The effects of using plant ingredients in Senegalese sole (
Solea senegalensis
) diet on immune competence and intestine morphology and microbial ecology are still controversial. Probiotics or ...immunostimulants can potentially alter the intestinal microbiota in a way that protects fish against pathogens. The current study aimed to examine the intestine histology and microbiota and humoral innate immune response in juvenile sole fed diets with low (35 %) or high (72 %) content of plant protein (PP) ingredients supplemented with a multispecies probiotic bacteria or autolysed yeast. Fish fed the probiotic diet had lower growth performance. Lysozyme and complement activities were significantly higher in fish fed PP72 diets than in their counterparts fed PP35 diets after 17 and 38 days of feeding. At 2 days of feeding, fish fed unsupplemented PP72 showed larger intestine section area and longer villus than fish fed unsupplemented PP35. At 17 days of feeding, fish fed unsupplemented PP72 showed more goblet cells than the other dietary groups, except the group fed yeast supplemented PP35 diet. High dietary PP level, acutely stimulate fish innate immune defence of the fish after 2 and 17 days of feeding. However, this effect does not occur after 73 days of feeding, suggesting a habituation to dietary treatments and/or immunosuppression, with a reduction in the number of the goblet cells. Fish fed for 38 days with diets supplemented with autolysed yeast showed longer intestinal villus. The predominant bacteria found in sole intestine were
Vibrio
sp. and dietary probiotic supplementation caused a reduction in
Vibrio
content, regardless of the PP level.
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.
•Shewanella putrefaciens Pdp11 enhances tolerance to crowding stress in Solea senegalensis.•Dietary administration of S. putrefaciens Pdp11 modulates immune gene transcription.•Some intestinal changes are related to microbiota modulation by S. putrefaciens Pdp11.
The use of lysed microalgae in the diet of carnivorous fish can increase the bioavailability of proteins and bioactive compounds, such as unsaturated fatty acids or vitamins in the digestive tract. ...These are essential molecules for the proper physiological development of fish in aquaculture. However, some antinutritional components and other undesirable molecules can be released from an excess of microalgae supplied, compromising the integrity of the intestine. The inclusion of small amounts of hydrolized microalgae in the fish diet can be a good strategy to avoid negative effects, improving the availability of beneficial compounds. Nannochloropsis gaditana is an interesting microalgae as it contains nutraceuticals. Previous studies reported beneficial effects after its inclusion in the diet of Sparus aurata, a widely cultured species in Europe and in all Mediterranean countries. However, administration of raw microalgae can produce intestinal inflammation, increased intestinal permeability, bacterial translocation and disturbance of digestion and absorption processes. The aim of this study was to evaluate changes in the intestinal microbiota and barrier stability of S. aurata fed with low inclusion (5%) hydrolysed N. gaditana. Intestinal microbiota was analyzed using Illumina MiSeq technology and libraries were constructed using variable regions V3-V4 of 16S rDNA molecules. Analysis were based in the identification, quantification and comparison of sequences. The predictive intestinal microbial functionality was analyzed with PICRUSt software. The results determined that the intestinal microbiota bacterial composition and the predictive intestinal microbiota functionality did not change statistically after the inclusion of N. gaditana on the diet. The study of gene expression showed that genes involved in intestinal permeability and integrity were not altered in fish treated with the experimental diet. The potential functionality and bacterial taxonomic composition of the intestinal microbiota, and the expression of integrity and permeability genes in the intestine of the carnivorous fish S. aurata were not affected by the inclusion of hydrolysed 5% N. gaditana microalgae.
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-β, 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 OTC and SpPdp11 on intestinal microbiota and gene expression in the liver.•OTC produced an increase of the gene expression related to apoptosis.•OTC + SpPdp11 up-regulated the gene expression of antiapoptotic activity in liver.•Changes in the intestinal microbiota are correlated with the liver gene expression.
The effects of dietary administration of probiotic Pdp11, a bacterial probiotic strain from the Alteromonadaceae family, on growth and stress tolerance to high stocking density was analyzed in ...juvenile gilthead sea bream
Sparus auratus. In the first trial, juvenile specimens were fed during 116
days with control feed or diets supplemented with Pdp11, and growth, serum immunological parameters as well as plasmatic, hepatic and muscular metabolic parameters were assessed. Growth performance improved in group receiving probiotic compared to control group. No differences were observed for the assessed immunological or metabolic parameters. In the second trial, specimens from both groups from the first experiment were submitted to the following conditions for 15
days: i) group fed with control food and either under low density (3
kg/m
3), or high density (30
kg/m
3) and ii) group fed with probiotic supplemented feed and either under low density, or high density. Our results indicated that administration of probiotic Pdp11 in the diet promoted growth and improved stress tolerance under high stocking density, suggesting its use will be beneficial for the aquaculture industry of this species.
•Two infection routes modulate iron metabolism genes, decreasing its availability.•Phdp IP infection induces long lasting response in iron metabolism genes.•Late immune gene response after Phdp ...infection is related to the infection route.•Kidney innate immune gene response is mainly modulated after Phdp IM infection.•Genes encoding HSP70 and HSP90AA are not induced after Phdp infection.
The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting cultured fish species including the flatfish Solea senegalensis. In the present work, transcription of iron metabolism related genes (TF, FERR-M, HP-1 and HAMP-1) as well as innate immune system components such as complement proteins (C3 and C7), lysozyme (LYS-G), TNF family (TNFα, TRAF-3), NCCRP-1 and heat shock protein encoding genes (HSP70, HSP90AA, HSP90AB and GP96) has been determined in the liver and kidney of S. senegalensis specimens after Phdp infection. Intraperitoneal injection (IP) and immersion (IM) routes have been used for infection. Fish developed specific antibodies in both cases, higher levels being detected in IP infected specimens. Both infection routes resulted in increased relative transcript levels of FERR-M, HP-1 and HAMP-1 genes and TF decreased relative transcription, conducting to lower iron availability for the pathogen. This response can be considered as a strategy to limit iron availability for Phdp, a pathogen capable to obtain iron from transferrin. Relative transcription of genes encoding lysozyme and complement factors C3 and C7 were also increased regardless the infection route; the liver was the main organ involved in the initial stages and the kidney in later stages of the infection. TNFα and TRAF-3 relative gene transcription increased 24h post-infection. TRAF-3 gene induction was detected 30 d post-infection, whilst no changes in TNFα were observed 72h or 30 d post-infection. NCCRP-1 changes were observed after IP infection in the liver and kidney; however, IM infection resulted only in slight changes in the kidney of infected fish. This different response observed maybe related to a lower number of invaded cells by the pathogen. Finally, changes in HSP90AB and GP96 have been detected after infection by both routes. Different late modulation has been observed in assayed genes depending on the route of infection. Thus, only LYS-G, TF, NCCRP-1, GP96 and HSP90AB gene transcription was modulated 30 d post-infection in the kidney of IM infected specimens; however, IP infected fish showed modulation in a higher number of genes both in liver and kidney tissues. The implications of these responses in resistance to infection by Phdp need to be elucidated.
The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection ...against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response.
Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis.
Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida, and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule.
Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related.
•Sole specimens were immunized with inactivated cells of P. damselae subsp. piscicida and ECP to obtain antiserum.•Specific immune response was detected by ELISA using P. damselae subsp. piscicida antigens.•2D Western blots of ECP and OMP, using sole immunized serum, identified five immunogenic proteins.•Two reactive antigens were identified from ECP: ATP-dependent metalloprotease and Telurite resistance proteins.•In OMP were detected three proteins: Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related.
This study evaluates the effects of the macroalgae Ulva ohnoi on growth and intestinal functionality and integrity of Solea senegalensis juveniles in a 90-day feeding trial. Ulva diet (U-5) included ...5% dry U. ohnoi biomass, whereas an algae-free diet was used as control. After being fed for 45 and 90 days, fish body weight was recorded for growth evaluation, and biological samples were taken. The inclusion of U. ohnoi (5%) significantly affected growth performance of sole juveniles, given that after being fed for 45 and 90 days fish showed lower body weight, specific growth and weight gain rates than fish fed with the control diet. Compared to fish fed with control diet, protease activities measured in the pancreatic secretion was also reduced when Ulva was included in the diet, but these fish also showed increased alkaline phosphatase activity in their intestinal mucosa. Quantitative PCR revealed changes in the relative expression of a set of genes coding for enzymes involved in lipid absorption and metabolism, several digestive enzymes, as well as for some stress response indicators. Electron microscopy revealed a lack of damage in the intestinal mucosa, as well as significantly increased mucosal absorptive surface in those fish fed with Ulva-supplemented diet for 45 days, compared to control fish. The results suggest that U. ohnoi at low inclusion level could protect the intestinal epithelium of fish, but a relatively short administration period is recommended with the aim of avoiding negative effects on Senegalese sole growth and proteolytic digestive enzyme activities.
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•The potential of Ulva ohnoi as functional additive for Solea senegalensis was evaluated.•Fish growth and pancreatic protease activity were reduced by U. ohnoi.•U. ohnoi improved the mucosal intestinal epithelium of Senegalese sole juveniles.•Diets including U. ohnoi modulated transcription of intestinal protein and lipid metabolism-related genes.
The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, ...activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of juvenile Senegalese sole. In addition, the profile of intestinal microbiota was studied by Denaturing Gradient Gel Electrophoresis. Growth and nutrient utilization were significantly higher in fish receiving probiotics than in those fed the control diet. No differences were observed in proximal composition between treatments, though higher lipid muscle content was measured in fish receiving Pdp13. Those fish also exhibited higher activities of AP when compared to Ppd11 and control groups. The profile of intestinal microbiota clearly separated those fish receiving probiotics from those of the control group. Microscopical examination revealed accumulation of lipid droplets in the enterocytes of fish receiving the control diet, but not in those fed on probiotics. Interactions between those structural changes and growth performance are discussed.
The effects of dietary administration of two probiotics,
Shewanella
putrefaciens Pdp11 and
Shewanella
baltica Pdp13, on growth, respiratory burst activity of phagocytes of Senegalese sole (
Solea
...senegalensis), and survival of fish challenged with
Photobacterium
damselae subsp.
piscicida have been studied. Fish were fed for 60 days with three different diets: one control, and two diets supplemented with 10
9 cfu g
−
1
of probiotics Pdp11 (Pdp11 diet) and Pdp13 (Pdp13 diet). Respiratory burst activity of phagocytes from fish fed with diet Pdp11 significantly increased after 60 days of feeding, whilst this significant increase was not detected in phagocytes from fish fed control and Pdp13 diets. On the other hand, the cumulative percentage of mortality after the challenges with
P.
damselae subsp.
piscicida, was 100% in the groups fed with control diet, whereas mortality rates observed in the groups fed with diets supplemented with Pdp11 and Pdp13 ranged from 75–100% and 65–80%, respectively. These results suggest that an increased respiratory burst activity of the phagocytes is not essential to increase the protection against the
P.
damselae subsp.
piscicida. On the other hand, the use of both probiotics improved the growth, and the survival against the pathogen, in comparison with those fish receiving the control diet.
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