SpPdp11 Administration in Diet Modified the Transcriptomic Response and Its Microbiota Associated in Mechanically Induced Wound Sparus aurata Skin.

Skin lesions are a frequent fact associated with intensive conditions affecting farmed fish. Knowing that the use of probiotics can improve fish skin health, SpPdp11 dietary administration has demonstrated beneficial effects for farmed fish, so its potential on the skin needs to be studied more deeply. The wounded specimens that received the diet with SpPdp11 showed a decrease in the abundance of Enterobacteriaceae, Photobacterium and Achromobacter related to bacterial biofilm formation, as well as the overexpression of genes involved in signaling mechanisms (itpr3), cell migration and differentiation (panxa, ttbk1a, smpd3, vamp5); and repression of genes related to cell proliferation (vstm4a, areg), consistent with a more efficient skin healing processes than that observed in the wounded control group. In addition, among the groups of damaged skin with different diets, Achromobacter, f_Ruminococcaceae, p_Bacteroidetes, Fluviicola and Flavobacterium genera with significant differences showed positive correlations with genes related to cell migration and negative correlations with inflammation and cell proliferation and may be the target of future studies.

Immunomodulation and skin microbiota perturbations during an episode of chronic stress in gilthead seabream.

Fish stress is a major concern in the aquaculture industry. Many stressors coming from routine practices can predispose fish to compromised growth, immunity and overall health. This study focuses on the characterization of the skin microbiota using next generation sequencing (NGS) platform by targeting a genomic marker 16S and to determine growth performance and immune status of gilthead seabream (Sparus aurata) during an episode of chronic stress. Two groups were established: control group and chronically stressed group. Stressed fish were subjected to 1 min air exposure twice a week for 4 weeks. Results showed that stress negatively affected fish growth performance. Cellular and humoral systemic immunity remained unaffected while local immunity in skin was positively stimulated (total IgM and peroxidase). Skin mucus microbial composition showed significant differences especially after 14 days. Stressed fish showed a decrease in the abundance of the genera Acinetobacter, NS3a_marine_group and Pseudomonas, while Pseudoalteromonas and Marinagarivorans increased significantly. In conclusion, air exposure stress was associated with alterations in skin mucosal immunity and microbial composition that may have been beneficial to the host favoring adaptation to stress.

Alteration of the Immune Response and the Microbiota of the Skin during a Natural Infection by Vibrio harveyi in European Seabass (Dicentrarchus labrax).

Disease outbreaks continue to represent one of the main bottlenecks for the sustainable development of the aquaculture industry. In marine aquaculture, many species from the Vibrio genus are serious opportunistic pathogens responsible for significant losses to producers. In this study, the effects on the immune response and the skin microbiota of European sea bass (Dicentrarchus labrax) were studied after a natural disease outbreak caused by V. harveyi. Data obtained from infected and non-infected fish were studied and compared. Regarding the local immune response (skin mucus) a decrease in the protease activity was observed in infected fish. Meanwhile, at a systemic level, a decrease in protease and lysozyme activity was reported while peroxidase activity showed a significant increase in serum from infected fish. A clear dysbiosis was observed in the skin mucus microbiota of infected fish in comparison with non-infected fish. Moreover, V. harveyi, was identified as a biomarker for the infected group and Rubritalea for healthy fish. This study highlights the importance of characterizing the mucosal surfaces and microbial composition of the skin mucus (as a non-invasive technique) to detect potential disease outbreaks in fish farms.

Diet, Immunity, and Microbiota Interactions: An Integrative Analysis of the Intestine Transcriptional Response and Microbiota Modulation in Gilthead Seabream (Sparus aurata) Fed an Essential Oils-Based Functional Diet.

Essential oils (EOs) are promising alternatives to chemotherapeutics in animal production due to their immunostimulant, antimicrobial, and antioxidant properties, without associated environmental or hazardous side effects. In the present study, the modulation of the transcriptional immune response (microarray analysis) and microbiota [16S Ribosomal RNA (rRNA) sequencing] in the intestine of the euryhaline fish gilthead seabream (Sparus aurata) fed a dietary supplementation of garlic, carvacrol, and thymol EOs was evaluated. The transcriptomic functional analysis showed the regulation of genes related to processes of proteolysis and inflammatory modulation, immunity, transport and secretion, response to cyclic compounds, symbiosis, and RNA metabolism in fish fed the EOs-supplemented diet. Particularly, the activation of leukocytes, such as acidophilic granulocytes, was suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the gut. Fish growth performance and gut microbiota alpha diversity indices were not affected, while dietary EOs promoted alterations in bacterial abundances in terms of phylum, class, and genus. Subtle, but significant alterations in microbiota composition, such as the decrease in Bacteroidia and Clostridia classes, were suggested to participate in the modulation of the intestine transcriptional immune profile observed in fish fed the EOs diet. Moreover, regarding microbiota functionality, increased bacterial sequences associated with glutathione and lipid metabolisms, among others, detected in fish fed the EOs supported the metabolic alterations suggested to potentially affect the observed immune-related transcriptional response. The overall results indicated that the tested dietary EOs may promote intestinal local immunity through the impact of the EOs on the host-microbial co-metabolism and consequent regulation of significant biological processes, evidencing the crosstalk between gut and microbiota in the inflammatory regulation upon administration of immunostimulant feed additives.

Solea senegalensis Bacterial Intestinal Microbiota Is Affected by Low Dietary Inclusion of Ulva ohnoi.

The inclusion of macroalgae in the diets of farmed fish offers the opportunity for an added-value dietary ingredient to the nutraceutical feed. The composition of algae varies greatly among species. Several Ulva species have been considered in aquafeed formulations for different farmed fish, and Ulva ohnoi is being applied recently. However, the effects of seaweed dietary inclusion on the host must be evaluated. Considering the important role of the host intestinal microbiota, the potential effects of U. ohnoi dietary inclusion need to be studied. In this study, the characterization of the intestinal microbiome of Solea senegalensis, a flatfish with high potential for aquaculture in South Europe, receiving U. ohnoi (5%)-supplemented diet for 90 days has been carried out. In addition, the functional profiles of bacterial communities have been determined by using PICRUSt, a computational approach to predict the functional composition of a metagenome by using marker gene data and a database of reference genomes. The results show that long-term dietary administration of U. ohnoi (5%)-supplemented feed modulates S. senegalensis intestinal microbiota, especially in the posterior intestinal section. Increased relative abundance of Vibrio jointly with decreased Stenotrophomonas genus has been detected in fish receiving Ulva diet compared to control-fed fish. The influence of the diet on the intestinal functionality of S. senegalensis has been studied for the first time. Changes in bacterial composition were accompanied by differences in predicted microbiota functionality. Increased abundance of predicted genes involved in xenobiotic biodegradation and metabolism were observed in the microbiota when U. ohnoi diet was used. On the contrary, predicted percentages of genes associated to penicillin and cephalosporin biosynthesis as well as beta-lactam resistance were reduced after feeding with Ulva diet.

Inclusion of dietary Ulva ohnoi 5% modulates Solea senegalensis immune response during Photobacterium damselae subsp. piscicida infection.

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf, cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi, compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp, modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp-1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out.

TarSynFlow, a workflow for bacterial genome comparisons that revealed genes putatively involved in the probiotic character of Shewanella putrefaciens strain Pdp11.

Probiotic microorganisms are of great interest in clinical, livestock and aquaculture. Knowledge of the genomic basis of probiotic characteristics can be a useful tool to understand why some strains can be pathogenic while others are probiotic in the same species. An automatized workflow called TarSynFlow (Targeted Synteny Workflow) has been then developed to compare finished or draft bacterial genomes based on a set of proteins. When used to analyze the finished genome of the probiotic strain Pdp11 of Shewanella putrefaciens and genome drafts from seven known non-probiotic strains of the same species obtained in this work, 15 genes were found exclusive of Pdp11. Their presence was confirmed by PCR using Pdp11-specific primers. Functional inspection of the 15 genes allowed us to hypothesize that Pdp11 underwent genome rearrangements spurred by plasmids and mobile elements. As a result, Pdp11 presents specific proteins for gut colonization, bile salt resistance and gut pathogen adhesion inhibition, which can explain some probiotic features of Pdp11.

Modulation of Intestinal Microbiota in Solea senegalensis Fed Low Dietary Level of Ulva ohnoi.

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi.

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.

Insights into the fecal microbiota of captive Iberian lynx (Lynx pardinus).

The Iberian lynx (Lynx pardinus) is an endangered species restricted to several areas of Spain and Portugal. Its low genetic diversity likely provokes immune depression and high susceptibility to infectious diseases. The intestinal microbiota is closely related to host health and nutrition. In order to contribute to the knowledge of the Iberian lynx intestinal microbiota, fecal microbiota of captive specimens from two breeding centers ("La Olivilla" and "El Acebuche"), located in Southern Spain, were studied by Denaturing Gradient Gel Electrophoresis (DGGE). Results grouped microbiota in two main clusters (I and III) which included DGGE patterns of 19 out of 36 specimens, cluster I being the most frequent in "La Olivilla" (50%) and cluster III in "El Acebuche" (55.55 %) specimens. Bacteroidetes, Firmicutes and Proteobacteria phyla were identified. Segregation of clusters I and III was attributed to different microorganism presence (Pseudomonas koreensis, Pseudomonas migulae, Carnobacterium sp., Arthrobacter, Robinsoniella peorensis and Ornithinibacillus sp.) and ability to use different carbon sources. Biolog EcoPlates® results indicate high functional diversity of fecal microbiota, it being higher in cluster III. The great impact of intestinal microbiota on host health supports the importance of its microbial composition understanding. This study is the first report of captive Iberian lynx fecal microbiota composition. [Int Microbiol 20(1): 31-41 (2017)].

Insights into the fecal microbiota of captive Iberian lynx (Lynx pardinus)

The Iberian lynx (Lynx pardinus) is an endangered species restricted to several areas of Spain and Portugal. Its low genetic diversity likely provokes immune depression and high susceptibility to infectious diseases. The intestinal microbiota is closely related to host health and nutrition. In order to contribute to the knowledge of the Iberian lynx intestinal microbiota, fecal microbiota of captive specimens from two breeding centers («La Olivilla» and «El Acebuche»), located in Southern Spain, were studied by Denaturing Gradient Gel Electrophoresis (DGGE). Results grouped microbiota in two main clusters (I and III) which included DGGE patterns of 19 out of 36 specimens, cluster I being the most frequent in «La Olivilla» (50%) and cluster III in «El Acebuche» (55.55 %) specimens. Bacteroidetes, Firmicutes and Proteobacteria phyla were identified. Segregation of clusters I and III was attributed to different microorganism presence (Pseudomonas koreensis, Pseudomonas migulae, Carnobacterium sp., Arthrobacter, Robinsoniella peorensis and Ornithinibacillus sp.) and ability to use different carbon sources. Biolog EcoPlates® results indicate high functional diversity of fecal microbiota, it being higher in cluster III. The great impact of intestinal microbiota on host health supports the importance of its microbial composition understanding. This study is the first report of captive Iberian lynx fecal microbiota composition (AU)

No disponible

Modulation of immunity and gut microbiota after dietary administration of alginate encapsulated Shewanella putrefaciens Pdp11 to gilthead seabream (Sparus aurata L.).

The potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream (Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10(8) cfu g(-1)) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes (il1ß, bd, mhcIIα, ighm and tcrß) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrß after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

Probiotic supplementation influences the diversity of the intestinal microbiota during early stages of farmed senegalese sole (Solea Senegalensis, Kaup 1858).

Ingestion of bacteria at early stages results in establishment of a primary intestinal microbiota which likely undergoes several stages along fish life. The role of this intestinal microbiota regulating body functions is crucial for larval development. Probiotics have been proved to modulate this microbiota and exert antagonistic effects against fish pathogens. In the present study, we aimed to determine bacterial diversity along different developmental stages of farmed Senegalese sole (Solea senegalensis) after feeding probiotic (Shewanella putrefaciens Pdp11) supplemented diet for a short period (10-30 days after hatching, DAH). Intestinal lumen contents of sole larvae fed control and probiotic diets were collected at 23, 56, 87, and 119 DAH and DNA was amplified using 16S rDNA bacterial domain-specific primers. Amplicons obtained were separated by denaturing gradient gel electrophoresis (DGGE), cloned, and resulting sequences compared to sequences in GenBank. Results suggest that Shewanella putrefaciens Pdp11 induces a modulation of the dominant bacterial taxa of the intestinal microbiota from 23 DAH. DGGE patterns of larvae fed the probiotic diet showed a core of bands related to Lactobacillus helveticus, Pseudomonas acephalitica, Vibrio parahaemolyticus, and Shewanella genus, together with increased Vibrio genus presence. In addition, decreased number of clones related to Photobacterium damselae subsp piscicida at 23 and 56 DAH was observed in probiotic-fed larvae. A band corresponding to Shewanella putrefaciens Pdp11 was sequenced as predominant from 23 to 119 DAH samples, confirming the colonization by the probiotics. Microbiota modulation obtained via probiotics addition emerges as an effective tool to improve Solea senegalensis larviculture.

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics.

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

Superoxide dismutase and catalase in Photobacterium damselae subsp. piscicida and their roles in resistance to reactive oxygen species.

Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pasteurellosis or pseudotuberculosis in warm water marine fish. Enzymes which neutralize reactive oxygen species, produced during aerobic metabolism or during respiratory burst in fish macrophages, are important virulence factors in many pathogens. This study characterizes a periplasmic superoxide dismutase (SOD) and a cytoplasmic catalase in P. damselae. Purification and partial amino-terminal sequencing confirmed the SOD to be iron-cofactored, with a high degree of homology to other bacterial FeSODs. The SOD was common to all strains analysed in terms of type, location and activity, whilst the catalase varied in activity between strains. The catalase was constitutively expressed, but the SOD appeared to be repressed under low oxygen conditions. In spite of the presence of a periplasmic SOD, P. damselae was susceptible to killing by exogenous superoxide anion generated in a cell-free system. Addition of exogenous SOD to this system did not abolish the bactericidal effect; however, addition of catalase was protective. These results suggest that lack of periplasmic catalase may be implicated in susceptiblity to killing by reactive oxygen species.