Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet.

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

Deciphering the Immunostimulatory Effects of ß-Glucan on a Rainbow Trout (Oncorhynchus mykiss) Macrophage-like Cell Line (RTS11) by Whole Transcriptome Analysis.

ß-glucans are a commonly used immunostimulant/prebiotic in many aquaculture applications for boosting the immune status in fish. However, the method of action as an immunostimulant has not been fully deciphered. To determine the immunomodulatory effects of ß-glucans on the innate immune response, we stimulated the rainbow trout spleen macrophage-like cell line (RTS11) with ß-1,3/1,6-glucans for 4 h. This study uses a whole transcriptomic approach to analyse the immunomodulatory properties of ß-glucans. Several proinflammatory pathways were found to be enriched after stimulation, demonstrating the immunomodulatory effects of ß-glucan supplementation. Several pathways relating to responses to bacteria were also found to be enriched. This study clearly demonstrates the immunomodulatory effects of the supplementation of ß-glucans within an aquaculture setting and further validates the use of cell lines as predictive models to interpret the responses caused by dietary intervention.

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut.

BACKGROUND: Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. RESULTS: We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area. CONCLUSION: The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

Gut Associated Lymphoid Tissue (GALT) primary cells and stable cell lines as predictive models for intestinal health in rainbow trout (Oncorhynchus mykiss).

The use of functional feeds for farmed fish is now regarded as a key factor in improving fish health and performance against infectious disease. However, the mechanisms by which these nutritional components modulate the immune response are not fully understood. The present study was undertaken to identify the suitability of both primary gut-associated lymphoid tissue (GALT) leucocyte cells and established rainbow trout cell lines as potential alternative methods to test functional feed ingredients prior to full fish feeding trials that can take months to complete. In addition to the primary GALT culture cells, the two rainbow cell lines RTS11 and RTgutGC which are from macrophage and gut epithelial cells, respectively. The cells were stimulated with a variety of pathogen associated molecular patterns (PAMPs) (PHA and Poly I:C) and recombinant rainbow trout IL-1ß (rIL-1ß), a proinflammatory cytokine, additionally two forms of ß-glucan, a prebiotic commonly used aquafeeds were used as stimulants. From this, the suitability of cell models as a health screen for functional feeds was assessed. GALT leucocytes were deemed most effective to act as a health screen over the 4hr time point demonstrating responses to Poly I:C, PHA, and rIL-1ß. RTS11 and RTgutGC also responded to the stimulants but did not give a strong T-cell response, most likely reflecting the nature of the cell type as opposed to the mixed cell populations from the primary GALT cell cultures. When stimulated with both forms of ß-glucan, GALT leucocytes demonstrated a strong proinflammatory and T-cell response.

Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health.

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.

Corrigendum: Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae.

[This corrects the article DOI: 10.3389/fmicb.2018.02588.].

Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae.

In the last decades, pollution of the environment by large scale use of antibiotics in agriculture and human medicine have led to increased antimicrobial resistance in both the environment and the host animal microbiome. Disturbances in the host microbiome can result in impaired immunity and reduced resilience of aquaculture species. Here, we investigated whether environmentally measured levels of the commonly used antibiotics ciprofloxacin and oxytetracycline influences the host microbiome and susceptibility toward saponin-induced immune stimulation in larval zebrafish. Firstly, neutrophil and macrophage reporter zebrafish larvae were exposed to different concentrations of soy saponin by immersion. A dose-dependent increase in neutrophil presence in the intestinal area was observed together with increased expression of immune genes il1b, tnfa, il22 and mmp9. To investigate the effect of antibiotics, larval zebrafish were immersed in ciprofloxacin or oxytetracycline in the presence or absence of a low dose of saponin. In vivo imaging revealed that antibiotic treatment did not reduce the number of neutrophils that were recruited to the intestinal area upon saponin exposure, although it did tend to lower pro-inflammatory cytokine levels. Microbial sequencing of whole larvae revealed that exposure to a low dose of saponin already shifted the microbial composition. The combination of oxytetracycline and saponin significantly increased α-diversity compared to the controls. In conclusion, the current study provides evidence that the combination of low levels of antibiotics with low levels of anti-nutritional factors (saponin) can induce inflammatory phenotypes and can modify the microbiota, which might lead to altered disease susceptibility.