopositivity patterns were mainly influenced by MOS supplementation and not by its combination with P. acidilactici. MHCII-ß, TCR-ß, CD4 and CD8-α positive cells distribution and incidence was not affected by diet. Fish fed HP dose presented a clear up-regulation of TNF-α, cyclooxygenase-2 (COX-2), CD4 and IL10, whereas P. acidilactici dietary supplementation increased the number of interleukin-1ß (IL1ß) and COX-2 gene transcripts. Synbiotic supplementation resulted in a reduction of MOS-induced gut humoral proinflammatory response by increasing the expression of some cellular-immune system related genes. Fish mortality after V. anguillarum infection was reduced in fish fed LPB and LP diets compared to fish fed the non-suppelmented diet after 90 days of feeding. Thus, overall pointing to the combination of a low dose of MOS and P. acidilactici as synbiont (LPB) as a viable tool to potentiate European sea bass juvenile's growth and disease resistance when supplemented in low FM and FO diets.

Supplementation of arachidonic acid rich oil in European sea bass juveniles (Dicentrarchus labrax) diets: effects on growth performance, tissue fatty acid profile and lipid metabolism.

The aim of this study was to evaluate the effects of increasing dietary arachidonic acid (ARA) levels (from 1 to 6% of total fatty acids) on European sea bass (Dicentrarchus labrax) juveniles' growth performance, tissue fatty acid profile, liver morphology as well as long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, triglyceride and cholesterol synthesis and lipid transport. A diet with total fish oil (FO) replacement and defatted fish meal (FM) containing a 0.1-g ARA g-1 diet was added to the experimental design as a negative control diet. Dietary ARA inclusion levels below 0.2 g ARA g-1 diet significantly worsened growth even only 30 days after the start of the feeding trial, whereas dietary ARA had no effect on fish survival. Liver, muscle and whole body fatty acid profile mainly reflected dietary contents and ARA content increased accordingly with ARA dietary levels. Tissue eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels were positively correlated among them. Hepatic lipid vacuolization increased with reduced dietary ARA levels. Expressions of fatty acyl desaturase 2 and 3-hydroxy-3-methylglutaryl-coenzyme genes were upregulated in fish fed the negative control diet compared to the rest of the dietary treatments denoting the influence of ARA on lipid metabolism. Results obtained highlight the need to include adequate n-6 levels and not only n-3 LC-PUFA levels in European sea bass diets.

Disease resistance and response against Vibrio anguillarum intestinal infection in European seabass (Dicentrarchus labrax) fed low fish meal and fish oil diets.

The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub-lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up-regulation of interleukin-1ß (IL-1ß) and TNFα, major histocompatibility complex-II (MHCII) and cyclooxygenase-2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24 h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti-iNOS and anti-TNFα presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti-TNFα isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti-iNOS but not to anti-TNFα. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up-regulates the expression of several posterior gut inflammation-related genes conditioning fish growth and GALT capacity of response after bacterial infection.

Supplementation of arachidonic acid rich oil in European sea bass juveniles (Dicentrarchus labrax) diets: Effects on leucocytes and plasma fatty acid profiles, selected immune parameters and circulating prostaglandins levels.

The main objective of this study was to assess the effects of graded levels of dietary arachidonic acid (ARA), supplemented from alternative sources, on fatty acid composition of plasma and head kidney leucocytes of European sea bass (Dicentrarchus labrax). For that purpose, sea bass juveniles were fed four diets containing graded levels of ARA as follows: 0.5% (ARA0.5), 1% (ARA1), 2% (ARA2) and 4% (ARA4) during 60 days. At the end of the feeding trial fatty acid profiles of plasma and head kidney leucocytes were analyzed. Besides, plasma prostaglandins levels, head kidney leucocytes respiratory burst activity; peroxidase activity and phagocytic index were assayed. Reducing dietary ARA levels below 1% markedly reduced European sea bass growth performance. However, fish fed diet ARA0.5 tried to compensate this dietary ARA deficiency by a selective deposition of ARA on plasma and head kidney leucocytes, reaching similar levels to those fish fed diet ARA1 after 60 days of feeding. Nevertheless, head kidney phagocytic capacity was reduced as dietary ARA content in relation not only to variations on membrane composition but also to changes on fish basal prostaglandins levels. Results obtained demonstrated the importance to supply the necessary quantity n-6 LC-PUFA, and not only n-3 LC-PUFA levels, in European sea bass diets, in relation to not only growth performance but also immune system function.

[Patient's beliefs about oral targeted therapies and impact on drug adherence in lung cancer: A pilot prospective study]. / Croyances des patients envers les thérapies ciblées orales et leur influence sur l'observance dans le cancer broncho-pulmonaire : une étude pilote prospective.

INTRODUCTION: Oral targeted therapies are a new option for lung cancer treatment. However, patient's belief about these drugs - which may interact with adherence - is poorly known in this setting. METHOD: Our study is a pilot prospective unicentric study. Inclusion criteria were: to have been diagnosed with a lung cancer; and to be prescribed with an oral targeted therapy in second line or more. The main objective was to assess patient's specific (SB) and general beliefs (GB) about these drugs according to the Beliefs about Medicines Questionnaire (BMQ). The declared adherence was assessed with the Morisky's test. All included patients underwent a semi-structured interview with a psychologist. RESULTS: Fifthteen patients were included: 12 underwent erlotinib treatment and 3 a crizotinib treatment. The mean score (±standard deviation) at BMQ was 54/85 (±6) overall; 34/50 (±5) for specific belief and 19/35 (±3) for general belief about drugs. During interview, 47% believed in efficacy of targeted oral therapy; 93% reported concerns about their drug; 80% considered that the information given by the physician about the drug was comprehensive; but 40% still required additional information about it. The mean score at Morisky's test was 3/4 (±2) and 53% reported to have forgotten at least once their antineoplastic drug. No correlation was found between belief and adherence. CONCLUSION: Belief about t anti-cancer targeted oral therapy is relatively fair but adherence is moderate in this pilot study. Interview shows the need for additional information about the prescribed drug.

Effects on mortality and stress response in European sea bass, Dicentrarchus labrax (L.), fed mannan oligosaccharides (MOS) after Vibrio anguillarum exposure.

The effects of dietary mannan oligosaccharides (MOS; 4 g kg(-1) ; Bio-Mos, Alltech Inc, USA) in diets for European sea bass, Dicentrarchus labrax (L.), juveniles in relation to disease and stress resistance, combining intestinal infection with Vibrio anguillarum and stress challenge by confinement, were assessed in this study. After 8 weeks of MOS supplementation, fish were exposed to a pathogen challenge test against V. anguillarum by direct gut inoculation combined with a confinement stressor panel. Cumulative mortality of fish fed MOS caused by anally inoculated V. anguillarum decreased from 66% to 12.5% and from 54.1% to 25% in infected and infected + stressed fish, respectively, compared to fish fed control diet. Results for European sea bass revealed a positive effect of MOS dietary inclusion on disease resistance, in terms of cumulative mortality, against gut inoculated V. anguillarum, as well as reduced effects of stress on microbiota diversity. Both of these findings, together with the enhanced innate immune response and the higher gut mucus production and density of eosinophil granulocytes in gut mucosa obtained in previous studies after MOS supplementation (Torrecillas et al. 2007, 2011a,b) suggest that general reinforcement of the innate immune system, and particularly of the intestinal barrier efficiency, is the main defence mechanism of European sea bass fed MOS against pathogenic microorganisms.

Replacement of dietary fish oil by vegetable oils affects humoral immunity and expression of pro-inflammatory cytokines genes in gilthead sea bream Sparus aurata.

Commercial gilthead sea bream feeds are highly energetic, fish oil traditionally being the main lipid source. But the decreased fish oil production together with the increased prices of this oil encourages its substitution by vegetable oils, imposing new nutritional habits to aquaculture species. Partial replacement of fish oil by vegetable oils in diets for marine species allows good feed utilization and growth but may affect fish health, since imbalances in dietary fatty acids may alter fish immunological status. The effect of dietary oils on different aspects of fish immune system has been reported for some species, but very little is known about the effect of dietary oils on immune-related genes expression in fish. Thus, the objective of this study was to elucidate the role of dietary oils on the expression of two pro-inflammatory cytokines, Tumor Necrosis Factor-α (TNF-α) and Interleukine 1ß (IL-1ß) on intestine and head kidney after exposure to the bacterial pathogen Photobacterium damselae sp. piscicida. For that purpose, 5 iso-nitrogenous and iso-lipidic diets (45% crude protein, 22% crude lipid content) were formulated. Anchovy oil was the only lipid source used in the control diet (FO), but in the other diets, fish oil was totally (100%) or partially (70%) substituted by linseed (rich in n-3 fatty acids) or soybean (rich in n-6 fatty acids) (100L, 100S, 70L, 70S). Fish were fed experimental diets during 80 days and after this period were exposed to an experimental intestinal infection with the pathogen. Serum and tissue samples were obtained at pre-infection and after 1, 3 and 7 days of infection. RNA was extracted and cDNA was synthesized by reverse transcription from intestine and head kidney and the level expression of TNF-α and IL-1ß were assayed by using quantitative real time PCR. The expression level of genes analysed was represented as relative value, using the comparative Ct method (2(-ΔΔCt)). Serum anti-bacterial activity was measured as serum bactericidal capacity and lysozyme activity. Reduction of FO tends to reduce basal (pre-infection) genetic expression of both cytokines. However, complete FO replacement caused an over expression of both pro-inflammatory cytokines, particularly after 3 days of induced infection in fish fed soybean oil based diets. On the other hand, fish fed diets with low content of n-6 fatty acids showed better serum bactericidal capacity after infection, suggesting that the substitution of fish oil by vegetable oils containing high levels of n-6 fatty acids may induce imbalances on fish immune response, leading to a lower potential response against infections.

Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

The objective of this study was to determine the effect of two levels of inclusion of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on growth, feed utilization, immune status and disease resistance of European sea bass (Dicentrarchus labrax). Specimens of 35 g at initial density of 3 kg/m3 were fed during 67 days at 0 per thousand, 2 per thousand and 4 per thousand dietary MOS level of inclusion in a commercial sea bass diet. Food conversion rate, specific growth rate, whole body biochemical composition, phagocyctic index of head kidney macrophages, NBT index, lysozyme and alternative complement pathway (ACP) activities as well as gut and liver histological structure were evaluated. Growth significantly increased at both MOS dietary inclusion levels. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients. No differences were found on gut histological evaluation. Statistical differences (P<0.05) on the phagocytic index were denoted with the inclusion of 4 per thousand Bio-Mos group. A positive correlation was found between the levels of lysozyme and alternative complement pathway activities in blood and the level of inclusion of MOS in diets. After the feeding trial, a cohabitation challenge test and direct gut inoculation were also performed with the pathogen Vibrio alginolyticus in a ratio 3:1. Twenty-one days post-challenge the number of cohabitant fish infected in the control group reached 33% comparing with none on the 0.4 per thousand MOS group. Finally, new fish were infected with V. alginolyticus by gut canalisation. After 24h post-infection no significant difference was denoted between groups and 48 h post-infection total infected fish in the control group was twice that of the 2 per thousand and 4 per thousand MOS groups.