The effect of spray‐dried porcine plasma on gilthead seabream (Sparus aurata) intestinal microbiota

The effect of spray‐dried porcine plasma (SDPP) on the intestinal histological organization and autochthonous microbiota composition was evaluated in Sparus aurata. Fish were fed a basal diet (51 g/kg protein, 17 g/kg fat, 20.6 MJ/kg gross energy) and a diet containing 3 g/kg SDPP for 95 days (initial body weight, BW = 9.5 ± 0.2g, mean ± SD). The inclusion of SDPP promoted growth (p .05) between both groups. Intestinal microbiota was dominated by Proteobacteria (>85%) and Firmicutes (5%–12%), whereas Bacteroidetes never represented more than 1.5%. γ‐Proteobacteria, and Bacilli and Clostridia were the predominant classes. The short administration of SDPP (20 days) resulted in changes in microbiota diversity and richness associated with an increase in the sequences of the genus Lactobacillus and to a decrease in the genus Vibrio, whereas these changes were reverted at 95 days. Intestinal goblet cell density was not correlated to microbiota diversity and richness changes rather than to the immunostimulatory effect of the SDPP.info:eu-repo/semantics/acceptedVersio

Pathogenic strains of Shewanella putrefaciens contain plasmids that are absent in the probiotic strain Pdp11

Shewanella putrefaciens Pdp11 is a strain described as a probiotic for use in aquaculture. However, S. putrefaciens includes strains reported to be pathogenic or saprophytic to fish. Although the probiotic trait has been related to the presence of a group of genes in its genome, the existence of plasmids that could determine the probiotic or pathogenic character of this bacterium is unknown. In the present work, we searched for plasmids in several strains of S. putrefaciens that differ in their pathogenic and probiotic character. Under the different conditions tested, plasmids were only found in two of the five pathogenic strains, but not in the probiotic strain nor in the two saprophytic strains tested. Using a workflow integrating Sanger and Illumina reads, the complete consensus sequences of the plasmids were obtained. Plasmids differed in one ORF and encoded a putative replication initiator protein of the repB family, as well as proteins related to plasmid stability and a toxin-antitoxin system. Phylogenetic analysis showed some similarity to functional repB proteins of other Shewanella species. The implication of these plasmids in the probiotic or pathogenic nature of S. putrefaciens is discussed

Respuesta inmune en mucosas de piel y branquias de Solea senegalensis tras la alimentación con una dieta conteniendo Ulva ohnoi (5%)

Abstract In the search of the improvement of the feeding of the fish in the aquaculture, macroalgae seem to be a promising source of nutrients and bioactive substances. In the case of the genus Ulva, the effect of its inclusion in feed on the growth, tissues composition and the stress response of some fish species has been evaluated. However, it is necessary to evaluate the effects on the immune system, as its inclusion could modify the resistance to pathogens. In the present work, we have studied the response of the immune system at lysozyme expression level, cellular immune response genes, and inflammatory response genes in mucous of skin and sole gills (Solea senegalensis) fed with a diet supplemented in Ulva ohnoi (5%). Results obtained indicate absence of significant changes in the expression of assayed immune genes. Resumen En la búsqueda de la mejora de la alimentación de los peces en la acuicultura, las algas se presentan como una prometedora fuente de nutrientes y sustancias bioactivas. En el caso del género Ulva, se ha publicado el efecto de su inclusión en piensos sobre el crecimiento, la composición de los tejidos y la respuesta al estrés de algunas especies de interés acuícola. Sin embargo, es necesario evaluar los efectos en el sistema inmune, puesto que su inclusión podría modificar la resistencia a patógenos. En el presente trabajo se ha estudiado la respuesta del sistema inmune a nivel de expresión de lisozimas, genes de respuesta inmune celular, y genes de la respuesta inflamatoria en piel y branquias de lenguados (Solea senegalensis) alimentados con una dieta suplementada en Ulva ohnoi (5%). Los resultados indican que no se produce un cambio significativo en la expresión de estos grupos de genes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación (INIA) y FEDER (Ref. RTA2014 00023 C0202

Debaryomyces hansenii supplementation in low fish meal diets promotes growth, modulates microbiota and enhances intestinal condition in juvenile marine fish

Background The development of a sustainable business model with social acceptance, makes necessary to develop new strategies to guarantee the growth, health, and well-being of farmed animals. Debaryomyces hansenii is a yeast species that can be used as a probiotic in aquaculture due to its capacity to i) promote cell proliferation and differentiation, ii) have immunostimulatory effects, iii) modulate gut microbiota, and/or iv) enhance the digestive function. To provide inside into the effects of D. hansenii on juveniles of gilthead seabream (Sparus aurata) condition, we integrated the evaluation of the main key performance indicators coupled with the integrative analysis of the intestine condition, through histological and microbiota state, and its transcriptomic profiling. Results After 70 days of a nutritional trial in which a diet with low levels of fishmeal (7%) was supplemented with 1.1% of D. hansenii (17.2 × 105 CFU), an increase of ca. 12% in somatic growth was observed together with an improvement in feed conversion in fish fed a yeast-supplemented diet. In terms of intestinal condition, this probiotic modulated gut microbiota without affecting the intestine cell organization, whereas an increase in the staining intensity of mucins rich in carboxylated and weakly sulphated glycoconjugates coupled with changes in the affinity for certain lectins were noted in goblet cells. Changes in microbiota were characterized by the reduction in abundance of several groups of Proteobacteria, especially those characterized as opportunistic groups. The microarrays-based transcriptomic analysis found 232 differential expressed genes in the anterior-mid intestine of S. aurata, that were mostly related to metabolic, antioxidant, immune, and symbiotic processes. Conclusions Dietary administration of D. hansenii enhanced somatic growth and improved feed efficiency parameters, results that were coupled to an improvement of intestinal condition as histochemical and transcriptomic tools indicated. This probiotic yeast stimulated host-microbiota interactions without altering the intestinal cell organization nor generating dysbiosis, which demonstrated its safety as a feed additive. At the transcriptomic level, D. hansenii promoted metabolic pathways, mainly protein-related, sphingolipid, and thymidylate pathways, in addition to enhance antioxidant-related intestinal mechanisms, and to regulate sentinel immune processes, potentiating the defensive capacity meanwhile maintaining the homeostatic status of the intestine.This work has been financed through the DIETAplus project of JACUMAR (Junta de Cultivos Marinos, MAPAMA; Spanish government), which is co-funded with FEMP funds (EU). Furthermore, this research was funded by means of grants from the Spanish Government: PID2019-106878RB-I00 and IS was granted with a Postdoctoral fellowship (FJC2020-043933-I). EVV and FERL thank the support of Fondecyt iniciación (project number 11221308) and Fondecyt regular (project number 11221308) grants (Agencia Nacional de Investigacióny Desarrollo de Chile, Government of Chile), respectively. Collaboration between Ibero-American researchers has been done under the framework of the network LARVAplus “Strategies for the development and im-provement of fish larvae production in Ibero-America” (117RT0521) funded by the Ibero-American Program of Science and Technology for Development (CYTED, Spain).info:eu-repo/semantics/publishedVersio

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group’s il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.Versión del edito

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

Variation in the molecular weight of Photobacterium damselae subsp. piscicida antigens when cultured under different conditions in vitro

The antigenicity of Photobacterium damselae (Ph. d.) subsp. piscicida, cultured in four different growth media [tryptone soya broth (TSB), glucose-rich medium (GRM), iron-depleted TSB (TSB + IR-), and iron-depleted GRM (GRM + IR-)] was compared by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using sera obtained from sea bass (Dicentrarchus labrax) raised against live or heat-killed Ph. d. subsp. piscicida. The antigenic expression of Ph. d. subsp. piscicida was found to differ depending on the culture medium used. A significantly higher antibody response was obtained with iron-depleted bacteria by ELISA compared with non-iron depleted bacteria obtained from the sera of sea bass raised against live Ph. d. subsp. piscicida. The sera from sea bass raised against live bacteria showed a band at 22 kDa in bacteria cultured in TSB + IR- or GRM+ IR- when bacteria that had been freshly isolated from fish were used for the screening, while bands at 24 and 47 kDa were observed with bacteria cultured in TSB or GRM. When bacteria were passaged several times on tryptic soya agar prior to culturing in the four different media, only bands at 24 and 47 kDa were recognized, regardless of the medium used to culture the bacteria. It would appear that the molecular weight of Ph. d. subsp. piscicida antigens change in the presence of iron restriction, and sera from sea bass infected with live bacteria are able to detect epitopes on the antigens after this shift in molecular weight

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium's structure and antigenicity when cultured in vivo is discussed

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