Implication of mucus-secreting cells, acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ-carrageenin to gilthead seabream (Sparus aurata) specimens.

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1ß, il-1ß) were up-regulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1ß and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Ultrasonography study of the skin wound healing process in gilthead seabream (Sparus aurata).

This work aimed to carry out an in vivo study of the skin healing process in gilthead seabream (Sparus aurata) after being experimentally wounded. Firstly, the structure of normal skin was studied by real-time ultrasonography (Vevo Lab, VisualSonics) and light microscopy. Besides this, experimental wounds were made on the left flank of each fish with a circular biopsy punch (8 mm diameter) below the lateral line. The healing process was assessed on live fish at 0, 6, 11 and 23 days post-wounding using the real-time ultrasonography in B-mode and Power Doppler mode (Vevo 3100 FUJIFILM, VisualSonics). Through the ultrasonography images, both the skin structure and the evolution of the changes that wounds originated in the surrounding tissues were studied in vivo over time. Concomitantly, the pattern of neovascularization in the wounded area was followed during the healing process and it was demonstrated that, although the neovascularization started very early after the skin damage, it was increased in wounded areas from day 11 post-wounding onwards. The results obtained proved the utility and power of using ultrasounds in fish to evaluate in vivo complex biological processes in real time, which are difficult to study by other methodologies. The present data shed some light on the reparation of external injuries in aquatic vertebrates.

The alleviation of skin wound-induced intestinal barrier dysfunction via modulation of TLR signalling using arginine in gilthead seabream (Sparus aurata L).

The present study sought to investigate the effect of arginine on the involvement of toll-like receptors (TLRs) in skin wound-induced intestinal barrier dysfunction in gilthead seabream (Sparus aurata L.). Two replicates of fish (n = 8) were fed a commercial diet (CON, total 2.75% arginine), CON diet enriched with 1% arginine (ARG1, total 3.65% arginine) and 2% arginine (ARG2, total 4.53% arginine) for 30 days. Half of the fish were sampled, whereas the others were injured and sampled 7 days post-wounding. The intestinal histology results showed that a more intense infiltration of mixed leucocytes was evident in the wounded fish, which was remarkably reduced in fish that were fed the ARG1 diet. Serum IgM levels were significantly higher in the ARG1 group than levels in the CON group at 7 days post-wounding. Compared with the fish in the CON group after wounding, dietary administration of 1% arginine markedly downregulated the gene expression of TLRs (TLR2 and TLR5), MyD88, and proinflammatory cytokines (CSF1R, IL-1ß, and TNFα), but significantly enhanced the gene expression of IκBα, the anti-inflammatory cytokine TGF-ß1, and tight junction proteins (tricellulin and occludin) in wounded fish. Furthermore, the ARG2 diet demonstrated no additional benefits on intestinal cells, compared to both the ARG1 and the CON diets, and it even appeared to induce negative effects. In summary, dietary administration of 1% arginine significantly inhibited intestinal inflammatory response and tight junction disruption in skin-wounded gilthead seabream by modulating TLR signalling in the intestine.

Radiological characterization of gilthead seabream (Sparus aurata) by X-ray computed tomography.

In recent years, the increasing use of fish as new animal models in scientific research and the growth of fish farming (mainly for human consumption) have highlighted the need for advanced technology to deepen our knowledge of fish biology. Hence, the present study was carried out to radiologically analyse the whole body of gilthead seabream (Sparus aurata) specimens using X-ray computed tomography (CT). Images were acquired in an Albira SPECT/PET/CT tri-modal preclinical-scanner. Segmentation, measurements and three-dimensional reconstruction were made using the Carestream Molecular imaging Albira CT system in conjunction with Pmod, AMIDE and Amira software packages. The results showed that the density values of gilthead seabream are in the range -700 to +2500 HU for the whole body. We also determined the density ranges that topographically coincide with the swim bladder, soft tissues, fat, skin and skeleton. This work describes, validates and demonstrates the application of a fully automated image analysis technique to study and quantify fish body composition, whether segmented or as a whole. In addition, the basis for applying this image technique in other in vivo studies is established.

Dietary administration of the probiotic Shewanella putrefaciens to experimentally wounded gilthead seabream (Sparus aurata L.) facilitates the skin wound healing.

The effect of the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) was studied on the skin healing of experimentally wounded gilthead seabream (Sparus aurata L.). Two replicates (n = 12) of fish were fed CON diet or SP diet for 30 days. Half of the fish were sampled while the others were injured and sampled 7 days post-wounding. Results by image analysis of wound areas showed that SpPdp11 inclusion facilitated wound closure. Compared with the CON group, fish in SP group sampled 7 days post-wounding had a significantly decreased serum AST and increased ALB/GLOB ratio. Furthermore, protease and peroxidase activities were significantly increased in skin mucus from fish in SP group sampled 7 days post-wounding, compared with those fed CON diet. Additionally, SP diet up-regulated the gene expression of antioxidant enzymes, anti-inflammatory cytokines, and re-epithelialization related genes in the fish skin. Furthermore, significant decreases in pro-inflammatory cytokines expression were detected in fish from SP group, respect to control ones. Overall, SpPdp11 inclusion facilitated the wound healing and the re-epithelialization of the damaged skin, alleviated the inflammatory response in the wound area through intensifying the antioxidant system, and enhancing the neo-vascularization and the synthesis of matrix proteins in the skin wound sites of fish.

Radiological characterization of gilthead seabream (Sparus aurata) fat by X-ray micro-computed tomography.

In fish, the fat content contributes to promoting the nutritional and organoleptic characteristics of the flesh, which is crucial for consumer acceptance. Methods to predict the fat in fish are important in nutritional and physiological research, where body content is traditionally determined by dissection followed by chemical analysis. However, X-ray micro-computed tomography (micro-CT) provides three-dimensional information in a non-destructive way. This work aims to characterize radiologically the fat, in situ, in a widely cultivated marine species, gilthead seabream (Sparus aurata). To validate the method changes in fat content in a control group (fed) and another group (unfed for 60 days) were assessed. Fish images were acquired on an Albira SPECT/PET/CT preclinical-scanner. Image analysis and measurements were performed using the Carestream Molecular Imaging Albira CT system in conjunction with Pmod and Amide packages. By micro-CT analysis the density values were determined for the whole fish body (- 1,000 to + 2,500 HU, Hounsfield units), and density ranges for the fat in S. aurata were established from - 115 to + 50 HU. As expected, significant differences were found between fed and starved groups at 60 days. The present study confirms the usefulness of high-resolution morphological analysis for evaluating the presence and distribution of fat in this important fish species.

Skin wound healing in gilthead seabream (Sparus aurata L.) fed diets supplemented with arginine.

Dietary administration of arginine on the wound healing process of gilthead seabream was studied. Two replicates of fish (n = 8) were fed with either a commercial diet [control diet (CON), no arginine added] and the CON diet supplemented with 1% arginine (ARG1) or with 2% arginine (ARG2) for 30 days. Afterward, half of the fish were sampled while the other half were injured and continued to be fed the same diet for an extra week. Results by image analysis showed that the wound closure rate was significantly improved in fish that were fed the ARG1 diet, compared with those in the CON group. After seven days of wound healing, the aminotransferase and creatine kinase levels in the serum and the protease and peroxidase activities in the skin mucus were down-regulated, while the immunoglobulin M level in the skin mucus was up-regulated in the ARG1 group after wounding and in the CON group before wounding. Compared with the CON diet, the ARG1 diet remarkedly depressed the gene expression of mpo, il-8, and tnf-α, and enhanced the gene expression of tgf-ß1, igf-1, pcna, krt2, mmp9, fn1α, and colIα and the antioxidant enzyme cat in the skin tissues after wounding. Furthermore, compared with both the ARG1 and the CON groups, negative effects of the ARG2 diet on wound healing were demonstrated. In conclusion, a 1% arginine supplementation facilitates skin wound healing and prevents a systemic inflammation reaction by alleviating the inflammatory response and enhancing the re-epithelialization and ECM biosynthesis in skin wound sites.

Bactericidal effect on skin mucosa of dietary guava (Psidium guajava L.) leaves in hybrid tilapia (Oreochromis niloticus × O. mossambicus).

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the intensification practices in global aquaculture, fish are often confined in small volumes, which can results in outbreak diseases. In this context, the use of antibiotics is very usual. Thus, looking for natural substance able to reduce the use of the antibiotics is imperative. Among them, there is a great interest at present in the study of medicinal plants such as guava (Psidium guajava L.). These plants could help to develop a more sustainable aquaculture all over the world. The application of guava in traditional medicine dates for centuries and it is widely used in tropical countries for the treatment of diseases in human and animals. AIM OF THE STUDY: The purpose of this work was to study the effects of the dietary administration of dried leaves of Psidium guajava on the skin mucosal immunity of hybrid tilapia (Oreochromis niloticus × O. mossambicus). Furthermore, the ability of this plant to inhibit the bacterial load in different tissues after an experimental infection with Vibrio harveyi was studied. MATERIALS AND METHODS: P. guajava leaves collection and the experimentation was carried out in Dominican Republic. Fish were fed with a commercial diet supplemented with guava leaf at different concentrations (0%, 1.5% and 3%) for 21 days before being intraperitoneally injected with V. harveyi (1 × 104 cells mL-1). Thereafter, several immune activities were measured in fish skin mucus and after 48 h of injection, the skin, spleen and liver were collected to analyse the bactericidal activity of guava leaf and the gene expression of some immune related genes. RESULTS: The administration of P. guajava leaves significantly modulated some immune-related enzymes (protease, antiprotease and peroxidase) in the skin mucus of hybrid tilapia. In addition, the bacterial load after V. harveyi infection in skin, spleen and liver significantly reduced in fish supplemented with guava leaves. Finally, the expression profile of hepcidin gene in skin and liver was modulated in fish feed with control diet after V. harveyi infection. CONCLUSION: These results demonstrate that the dietary intake of guava leaves increases the skin mucosal barrier defences of hybrid tilapia and confers protection against V. harveyi colonization.

Influence of skin wounds on the intestinal inflammatory response and barrier function: Protective role of dietary Shewanella putrefaciens SpPdp11 administration to gilthead seabream (Sparus aurata L.).

The effects of skin wounds on the intestinal barrier function and the beneficial effects of the dietary administration of Shewanella putrefaciens (known as SpPdp11) in gilthead seabream (Sparus aurata L.) were studied. Two replicates of fish were fed a commercial diet (control, CON) or CON diet enriched with 109 cfu g-1 SpPdp11 (SP diet) for 30 days. After this time, half of the fish were sampled, while the others were injured below the lateral line (wounded fish, W) and fed the same diets for an extra week before sampling (CON + W and SP + W groups). The intestinal histology and gene expression of different genes relevant for the intestinal barrier function were studied. The results showed that injured fish had a disordered enterocyte nucleus disposition, a more intense infiltration of mixed leucocytes and a thicker lamina propria in the intestine compared to the control fish. However, the fish in the SP + W group did not present these pathological symptoms in the intestine. No significant variations in the number of goblet cells were detected among the different experimental groups. Pro-inflammatory cytokines (colony-stimulating factor receptor 1, CSF1R, myeloperoxidase, MPO and interleukin-1ß, IL-1ß), mucins (intestinal mucin, IMUC and mucin 2, MUC2), and immunoglobulin T heavy chain (IGHT) were up-regulated, while tight junction protein occludin was down-regulated in the intestine from fish of the CON + W group. Similarly, the dietary administration of SpPdp11 markedly depressed the gene expression of pro-inflammatory cytokines, MUC2 and IGHT, but increased the gene expression of anti-inflammatory cytokine transforming growth factor-ß1 (TGF-ß1) and the tight junction proteins tricellulin and occluding after wounding. In brief, the skin wounds provoked an intestinal inflammatory response that included changes in the mucus layer and tight junction disruptions. Besides this, preventive administration of SpPdp11 alleviated the intestinal dysfunctions caused by skin wounds in gilthead seabream.

Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-ß), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Effects of dietary inclusion of Moringa oleifera leaves on growth and some systemic and mucosal immune parameters of seabream.

The effect of the dietary incorporation of drumstick, Moringa oleifera, leaf meal (MOL; 0, 5, 10 and 15%) on the growth, feed utilization, some skin mucus and systemic immune parameters and intestinal immune-related gene expression in gilthead seabream (Sparus aurata) specimens. The experiment lasted 4 weeks. The results revealed that MOL can be incorporated in S. aurata diet up to 10% with no significant negative effect on growth and feed utilization. However, there was a significant decrease with MOL at a level of 15% after 2 weeks of feeding. The systemic immune status of fish fed with the different levels of MOL showed an improvement in head kidney leucocyte phagocytosis, respiratory burst and peroxidase activities. Also, serum humoral components, including protease, ACH50 and lysozyme activities and IgM level, increased with MOL inclusion especially at the 5% level. MOL at 5% improved skin-mucosal immunity such as protease, antiprotease, peroxidase and lysozyme activities. Moreover, the feeding of MOL revealed an upregulation of the intestinal mucosal immunity genes (lyso and c3), tight junction proteins (occludin and zo-1) and anti-inflammatory cytokines (tgf-ß) with a downregulation of pro-inflammatory cytokine (tnf-α). Therefore, it is recommended to incorporate MOL in S. aurata diets at a level of 5% for the best immune status or 10% for the high growth performance and acceptable immune surveillance. Graphical abstract ᅟ.

Mucus glycosylation, immunity and bacterial microbiota associated to the skin of experimentally ulcered gilthead seabream (Sparus aurata).

Interest in fish skin immunity and its associated microbiota has greatly increased among immunologists. The objective of this study is to know if skin ulcers may be associated with changes in the mucus composition and microbial diversity. The abundance of terminal carbohydrates, several enzymes (protease, antiprotease, peroxidase, lysozyme) and total immunoglobulin M levels were evaluated in skin mucus of experimentally ulcered gilthead seabream (Sparus aurata L.). Furthermore, the composition of the microbiota of ulcered and non-ulcered skin has been determined using Illumina Miseq technology. Significant decreases of terminal abundance of α-D-mannose, α-D-glucose and N-acetyl-galactosamine in skin mucus of ulcered fish, compared to control fish were detected. The levels of IgM and all the tested enzymes in mucus were decreased in ulcered fish (compared to control fish) although the observed decreases were only statistically significant for proteases and antiproteases. Concomitantly, the analysis of the composition of the skin microbiota showed clear differences between ulcered and non-ulcered areas. The genus taxonomic analysis showed that Staphylococcus and Lactobacillus were more abundant in non-ulcered skin whereas in ulcered area were Streptococcus and Granulicatella. Important decreases of the number of sequences related to Alteromonas, Thalassabius and Winogradskyella were detected in ulcered skin whilst slight increases of sequences related to Flavobacterium, Chryseobacterium and Tenacibaculum genera were observed. Overall these results demonstrated that the presence of skin ulcers provide microenvironments that perturb both the mucus composition and microbial biodiversity of this important external surface which seem to be more vulnerable to diseases.

Humoral immune parameters in serum of gilthead seabream (Sparus aurata L.) after induced skin injury.

Skin lesions contribute to significant rates of morbidity and mortality in fish because they permit the entry of pathogens. Different seric immune parameters (IgM level, protease, antiprotease, peroxidase and lysozyme) and bactericidal activity (against Vibrio harveyi and Photobacterium damsealae) of gilthead seabream (Sparus aurata L.) specimens were evaluated after experimentally wounding fish in two body locations (above or below the lateral line). The results demonstrate that the level of several immune parameters present in fish serum (IgM, proteases, peroxidase and bactericidal activity) showed statistically significant variations depending on the site of the skin wound and the time post-wounding (from 0 to 7 days). However, other parameters (such as antiproteases or lysozyme) remained unaltered during the experiment and did not differs from the values recorded on control fish (non-wounded). The highest activities recorded coincided with the inflammatory healing phase. Moreover, many more significant variations were observed in fish wounded below the lateral line than in those wounded above the lateral line. The present results demonstrate the importance of skin integrity in the maintenance of fish body homeostasis.

Healing and mucosal immunity in the skin of experimentally wounded gilthead seabream (Sparus aurata L).

Skin lesions are very common in fisheries, increasing the risk of pathogens entering through the wounded skin of the fish. In the present assay, the progression of wound healing was studied over a 7 day period in gilthead seabream (Sparus aurata L.) after making experimental wounds in two different locations: above (group A) or below (group B) the lateral line. Macroscopic observation confirmed faster wound healing of the wounds of fish from group B. Furthermore, several immune-related components were studied in the skin mucus of wounded fish to ascertain whether wounding altered the mucus composition compared with the values obtained from non-wounded fish (group C, control). Significant variations were detected depending on both the site of the wound and the studied parameter. At the same time, the gene expression profile of several immune-relevant genes, including pro-inflammatory (il1b,il6, tnfa), anti-inflamamtory (tgfb, il10), immunoglobulins (ighm, ight), involved in oxidative stress (sod, cat) and in skin regeneration (krt1and grhl1) were studied in the three groups of fish (A, B and C). The results throw further light on the complex process of skin wound healing in fish, since substantial changes in the skin mucus and in the skin gene expression originated by the presence of wounds were observed. This work underline some important differences depending on the place of the fish body where the wound is located. Of particular note was the fact that such changes depended on the site of the wound.

Dorso-ventral skin characterization of the farmed fish gilthead seabream (Sparus aurata).

The skin is the first barrier of defence in fish, protecting against any external stressor and preserving the integrity and homeostasis of the fish body. The aim of this study was to characterise gilthead seabream skin by isolating cells and studying the cell cycle by flow cytometry, to study the skin histology by scanning electron microscopy and the transcription level of some immune-relevant genes by RT-PCR. Furthermore, the results obtained from samples taken from the dorsal and the ventral part of the specimens are compared. No differences were observed in the cell cycle of cells isolated from the dorsal and ventral zones of the skin or in the gene expression of the genes studied in both epidermal zones. However, the epidermis thickness of the ventral skin was higher than that of the dorsal skin, as demonstrated by image analysis using light microscopy. Besides, scanning electron microscopy pointed to a greater cell size and area of microridges in the apical part of the dorsal epidermal cells compared with ventral skin epidermal cells. This study represents a step forward in our knowledge of the skin structure of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide. Furthermore, for functional characterization, experimental wounds were carried out comparing the wound healing rate between the dorsal and ventral regions of skin over the time. The results showed higher ratio of wound healing in the ventral region, whose wounds were closed after 15 days, compared to dorsal region of skin. Taking into account all together, this study represents a step forward in our knowledge of the skin structure and skin regeneration of an important farmed teleost, gilthead seabream, one of the most commonly farmed fish worldwide.

Dietary yeast Sterigmatomyces halophilus enhances mucosal immunity of gilthead seabream (Sparus aurata L.).

A yeast was isolated from hypersaline sediments, grown and phylogenetically characterized as Sterigmatomyces halophilus strainN16. The dietary administration of this yeast was studied for its effect on skin mucosal immune and antioxidant status of gilthead seabream (Sparus aurata L.). Fish were fed a commercial diet (control, non-supplemented diet), or the same commercial diet supplemented with 0.55% or 1.1% of yeast for 15 and 30 days. One month after the end of the trial, fish from all treatments were intraperitoneally injected with pathogenic Vibrio parahaemolyticus and further fed with the same diets for one week, after which fish were also sampled. Significant increases were observed in the immune activities determined in the fish fed the yeast supplemented diets compared with the values recorded in mucus of fish from the control group. The expression levels of trypsin (one of the main digestive enzymes) and several immune-related genes (IL-1ß, TNF-α, IgM, C3 and lysozyme) were also evaluated by real-time PCR in intestine and skin. Interestingly, trypsin gene expression in intestine was up regulated in both experimental diets compared with the control group, particularly in fish fed with 0.55% of S. halophilus at any time of the experimental trial. Immune-related genes in intestine and skin were strongly expressed principally in fish fed with 0.55% of S. halophilus for 15 days and 1.1% for 30 days and after infection, respectively. The present results suggest that the yeast S. halophilus can be considered as a novel fish immunostimulant. The excellent potential of marine microorganisms isolated from extreme environments with beneficial properties for fish is discussed.

Enhancing gilthead seabream immune status and protection against bacterial challenge by means of antigens derived from Vibrio parahaemolyticus.

In an attempt to control the proliferation of the pathogenic bacterium Vibrio parahaemolyticus in gilthead seabream (Sparus aurata), the immunostimulant effect of lysate and ToxA from this bacterium was evaluated. Fish were intraperitoneally injected twice (first injection, day 1 of the experiment; second injection, day 7) and sampled after one week (on days 8 and 15). Afterwards, all fish specimens were experimentally infected with V. parahaemolyticus and mortality was recovered for 1 week. Fish injected with lysate, ToxA and phosphate buffer saline (control) showed 100%, 50% and 0% survival, respectively, when challenged with the pathogen. Skin mucus immune parameters and immune-related gene expression in skin and spleen were also evaluated. The results showed that mucus immune parameters were enhanced in the lysate and ToxA groups compared with the values obtained for fish from the control group. Expression of IL-1ß, TNF-α, C3 and IgM genes was significantly up-regulated in the lysate and ToxA groups, principally after infection with the bacterium. Interestingly, TLR5 gene expression increased in fish immunized with lysate. The most prominent histological characteristic in gut from infected fish was the presence of a great number of intraepithelial leucocytes as well as inflammation of the submucosa, while severe hydropic degeneration and hemosiderosis were detected in liver from infected fish. Injection of lysate or ToxA had a protective effect against the deleterious consequences of subsequent infection with V. parahaemolyticus in gut and liver. The findings underline the potential of lysate and ToxA as potent preventive antigens against this kind of vibriosis.