Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies.

In the present study, we propose the use of swabs in non-lethal sampling procedures to collect the mucosa-adhered gut microbiota from the posterior intestine of fish, and therefore, we compare the bacterial communities collected by conventional scraping and by swabbing methods. For this purpose, samples of the posterior intestine of rainbow trout (Oncorhynchus mykiss) were collected first using the swabbing approach, and after fish euthanasia, by mucosa scraping. Finally, bacterial communities were compared by 16S rRNA gene Illumina sequencing. Results from the current study revealed that similar values of bacterial richness and diversity were found for both sampling procedures. Similarly, there were no differences between procedures when using qualitative metrics (Jaccard and unweighted UniFrac) for estimating inter-individual diversity, but the quantitative metrics (Bray-Curtis and weighted UniFrac) showed a higher dispersion when samples were obtained by swabbing compared to scraping. In terms of bacterial composition, there were differences in abundance for the phyla Firmicutes and Proteobacteria. The cause of these differential abundances may be the inability of the swab to access to certain areas, such as the basal region of the intestinal villi. Moreover, swabbing allowed a higher representation of low abundant taxa, which may also have an important role in host microbiome regardless of their low abundance. Overall, our results demonstrate that the sampling method is a factor to be considered in experimental design when studying gut bacterial communities to avoid potential biases in the interpretation or comparison of results from different studies. In addition, the advantages and disadvantages of each procedure (swabbing vs scraping) are discussed in detail, concluding that swabbing can be implemented as a reliable and non-lethal procedure for posterior gut microbiota studies, which is of particular interest for animal welfare and the 3Rs principle, and may offer a wide range of novel applications.

Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model.

The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species.

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.

More Than an Antioxidant: Role of Dietary Astaxanthin on Lipid and Glucose Metabolism in the Liver of Rainbow Trout (Oncorhynchus mykiss).

This study investigated the influence of dietary astaxanthin (AX) on glucose and lipid metabolism in rainbow trout liver. Two iso-nitrogenous and iso-lipidic diets were tested for 12 weeks in rainbow trout with an initial mean weight of 309 g. The S-ASTA diet was supplemented with 100 mg of synthetic AX per kg of feed, whereas the control diet (CTRL) had no AX. Fish fed the S-ASTA diet displayed lower neutral and higher polar lipids in the liver, associated with smaller hepatocytes and lower cytoplasm vacuolization. Dietary AX upregulated adipose triglyceride lipase (atgl), hormone-sensitive lipase (hsl2) and 1,2-diacylglycerol choline phosphotransferase (chpt), and downregulated diacylglycerol acyltransferase (dgat2), suggesting the AX's role in triacylglycerol (TAG) turnover and phospholipid (PL) synthesis. Dietary AX may also affect beta-oxidation with the upregulation of carnitine palmitoyltransferase 1 (cpt1α2). Although hepatic cholesterol levels were not affected, dietary AX increased gene expression of sterol regulatory element-binding protein 2 (srebp2). Dietary AX upregulated the expression of 6-phosphogluconate dehydrogenase (6pgdh) and downregulated pyruvate kinase (pkl). Overall, results suggest that dietary AX modulates the oxidative phase of the pentose phosphate pathway and the last step of glycolysis, affecting TAG turnover, ß-oxidation, PL and cholesterol synthesis in rainbow trout liver.

Genetics and Nutrition Drive the Gut Microbiota Succession and Host-Transcriptome Interactions through the Gilthead Sea Bream (Sparus aurata) Production Cycle.

Fish genetically selected for growth (GS) and reference (REF) fish were fed with CTRL (15% FM, 5-7% FO) or FUTURE (7.5% FM, 10% poultry meal, 2.2% poultry oil + 2.5% DHA-algae oil) diets during a 12-months production cycle. Samples from initial (t0; November 2019), intermediate (t1; July 2020) and final (t2; November 2020) sampling points were used for Illumina 16S rRNA gene amplicon sequencing of the adherent microbiota of anterior intestine (AI). Samples from the same individuals (t1) were also used for the gene expression profiling of AI by RNA-seq, and subsequent correlation analyses with microbiota abundances. Discriminant analyses indicated the gut bacterial succession along the production cycle with the proliferation of some valuable taxa for facing seasonality and different developmental stages. An effect of genetic background was evidenced along time, decreasing through the progression of the trial, namely the gut microbiota of GS fish was less influenced by changes in diet composition. At the same time, these fish showed wider transcriptomic landmarks in the AI to cope with these changes. Our results highlighted an enhanced intestinal sphingolipid and phospholipid metabolism, epithelial turnover and intestinal motility in GS fish, which would favour their improved performance despite the lack of association with changes in gut microbiota composition. Furthermore, in GS fish, correlation analyses supported the involvement of different taxa with the down-regulated expression of pro-inflammatory markers and the boosting of markers of extracellular remodelling and response to bacterium. Altogether, these findings support the combined action of the gut microbiome and host transcriptionally mediated effects to preserve and improve gut health and function in a scenario of different growth performance and potentiality.

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.

Impact of polypropylene microplastics and chemical pollutants on European sea bass (Dicentrarchus labrax) gut microbiota and health.

Plastic pollution has become a global problem for marine ecosystems. Microplastics (MPs) are consumed by several marine organisms, including benthic and pelagic fish species that confuse them with food sources, thus contributing to bioaccumulation along the food chain. In addition to structural intestinal damage, ingestion of MPs represents a pathway for fish exposure to potentially hazardous chemicals, too. Most of them are endocrine disrupters, genotoxic or induce immune depression in fish. Accordingly, we assessed the combined toxicological effects of microplastics (MPs) and adsorbed pollutants by adding them to marine fish diet. European sea bass (Dicentrarchus labrax) juveniles were fed for 60 days with feeds containing polypropylene MPs, either virgin or contaminated with chemical pollutants (a blend of dichlorodiphenyldichloroethylene, chlorpyrifos, and benzophenone-3). The data demonstrated a synergic action of MPs and chemical pollutants to induce an inflammatory-like response in distal intestine of sea bass as shown by the up regulation of cytokine il-6 and tnf-α expression. Morphological analysis detected the presence of a focus of lymphocytes in anterior and posterior intestinal segments of fish fed with contaminants in the diet. With regard to microbiota, significant changes in bacterial species richness, beta diversity, and composition of gut microbiota were observed as a consequence of both pollutants and polluted MPs ingestion. These perturbations in gut microbial communities, including the reduction of beneficial lactic acid bacteria and the increase in potential pathogenic microorganism (Proteobacteria and Vibrionales), were undeniable signs of intestinal dysbiosis, which in turn confirmed the signs of inflammation caused by pollutants, especially when combined with MPs. The results obtained in this study provide, therefore, new insights into the potential risks of ingesting MPs as pollutant carriers in marine fish.

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.

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.

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.

Short-Term Supplementation of Dietary Arginine and Citrulline Modulates Gilthead Seabream (Sparus aurata) Immune Status.

Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream (Sparus aurata) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level.

Assessment of dietary supplementation with galactomannan oligosaccharides and phytogenics on gut microbiota of European sea bass (Dicentrarchus Labrax) fed low fishmeal and fish oil based diet.

There is an increasing interest from the aquafeed industry in functional feeds containing selected additives that improve fish growth performance and health status. Functional feed additives include probiotics, prebiotics, organic acids, and phytogenics (substances derived from plants and their extracts). This study evaluated the effects of dietary inclusion of a mucilage extract rich in galactomannan oligosaccharides (GMOS), a mixture of garlic and labiatae-plants oils (PHYTO), and a combination of them (GMOSPHYTO), on gut microbiota composition of European sea bass (Dicentrarchus labrax) fed with a low fishmeal (FM) and fish oil (FO) diet. Three experimental diets and a control diet (plant-based formulation with 10% FM and 6% FO) were tested in a 63-days feeding trial. To analyze the microbiota associated to feeds and the intestinal autochthonous (mucosa-adhered) and allochthonous (transient) microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME2 pipeline were used. Metabarcoding analysis of feed-associated bacteria showed that the microbial communities of control (CTRL) feed deeply differed from those of experimental diets. The number of reads was significantly lower in CTRL feed than in other feeds. The OTU (operational taxonomic unit) number was instead similar between the feeds, ranging from 42 to 50 OTUs. The variation of resident gut microbiota induced by diet was lower than the variation of transient intestinal microbiota, because feedstuffs are a major source of allochthonous bacteria, which can temporarily integrate into the gut transient microbiome. However, the composition of transient bacterial communities was not simply a mirror of feed-borne bacteria. Indeed, the microbial profile of feeds was different from both faecal and mucosa profiles. Our findings suggest that the dietary inclusion of GMOS (0.5%) and PHYTO (0.02%) in a low FM and FO diet induces changes in gut microbiota composition of European sea bass. However, if on allochthonous microbiota the combined inclusion of GMOS and PHYTO showed an antagonistic effect on bactericidal activity against Vibrionales, at mucosa level, only GMOSPHYTO diet increased the relative abundance of Bacteroidales, Lactobacillales, and Clostridiales resident bacterial orders. The main beneficial effects of GMOS and PHYTO on gut microbiota are the reduction of coliforms and Vibrionales bacteria, which include several potentially pathogenic species for fish, and the enrichment of gut microbiota composition with butyrate producer taxa. Therefore, these functional ingredients have a great potential to be used as health-promoting agents in the farming of European sea bass and other marine fish.

Estudio de una cohorte de paciente con fibrosis quística y Scedosporium spp / Study of a Cohort of Patients With Cystic Fibrosis and Isolation of Scedosporium spp

Introducción: En los últimos años se observa un aumento de la prevalencia de colonización e infección por Scedosporium spp. en pacientes con fibrosis quística (FQ). En el presente estudio se registra la frecuencia de aislamiento de Scedosporium spp. en una Unidad de FQ de adultos, analizándose las características de los pacientes y los factores predisponentes. Métodos: Se realizó un estudio observacional retrospectivo en 87 pacientes adultos con FQ en los que se valoró la presencia de cultivo positivo para Scedosporium spp. durante 5 años (enero de 2012-julio de 2017). Se recogieron las siguientes variables clínicas: edad, sexo, índice de masa corporal, genotipo, presencia de insuficiencia pancreática, colonizaciones bacterianas, función pulmonar, complicaciones, exacerbaciones y tratamiento, así como puntuación Bhalla modificada de la última tomografía computarizada axial de alta resolución. Los resultados se analizaron con el paquete estadístico IBM SPSS Statistics Version 22.0. Resultados: En un 25,3% de los pacientes se aisló Scedosporium spp. En el análisis bivariante se observó en estos enfermos más frecuencia de Pseudomonas aeruginosa, peor puntuación en la clasificación de Bhalla (destacando los ítems presencia de bronquiectasias, tapones mucosos y generaciones bronquiales), un descenso leve en la capacidad de difusión pulmonar (DLCO) y que recibían con más frecuencia antibioterapia inhalada. En el análisis multivariante de regresión logística únicamente el ítem generaciones bronquiales fue significativo. Conclusiones: Scedosporium spp. debe considerarse un patógeno oportunista emergente en pacientes con FQ del que se desconoce su implicación clínica, factores de riesgo o necesidad de tratamiento

Introduction: In recent years an increase in the prevalence of colonization and infection by Scedosporium spp. in patients with cystic fibrosis (CF) has been observed. In this article, we study the frequency of isolation of Scedosporium spp. in an adult CF Unit, analyzing characteristics of the patients and predisposing factors. Methods: A retrospective observational study was conducted in 87 adult CF patients in whom the presence of positive culture for Scedosporium spp. was tested for a 5-year period (January 2012-July 2017). We recorded the following clinical variables: age, sex, body mass index, genotype, presence of pancreatic insufficiency, bacterial colonization, lung function, other complications, exacerbations and treatment, and the modified Bhalla score from the last high-resolution computed tomography. Results were analyzed with IBM SPSS Statistics Version 22.0 software. Results: Scedosporium spp. was isolated in 25.3% of patients. In the bivariate analysis, these patients showed a higher rate of Pseudomonas aeruginosa infection, worse score in the Bhalla classification (highlighting the following items: bronchiectasis, mucus plugs and bronchial generations), a slight decrease in the lung diffusion capacity and more frequently received inhaled antibiotics. In the logistic regression multivariate analysis, only the bronchial generations item was significant. Conclusion: Scedosporium spp. must be considered an emerging opportunistic pathogen in patients with CF whose clinical involvement, risk factors or need for treatment is unknown

Dietary phytogenics and galactomannan oligosaccharides in low fish meal and fish oil-based diets for European sea bass (Dicentrarchus labrax) juveniles: Effects on gut health and implications on in vivo gut bacterial translocation.

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

An insight into piscidins: The discovery, modulation and bioactivity of greater amberjack, Seriola dumerili, piscidin.

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.

Study of a Cohort of Patients With Cystic Fibrosis and Isolation of Scedosporium spp. / Estudio de una cohorte de paciente con fibrosis quística y Scedosporium spp.

INTRODUCTION: In recent years an increase in the prevalence of colonization and infection by Scedosporium spp. in patients with cystic fibrosis (CF) has been observed. In this article, we study the frequency of isolation of Scedosporium spp. in an adult CF Unit, analyzing characteristics of the patients and predisposing factors. METHODS: A retrospective observational study was conducted in 87 adult CF patients in whom the presence of positive culture for Scedosporium spp. was tested for a 5-year period (January 2012-July 2017). We recorded the following clinical variables: age, sex, body mass index, genotype, presence of pancreatic insufficiency, bacterial colonization, lung function, other complications, exacerbations and treatment, and the modified Bhalla score from the last high-resolution computed tomography. Results were analyzed with IBM SPSS Statistics Version 22.0 software. RESULTS: Scedosporium spp. was isolated in 25.3% of patients. In the bivariate analysis, these patients showed a higher rate of Pseudomonas aeruginosa infection, worse score in the Bhalla classification (highlighting the following items: bronchiectasis, mucus plugs and bronchial generations), a slight decrease in the lung diffusion capacity and more frequently received inhaled antibiotics. In the logistic regression multivariate analysis, only the bronchial generations item was significant. CONCLUSION: Scedosporium spp. must be considered an emerging opportunistic pathogen in patients with CF whose clinical involvement, risk factors or need for treatment is unknown.

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.

Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758).

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses.

The present study examined the short and mid-term effects of a rise in temperature from 18°C to 24°C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6°C, after 1month of acclimation at 18°C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1h, 24h, 3days and 1week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration. Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1h as a short-term response, and a consecutive increase after one week, as a mid-term response. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis. Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol.

Modulation of the Expression of Components of the Stress Response by Dietary Arachidonic Acid in European Sea Bass (Dicentrarchus labrax) Larvae.

This study reports for the first time on European sea bass, Dicentrarchus labrax (L.), larvae, the effect of different levels of dietary arachidonic acid (ARA; 20:4n-6) on the expression of genes related to the fish stress response. Copies of mRNA from genes related to steroidogenesis [StAR (steroidogenic acute regulatory protein), c-Fos, and CYP11ß (11ß-hydroxylase gene)], glucocorticoid receptor complex [GR (glucocorticoid receptor) and HSP (heat shock proteins) 70 and 90) and antioxidative stress (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase] were quantified. Eighteen day-old larvae were fed for 14 days with three experimental diets with increasing levels of ARA (0.3, 0.6 and 1.2% d.w.) and similar levels of docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acids (5 and 3%, respectively). The quantification of stress-related genes transcripts was conducted by One-Step TaqMan real time RT-PCR with the standard curve method (absolute quantification). Increase dietary levels of ARA induced a significantly (p < 0.05) down-regulation of genes related to cortisol synthesis, such as StAR and CYP11ß and up-regulated genes related to glucocorticoid receptor complex, such as HSP70 and GR. No effects were observed on antioxidant enzymes gene expression. These results revealed the regulatory role of dietary ARA on the expression of stress-related genes in European sea bass larvae.