Mucosal and systemic immune responses in Senegalese sole (Solea senegalensis Kaup) bath challenged with Tenacibaculum maritimum: A time-course study.

Tenacibaculosis, caused by Tenacibaculum maritimum, continues to inflict substantial losses among cultured marine species, particularly in the Senegalese sole. However, the immune mechanisms in fish involved in fighting against this disease are still poorly understood. Thus, the present study aimed to investigate the skin mucus's terminal carbohydrate composition, several immune-related enzymes (i.e. lysozyme, peroxidase, proteases and antiproteases), the haemolytic activity of complement and the bactericidal activity in the skin mucus and plasma of the Senegalese sole in a time-course study following a bath challenge with T. maritimum. The haematological profile and the kinetics of cell migration post-infection were also considered. The bath challenge induced slight variations in the terminal carbohydrate composition of Senegalese sole skin mucus. In general, results from this study showed a delay in the mucosal immune response compared to that found at the systemic level (i.e. blood and plasma). For instance, a significant increase in the skin mucus's lysozyme, complement, protease and antiprotease activities were observed at the end of the experiment (14 d post-challenge). Interestingly, the higher activity of these enzymes could be related to the skin mucus's bactericidal capacity and haemolytic complement activity, suggesting that these enzymes play an important role in the defence against Gram-negative bacteria. The haematological profile revealed a significant increase in circulating neutrophils in challenged fish after 48 and 72 h, which was positively correlated to the increments observed in peroxidase and lysozyme activities, respectively, in the plasma of challenged fish at the same time. Although the route of entry and the survival strategy of T. maritimum are still not fully elucidated, results from the present study will contribute to this endeavour through the study of the mucosal immune responses of fish against this particular pathogen.

Effects of dietary administration of fenugreek seeds on metabolic parameters and immune status of gilthead seabream (Sparus aurata L.).

Medical plants could be used as a prophylactic method in aquaculture because they are considered safe and so very promising alternatives to the use of chemicals. The aim of the present work was to examine the effects of dietary fenugreek (Trigonella foenum graecum) seeds administered for 8 weeks on the metabolic and immune status of gilthead seabream (Sparus aurata L.). Four experimental groups were designated: one receiving a basal diet (control) and three fed powdered fenugreek seeds incorporated in the fish feed at 1%, 5% and 10%. The results show that significant decreases in aspartate aminotransferase, creatine kinase, potassium and the albumin/globulin ratio were detected in the serum of fish fed 10% fenugreek compared with the values recorded in control fish. As regards the immune status, fish fed the 5% supplemented diet had higher haemolytic complement and peroxidase activities than the control fish whilst antiprotease activity was higher in fish fed the 1% fenugreek level respect to control fish and the fish fed the highest fenugreek supplementation rate. Interestingly, the results also revealed a significant enhancement of most of the cellular immune parameters studied, especially in fish fed the highest level of fenugreek (10%). However, the bacteriostatic activity of serum against fish pathogenic and non-pathogenic bacteria was non-affected to any significant extent in fish fed the supplemented diets. Overall, the results suggest that the high level of dietary fenugreek tested in this work (10%) did not negatively affect any of the metabolic parameters measured in serum but increased some of them. In addition, the inclusion of fenugreek seeds in the gilthead seabream diet at 5% or 10% improved the humoral and cellular immune activities, respectively. Further studies are needed to better understand the effects of this natural product, which may be suitable for use as a feed additive in fish aquaculture.

A time course study of glucose levels and innate immune response in gilthead seabream (Sparus aurata L.) after exposure to clove oil-eugenol derived anaesthetic.

Clove oil is used as an anaesthetic for many species of fish worldwide; however, relatively few studies have assessed its effects on the innate immune response on these species. The present work aimed to investigate the effects of clove oil-eugenol derived anaesthetic on some humoral and cellular immune response in gilthead seabream (Sparus aurata L.). To compare with an unexposed control group, fish were exposed to 55 ppm clove oil for 5 min, before being sampled at 1, 24 and 48 h post-exposure. Serum glucose level was also measured to obtain information on the fish physiological response after clove oil anaesthesia. One hour after exposure the haemolytic complement activity of fish was lower than in the unexposed group. By contrast, the leucocyte peroxidase activity in head-kidney was significantly stimulated 24 h after exposure to clove oil-eugenol. The rest of innate immune parameters evaluated and the glucose levels not were affected by clove oil exposure at any sampling point. Overall, the use of clove oil at 55 ppm as anaesthetic did not seem to alter the innate immune response and neither did it trigger a stress response. The use of clove oil-eugenol derived had become common practice in aquaculture, and its use with gilthead seabream can be considered safe as it does not cause immunodepression in anesthetized fish.

Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata L.).

Improving fish defense through the preventive administration of immunostimulants has an important role in controlling the outbreak of the disease in aquaculture. As a continuity of our previous studies, this paper describes the effects of dietary fenugreek (Trigonella foenum graecum) seeds on the skin mucosal antioxidant status and immune response of gilthead seabream (Sparus aurata L.). Fish were fed with four experimental diets: one a basal diet (control) and three diets with powdered fenugreek seeds incorporated in the fish feed at 1%, 5%, and 10%. After eight weeks of feeding, free radicals scavenging and antioxidant assays were assessed in skin mucus by measuring the peroxidation of phospholipid liposomes, hydroxyl radical and hydrogen peroxide scavenging, measurement of total antioxidant activity and the determination of antioxidant activity in a linoleic acid system. The skin mucosal immune response was evaluated by measuring the IgM levels and some enzymatic activities (peroxidase, antiprotease, protease, esterase, and ceruloplasmin). Our results demonstrated that fenugreek inclusion improved the hydroxyl radical scavenging capacity and conferred very high antioxidant activity. Besides, only the highest supplementation level (10%) was able to augment the peroxidase and protease activities confronted by a general decrement in the antiprotease activity in the experimental fed groups with 1% and 10%. These results suggest that the dietary administration of fenugreek at the higher inclusion dose enhances the skin mucosal immunity response and the antioxidant status of gilthead seabream a species with one of the highest rates of production in marine aquaculture.

Evaluation of silver nanospheres on viability and innate cellular parameters of gilthead seabream (Sparus aurata L.) head-kidney leucocytes.

The increasing use of nanomaterials, e.g. nanosilver, has lead to concerns about environmental contamination and possible toxic effects on aquatic organisms. Here, we present evidence for the impact of silver nanospheres (AgNSs) on fish innate immune cells after in vitro exposure. AgNSs of 20, 50 or 100 nm in diameter were tested with the smallest ones (20 nm) clearly having the most deleterious effects, after an exposure period of 30 min, followed by the medium-sized ones; the NSs of 100 nm had no impact. The effective concentration was determined at 10 µg ml-1 while lower concentrations (1, 2.5 or 5 µg ml-1) were ineffective. Head-kidney mixed leucocyte population showed significant viability reduction which was attributable to diminished viability of macrophages/monocytes and lymphocytes only whereas granulocytes' viability was not affected at the above exposure regime. Furthermore, cellular respiratory burst activity, phagocytic capacity and phagocytic ability were all reduced, with the first two parameters exhibiting the sharper reductions. Finally, transmission electron microscopy revealed that the AgNSs' internalization was brought about via phagocytosis, pinocytosis, receptor-mediated endocytosis and macropinocytosis; also, that cell death could be effected in either an apoptotic or a necrotic manner. It is concluded that AgNSs are potentially very noxious for the teleost fish immune system as they can adversely affect the function and viability of the head-kidney leucocytes.

Effects of dietary supplementation with fenugreek seeds, alone or in combination with probiotics, on gilthead seabream (Sparus aurata L.) skin mucosal immunity.

Despite increasing interest in modulating the immune response of fish, providing a combination of probiotics and herbal immunostimulants in aquafeed has rarely has been studied. The effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds alone (FE), or combined with one of the following probiotic strains: Bacillus licheniformis (FEBL), Lactobacillus plantarum (FELP) or Bacillus subtilis (FEBS) were evaluated. Fish were fed a control or one of the supplemented diets for 3 weeks. After 2 and 3 weeks of the feeding trial, the abundance of terminal carbohydrates, IgM levels, enzymatic activities (proteases, alkaline phosphatase, esterase and ceruloplasmin) and bactericidal activity were determined in skin mucus. Our results demonstrated that the dietary administration of FE in combination with L. plantarum, particularly, increased carbohydrate abundance, the activity of certain enzymes such as ceruloplasmin, and bactericidal activity against the pathogenic bacterium Photobacterium damselae and the non-pathogenic bacterium B. subtilis in skin mucus at the end of the trial. The carbohydrates most affected by the FELP diet were mannose/glucose, N-acetyl-d-galactosamine and N-acetyl-ß-d-glucosamine. Interestingly, IgM levels were significantly higher in fish fed the FELP and FEBS diets whilst protease activity generally increased in all supplemented diets, which could suggests that the main effect in this activity was to the result of FE supplementation although that fact cannot be confirmed because the effects of probiotics addition alone were not studied. These results suggest that the combined dietary administration of fenugreek and L. plantarum will best enhance the skin mucosal immunity response of gilthead seabream.

Quality and antioxidant response of gilthead seabream (Sparus aurata L.) to dietary supplements of fenugreek (Trigonella foenum graecum) alone or combined with probiotic strains.

The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.).

The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune-relevant genes (igm, tcr-ß, csfr1 and bd) were measured in the head-kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune-associated genes in the head-kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture.

Local immune response of two mucosal surfaces of the European seabass, Dicentrarchus labrax, fed tryptophan- or methionine-supplemented diets.

Immune responses relies on an adequate provision of multiple nutrients that sustain the synthesis of key effector molecules. These needs are depicted in the already reported increase of circulating free amino acids in fish under stressful conditions. Since aquaculture and the inherent fish welfare are an emergent call, the immunomodulatory effects of amino acids on gut- and skin-associated lymphoid tissues of the European seabass (Dicentrarchus labrax) were studied under unstressed conditions and after an inflammatory insult. To achieve this goal, fish were distributed in duplicate tanks (fifteen fish per tank) and were fed for 14 days with methionine or tryptophan-supplemented diets at 2× dietary requirement level (MET and TRP, respectively) or a control diet meeting the amino acids requirement levels (CTRL). Afterwards, samples of skin and posterior gut were collected from 6 fish per dietary treatment for the assessment of the immune status while the remaining animals were intraperitoneally-injected with inactivated Photobacterium damselae subsp. piscicida and subsequently sampled either 4 or 24 h post-injection. The immune status of both mucosal surfaces was poorly affected, although a tryptophan effect was denoted after bacterial inoculation, with several immune-related genes up-regulated in the gut at 4 h post-injection, which seems to suggest a neuroendocrine-immune systems interaction. In contrast, skin mucosal immunity was inhibited by tryptophan dietary supplementation. Regarding methionine, results were often statistically non-significant, though increasing trends were denoted in a few parameters. Overall, dietary methionine did not significantly affect neither gut nor skin immunity, whereas tryptophan supplementation seems to induce modulatory mechanisms that might be tissue-specific.

Vitamin D3 affects innate immune status of European sea bass (Dicentrarchus labrax L.).

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500 U kg-1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2 weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets after 4 weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total IgM level. At gene level, fbl and rbl transcripts were up-regulated in HK in fish fed with the highest concentration of vD3-supplemented diets after 4 weeks, while in the gut, an up-regulation of hep gene was observed in fish fed with the different doses of vD3. These results suggest that vD3 may be of great interest for immunostimulatory purposes in fish farms.

Impact of date palm fruits extracts and probiotic enriched diet on antioxidant status, innate immune response and immune-related gene expression of European seabass (Dicentrarchus labrax).

The application of additives in the diet as plants or extracts of plants as natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics and immunoprophylactics. The aim of the current study was to evaluate the potential effects of dietary supplementation of date palm fruit extracts alone or in combination with Pdp11 probiotic on serum antioxidant status, on the humoral and cellular innate immune status, as well as, on the expression levels of some immune-related genes in head-kidney and gut of European sea bass (Dicentrarchus labrax) after 2 and 4 weeks of administration. This study showed for the first time in European sea bass an immunostimulation in several of the parameters evaluated in fish fed with date palm fruits extracts enriched diet or fed with this substance in combination with Pdp 11 probiotic, mainly after 4 weeks of treatment. In the same way, dietary supplementation of mixture diet has positive effects on the expression levels of immune-related genes, chiefly in head-kidney of Dicentrarchus labrax. Therefore, the combination of both could be considered of great interest as potential additives for farmed fish.

Characterization of the gilthead seabream (Sparus aurata L.) immune response under a natural lymphocystis disease virus outbreak.

Lymphocystis or lymphocystis disease virus (LCDV) is distributed worldwide and affects many fresh and marine water fish species. LCDV is commonly found in aquaria fish species but also in farmed fish species, among them the gilthead seabream (Sparus aurata L.). The immune status of gilthead seabream (S. aurata) specimens under a natural outbreak of LCDV was studied. The replication of the virus was demonstrated in infected fish, but not in control fish. The results showed decreased total serum IgM levels and increased innate cellular immune response (peroxidase and respiratory burst activities) of head kidney leucocytes in LCDV-infected fish, compared to the values obtained in uninfected specimens. In addition, transcription of antiviral genes (ifn and irf3) was down-regulated in the skin of LCDV-positive fish as well as genes involved in cellular immunity (csf1r, mhc2a, tcra and ighm) that were down-regulated in skin and head kidney of infected fish. By contrast, the transcription of nccrp1 was up-regulated in head kidney after LCDV infection. These present results show that head kidney leucocytes are activated to encounter the virus at the sites of replication.

Interactions between candidate probiotics and the immune and antioxidative responses of European sea bass (Dicentrarchus labrax) larvae.

The use of lactic acid bacteria (LAB) as probiotics in aquaculture may improve the quality of seed production and limit the use of antibiotics in fish hatcheries. This study attempted to further characterize the candidate probiotic Lactobacillus casei X2, and the immune and physiological responses of the sea bass larvae. L. casei X2 was confirmed as a good candidate, due to its wide antibacterial spectrum against both Gram-positive and Gram-negative bacteria, and its free radical scavenging activity. In addition, if the strain did not seem able to form biofilm on abiotic surfaces, it adhered strongly to Hep-2 cells. However, these characteristics did not seem efficient in vivo. At 20 days post-hatch (dph), the expression level of CAT gene was significantly different between group fed without probiotic and the two groups treated with either Pediococcus acidilactici or L. casei. This gene was upregulated in the group treated with strain X2 and downregulated in the group with a commercial probiotic strain P. acidilactici, suggesting a better antioxidant activity with the later strain. At the same sampling date, the IL-1ß gene was upregulated in the group treated with P. acidilactici, and the HSP70 gene was overexpressed at 41 dph. As the stimulation of these two last genes, such transcriptomic indicators must be cautiously interpreted.

Mercury Accumulation, Structural Damages, and Antioxidant and Immune Status Changes in the Gilthead Seabream (Sparus aurata L.) Exposed to Methylmercury.

In aquatic systems, mercury (Hg) is an environmental contaminant that causes acute and chronic damage to multiple organs. In fish, practically all of the organic Hg found is in the form of methylmercury (MeHg), which has been associated with animal and human health problems. This study evaluates the impact of waterborne-exposure to sublethal concentrations of MeHg (10 µg L(-1)) in gilthead seabream (Sparus aurata). Hg was seen to accumulate in liver and muscle, and histopathological damage to skin and liver was detected. Fish exposed to MeHg showed a decreased biological antioxidant potential and increased levels of the reactive oxygen molecules compared with the values found in control fish (nonexposed). Increased liver antioxidant enzyme activities (superoxide dismutase and catalase) were detected in 2 day-exposed fish with respect to the values of control fish. However, fish exposed to MeHg for 10 days showed liver antioxidant enzyme levels similar to those of the control fish but had increased hepato-somatic index and histopathological alterations in liver and skin. Serum complement levels were higher in fish exposed to MeHg for 30 days than in control fish. Moreover, head-kidney leukocyte activities increased, although only phagocytosis and peroxidase activities showed a significant increase after 10 and 30 days, respectively. The data show that 30 days of exposure to waterborne MeHg provokes more significant changes in fish than a short-term exposure of 2 or 10 days.

Dietary administration of the probiotic SpPdp11: Effects on the intestinal microbiota and immune-related gene expression of farmed Solea senegalensis treated with oxytetracycline.

Few antimicrobials are currently authorised in the aquaculture industry to treat infectious diseases. Among them, oxytetracycline (OTC) is one of the first-choice drugs for nearly all bacterial diseases. The objective of this study was to evaluate the effect of the dietary administration of OTC both alone and jointly with the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) on the intestinal microbiota and hepatic expression of genes related to immunity in Senegalese sole (Solea senegalensis) juveniles. The results demonstrated that the richness and diversity of the intestinal microbiota of fish treated with OTC decreased compared with those of the control group but that these effects were lessened by the simultaneous administration of SpPdp11. In addition, specimens that received OTC and SpPdp11 jointly showed a decreased intensity of the Denaturing Gradient Gel Electrophoresis (DGGE) bands related to Vibrio genus and the presence of DGGE bands related to Lactobacillus and Shewanella genera. The relationship among the intestinal microbiota of fish fed with control and OTC diets and the expression of the NADPH oxidase and CASPASE-6 genes was demonstrated by a Principal Components Analysis (PCA) carried out in this study. In contrast, a close relationship between the transcription of genes, such as NKEF, IGF-ß, HSP70 and GP96, and the DGGE bands of fish treated jointly with OTC and SpPdp11 was observed in the PCA study. In summary, the results obtained in this study demonstrate that the administration of OTC results in the up-regulation of genes related to apoptosis but that the joint administration of OTC and S. putrefaciens Pdp11 increases the transcription of genes related to antiapoptotic effects and oxidative stress regulation. Further, a clear relationship between these changes and those detected in the intestinal microbiota is established.

Effect of dietary supplementation of probiotics and palm fruits extracts on the antioxidant enzyme gene expression in the mucosae of gilthead seabream (Sparus aurata L.).

Antioxidant activity is particularly important, since oxidation is an unavoidable reaction in all living bodies. At present, natural antioxidants to be used on food as an alternative to synthetic ones are being sought. Gilthead seabream (Sparus aurata L.) specimens were fed for 4 weeks with diets enriched with bacterial probiotics (Shewanella putrefaciens Pdp11 and Bacillus sp), single or in combination with Tunisian dates palm fruit extracts. The expression of the main antioxidant enzyme genes (superoxide dismutase, catalase and glutathione reductase) in the mucosae (gut, skin and gill) was evaluated after 2 and 4 weeks. Previously, free radical scavenging and several antioxidant assays were developed to know the antioxidant properties present on the palm fruits extracts. The results demonstrated that experimental diets alter the expression of the studied antioxidant genes, primarily in the gill and skin. Furthermore, the tested probiotics and mainly, the aqueous date palm fruits extracts had significant antioxidant properties based on their protective effect against the levels of intracellular reactive oxygen species, especially when administering during 4 weeks. For this reason, probiotics and date palm fruit extracts may serve as good natural antioxidants and could potentially be considered as a functional food ingredient for fish in farms.

Modulatory effects of deltamethrin-exposure on the immune status, metabolism and oxidative stress in gilthead seabream (Sparus aurata L.).

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

Accumulation, histopathology and immunotoxicological effects of waterborne cadmium on gilthead seabream (Sparus aurata).

Studies in fish have demonstrated that Cd-exposure produce skeletal deformities and alterations in tissue morphology, enzyme activities, stress response, ion regulation and immune response. In the present work, gilthead seabream (Sparus aurata) specimens were exposed to waterborne Cd (5 µM CdCl2 or 1 mg L(-1)) for 2, 10 or 30 days. Organo-somatic changes, Cd accumulation, liver histology and humoral and cellular immune responses were determined. Results showed that exposure of seabream specimens to Cd induced no alterations on spleen and liver organo-somatic indexes whilst produced progressive deleterious morphological alterations in liver and exocrine pancreas that correlated with the hepatic Cd-accumulation. Regarding the immunotoxicological potential, strikingly, Cd-exposure produced a reduction in the serum complement activity and leucocyte respiratory burst to a significant extent after 10 and 30 days whilst the serum peroxidase activity and leucocyte phagocytosis were increased at different sampling times. On the other hand, serum IgM levels and leucocyte peroxidase activity resulted unaltered. The present results seem to indicate that seabream exposed to Cd in the present conditions suffer toxicity.