Yeast cell wall extracts from Saccharomyces cerevisiae varying in structure and composition differentially shape the innate immunity and mucosal tissue responses of the intestine of zebrafish (Danio rerio).

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and ß-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.

Selection of carbohydrate-active probiotics from the gut of carnivorous fish fed plant-based diets.

The gastrointestinal microbiota plays a critical role on host health and metabolism. This is particularly important in teleost nutrition, because fish do not possess some of the necessary enzymes to cope with the dietary challenges of aquaculture production. A main difficulty within fish nutrition is its dependence on fish meal, an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fish meal are plant feedstuffs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSP), which are not metabolized by fish. The composition of fish-gut microbial communities have been demonstrated to adapt when the host is fed different ingredients. Thus, we hypothesized that a selective pressure of plant-based diets on fish gut microbiota, could be a beneficial strategy for an enrichment of bacteria with a secretome able to mobilize dietary NSP. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass, we have obtained isolates with high probiotic potential. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, we identified the best two candidates to become industrially valuable probiotics. This potential was confirmed in vivo, since one of the select isolates lead to a better growth and feed utilization efficiency in fish fed probiotic-supplemented plant-based diets, thus contributing for sustainable and more cost-effective aquaculture practices.

Influence of Probiotics Administration on Gut Microbiota Core: A Review on the Effects on Appetite Control, Glucose, and Lipid Metabolism.

An increasing number of studies has shown that dietary probiotics exert beneficial health effects in both humans and animals. It is well established that gut microbiota play a pivotal role in regulating host metabolism, and a growing number of studies has elucidated that probiotics positively interfere with gut microbiota. Accumulating evidence shows that probiotics, through their metabolic activity, produce metabolites that in turn contribute to positively affect host physiology. For these reasons, probiotics have shown significant potential as a therapeutic tool for a diversity of diseases, but the mechanisms through which probiotics act has not been fully elucidated yet. The goal of this review was to provide evidence on the effects of probiotics on gut microbiota changes associated with host metabolic variations, specifically focusing on feed intake and lipid and glucose metabolism. In addition, we review probiotic interaction with the gut microbiota. The information collected here will give further insight into the effects of probiotics on the gut microbiota and their action on metabolite release, energy metabolism, and appetite. This information will help to improve knowledge to find better probiotic therapeutic strategies for obesity and eating disorders.

Correction to: Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles.

The original version of this article unfortunately contained mistakes in Authors First name/Family name order. Please refer to this paper as Gioacchini et al. and not as Giorgia et al.

Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles.

In the present study, Nile tilapia Oreochromis niloticus was used as experimental model to study the molecular effects of a new probiotic preparation, Lactogen 13 (Lactobacillus rhamnosus IMC 501® encapsulated with vegetable fat matrices by spray chilling and further indicated as probiotic microgranules), on growth and appetite during larval development. Probiotic microgranules were administered for 30 days to tilapia larvae starting from first feeding. Molecular analysis using high-throughput sequencing revealed that the probiotic could populate the gastrointestinal tract and modulate the microbial communities by significantly increasing the proportion of Lactobacillus as well as reducing the proportion of potential pathogens such as members of the Family Microbacteriaceae, Legionellaceae, and Weeksellaceae. Morphometric analysis evidenced that body weight and total length significantly increased after probiotic treatment. This increase coincided with the modulation of genes belonging to the insulin-like growth factors (igfs) system and genes involved on myogenesis, such as myogenin, and myogenic differentiation (myod). Alongside the improvement of growth, an increase of feed intake was evidenced at 40 days post-fertilization (dpf) in treated larvae. Gene codifying for signals belonging to the most prominent systems involved in appetite regulation, such as neuropeptide y (npy), agouti-related protein (agrp), leptin, and ghrelin were significantly modulated. These results support the hypothesis that gastrointestinal (GI) microbiota changes due to probiotic administration modulate growth and appetite control, activating the endocrine system of tilapia larvae.

Dietary lipid content reorganizes gut microbiota and probiotic L. rhamnosus attenuates obesity and enhances catabolic hormonal milieu in zebrafish.

In the present study, we explored whether dietary lipid content influences the gut microbiome in adult zebrafish. Diets containing three different lipid levels (high [HFD], medium [MFD], and low [LFD]) were administered with or without the supplementation of Lactobacillus rhamnosus (P) to zebrafish in order to explore how the dietary lipid content may influence the gut microbiome. Dietary lipid content shifted the gut microbiome structure. The addition of L. rhamnosus in the diets, induced transcriptional reduction of orexigenic genes, upregulation of anorexigenic genes, and transcriptional decrease of genes involved in cholesterol and triglyceride (TAG) metabolism, concomitantly with lower content of cholesterol and TAG. Probiotic feeding also decreased nesfatin-1 peptide in HFD-P and attenuated weight gain in HFD-P and MFD-P fed zebrafish, but not in LFD-P group. Intestinal ultrastructure was not affected by dietary fat level or probiotic inclusion. In conclusion, these findings underline the role of fat content in the diet in altering gut microbiota community by shifting phylotype composition and highlight the potential of probiotics to attenuate high-fat diet-related metabolic disorder.

Alternative Protein Sources in the Diet Modulate Microbiota and Functionality in the Distal Intestine of Atlantic Salmon (Salmo salar).

The present study aimed to investigate whether alternative dietary protein sources modulate the microbial communities in the distal intestine (DI) of Atlantic salmon, and whether alterations in microbiota profiles are reflected in modifications in host intestinal function and health status. A 48-day feeding trial was conducted, in which groups of fish received one of five diets: a reference diet in which fishmeal (diet FM) was the only protein source and four experimental diets with commercially relevant compositions containing alternative ingredients as partial replacements of fishmeal, i.e., poultry meal (diet PM), a mix of soybean meal and wheat gluten (diet SBMWG), a mix of soy protein concentrate and poultry meal (diet SPCPM), and guar meal and wheat gluten (diet GMWG). Samples were taken of DI digesta and mucosa for microbial profiling using high-throughput sequencing and from DI whole tissue for immunohistochemistry and expression profiling of marker genes for gut health. Regardless of diet, there were significant differences between the microbial populations in the digesta and the mucosa in the salmon DI. Microbial richness was higher in the digesta than the mucosa. The digesta-associated bacterial communities were more affected by the diet than the mucosa-associated microbiota. Interestingly, both legume-based diets (SBMWG and GMWG) presented high relative abundance of lactic acid bacteria in addition to alteration in the expression of a salmon gene related to cell proliferation (pcna). It was, however, not possible to ascertain the cause-effect relationship between changes in bacterial communities and the host's intestinal responses to the diets.IMPORTANCE The intestine of cultivated Atlantic salmon shows symptoms of compromised function, which are most likely caused by imbalances related to the use of new feed ingredients. Intestinal microbiota profiling may become in the future a valuable endpoint measurement in order to assess fish intestinal health status and effects of diet. The present study aimed to gain information about whether alternative dietary protein sources modulate the microbial communities in the Atlantic salmon intestine and whether alterations in microbiota profiles are reflected in alterations in host intestinal function and health status. We demonstrate here that there are substantial differences between the intestinal digesta and mucosa in the presence and abundance of bacteria. The digesta-associated microbiota showed clear dependence on the diet composition, whereas mucosa-associated microbiota appeared to be less affected by diet composition. Most important, the study identified bacterial groups associated with diet-induced gut dysfunction that may be utilized as microbial markers of gut health status in fish.

A high-resolution map of the gut microbiota in Atlantic salmon (Salmo salar): A basis for comparative gut microbial research.

Gut health challenges, possibly related to alterations in gut microbiota, caused by plant ingredients in the diets, cause losses in Atlantic salmon production. To investigate the role of the microbiota for gut function and health, detailed characterization of the gut microbiota is needed. We present the first in-depth characterization of salmon gut microbiota based on high-throughput sequencing of the 16S rRNA gene's V1-V2 region. Samples were taken from five intestinal compartments: digesta from proximal, mid and distal intestine and of mucosa from mid and distal intestine of 67.3 g salmon kept in seawater (12-14 °C) and fed a commercial diet for 4 weeks. Microbial richness and diversity differed significantly and were higher in the digesta than the mucosa. In mucosa, Proteobacteria dominated the microbiota (90%), whereas in digesta both Proteobacteria (47%) and Firmicutes (38%) showed high abundance. Future studies of diet and environmental impacts on gut microbiota should therefore differentiate between effects on mucosa and digesta in the proximal, mid and the distal intestine. A core microbiota, represented by 22 OTUs, was found in 80% of the samples. The gut microbiota of Atlantic salmon showed similarities with that of mammals.

Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism.

The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders.

The influence of probiotics on zebrafish Danio rerio innate immunity and hepatic stress.

In this study, the effects of probiotic administration on zebrafish Danio rerio intestinal innate immunity and hepatic stress were evaluated. Zebrafish adults were treated for 10 days with the probiotic Lactobacillus rhamnosus IMC 501(®). To assess the effects at the molecular level, the mRNA levels of genes involved in the innate immune system, stress response, oxidative stress, and apoptosis were quantified by real-time polymerase chain reaction. An increase of biomarkers related to innate immune responses was observed in intestinal tissue from the probiotic-treated fish compared with the control fish. In addition, a decrease in the abundance of stress and apoptotic-related genes was observed in the liver of the probiotic-fed fish. Finally, imaging Fourier transform infrared analysis was conducted on liver sections and the data obtained confirmed that probiotic administration decreased oxidative stress levels, decreased DNA damage, and increased lipid saturation levels. Overall, the results show that probiotic administration may enhance zebrafish welfare by modulating the innate immune response and improving hepatic stress tolerance.

Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish (Danio rerio).

Nanoparticles (NPs) can be ingested by organisms, and NPs with antimicrobial properties may disrupt beneficial endogenous microbial communities and affect organism health. Zebrafish were fed diets containing Cu-NPs or Ag-NPs (500 mg kg(-1) food), or an appropriate control for 14 d. Intestinal epithelium integrity was examined by transmission electron microscopy, and microbial community structure within the intestine was assessed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA. No lesions were observed in intestinal epithelia; however, presence of NPs in diets changed intestinal microbial community structure. In particular, some beneficial bacterial strains (e.g., Cetobacterium somerae) were suppressed to non-detectable levels by Cu-NP exposure, and two unidentified bacterial clones from the Firmicutes phylum were sensitive (not detected) to Cu, but were present in Ag and control fish. Unique changes in zebrafish microbiome caused by exposure to Ag-NP and Cu-NP indicate that NP ingestion could affect digestive system function and organism health.

Dietary supplementation of fructooligosaccharide (FOS) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach (Rutilus rutilus) fry.

The present study investigated the effects of prebiotic fructooligosaccharide (FOS) on the innate immune response, stress resistance, digestive enzyme activities, growth factors and survival of Caspian Roach (Rutilus rutilus) fry. After acclimation, fish (0.67 ± 0.03 g) were allocated into 12 tanks (50 fish per tank) and triplicate groups were fed a control diet or diets containing 1%, 2% or 3% FOS. At the end of the trial (7 weeks), humoral innate immune parameters (serum Ig levels, lysozyme activity and alternative complement activity (ACH50)), resistance to salinity stress (150 g L(-1)), digestive enzyme activities (amylase, lipase and protease) and growth factors (final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR), and condition factor) were assessed. At the end of the study the innate immune responses (Ig levels, lysozyme activity and ACH50) were significantly higher in 2% and 3% FOS fed fish (P < 0.05), whereas, 1% dietary FOS only elevated serum lysozyme activity. All dietary FOS levels significantly increased resistance to a salinity stress challenge (P < 0.05) and highest survival was observed in the 3% FOS group. Similarly, digestive enzyme activities were significantly elevated with increasing levels of dietary FOS (P < 0.05). Subsequently, elevated growth performance (final weight, SGR and FCR) was observed in roach fed 2% and 3% FOS compared to the control group (P < 0.05). These results indicate that FOS can be considered as a beneficial dietary supplement for improving the immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach fry.

Probiotics can induce follicle maturational competence: the Danio rerio case.

In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase.

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection.

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 106 CFU g⁻¹ feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1ß, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1ß, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.

Effect of dietary alginic acid on juvenile tilapia (Oreochromis niloticus) intestinal microbial balance, intestinal histology and growth performance.

The aim of the present study was to assess the effect of a commercial alginic acid source (Ergosan) on tilapia Oreochromis niloticus intestinal microbial balance, intestinal morphology, and growth parameters. Fish were fed a basal control diet or the basal diet plus a source of alginic acid (5 g kg(-1) Ergosan; Schering-Plough Aquaculture, UK) for 9 weeks. At the end of the trial, light and electron microscopy demonstrated that the morphology of the intestinal tract at the gross and ultra-structural level was not affected by dietary alginic acid inclusion. Both groups of fish displayed healthy, normal morphology with no signs of disease, cell or tissue damage. Intestinal epithelial leucocyte infiltration was not affected by dietary alginic acid. Molecular bacterial profiles derived from PCR-DGGE illustrated highly similar microbial communities (both within the lumen and associated with the intestinal mucosa) in the respective treatment groups. Microbial ecological parameters (e.g. species diversity and richness) also remained unaffected. Although not significant, trends towards elevated survival and body protein content were observed in the alginic acid-fed fish. These results are suggestive that alginic acid does not adversely impact the indigenous gastrointestinal microbial balance and subsequently does not impact upon the epithelial brush border integrity. Validation of non-detrimental impacts of immunostimulatory products on gastric microbiota and epithelial integrity should be pursued in future studies as maintaining microbial balance and epithelial integrity is essential for proper gut functionality.

Microbial manipulations to improve fish health and production--a Mediterranean perspective.

The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.

The study of some haematological and serum biochemical parameters of juvenile beluga (Huso huso) fed oligofructose.

A study was conducted to investigate the effects of dietary oligofructose (1, 2 and 3%) on the blood profiles of beluga (Huso huso) juveniles (18.77 ± 0.76 g) compared to fish fed an un-supplemented diet. After 7 weeks of feeding on the experimental diets, haematological parameters, metabolic products (cholesterol, glucose and total protein) and serum enzymes (lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase) were measured. Compared to the control group (0% oligofructose), dietary oligofructose had no effect on red blood cell counts (RBC), mean corpuscular volume (MCV), mean cellular haemoglobin (MCH) or mean cell haemoglobin concentration (MCHC) (P > 0.05). However, haemoglobin (Hb) concentration, leucocyte (WBC) levels and the proportion of lymphocytes were significantly higher (P > 0.05) in the 2% oligofructose fed fish than in the 3% oligofructose fed fish. Additionally, haematocrit (Hct) values (P = 0.049) and the proportion of lymphocytes (P ≤ 0.01) were significantly higher in the 2% oligofructose group than in the control group. Although serum glucose and total protein remained unaffected, serum cholesterol was significantly lower in the 2% oligofructose group than in the control and 3% oligofructose group (P < 0.05). The results of the present study showed that oligofructose had no significant effects on serum lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. These results indicate that fish blood profiles could be affected by prebiotics, which should be taken into account in future studies.