Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

Technical pipeline for screening microbial communities as a function of substrate specificity through fluorescent labelling.

The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

Fishmeal Dietary Replacement Up to 50%: A Comparative Study of Two Insect Meals for Rainbow Trout (Oncorhynchus mykiss).

The demand of optimal protein for human consumption is growing. The Food and Agriculture Organization (FAO) has highlighted aquaculture as one of the most promising alternatives for this protein supply gap due to the high efficiency of fish growth. However, aquaculture has been facing its own sustainability problem, because its high demand for protein has been traditionally satisfied with the use of fishmeal (FM) as the main source. Some of the most promising and sustainable protein substitutes for FM come from insects. The present manuscript provides insight into an experiment carried out on rainbow trout (Oncorhynchus mykiss) with a 50% replacement of FM with different larvae insect meals: Hermetia illucens (HI), and Tenebrio molitor (TM). TM showed better results for growth, protein utilization and more active digestive function, supported by intestinal histological changes. Liver histology and intermediary metabolism did not show relevant changes between insect meals, while other parameters such as antioxidant enzyme activities and tissue damage indicators showed the potential of insect meals as functional ingredients.

Modulation of Atlantic salmon (Salmo salar) gut microbiota composition and predicted metabolic capacity by feeding diets with processed black soldier fly (Hermetia illucens) larvae meals and fractions.

BACKGROUND: Black soldier fly (Hermetia illucens) is a promising insect species to use as a novel ingredient in fish feeds. Black soldier fly larvae consists of three major fractions, namely protein, lipid, and exoskeleton. These fractions contain bioactive compounds that can modulate the gut microbiota in fish such as antimicrobial peptides, lauric acid, and chitin. However, it is not certain how, or which fractions of black solider fly would affect gut microbiota in fish. In the present study, black soldier fly larvae were processed into three different meals (full-fat, defatted and de-chitinized) and two fractions (oil and exoskeleton), and included in diets for Atlantic salmon (Salmo salar). Atlantic salmon pre-smolts were fed with these diets in comparison with a commercial-like control diet for eight weeks to investigate the effects of insect meals and fractions on the composition and predicted metabolic capacity of gut microbiota. The gut microbiota was profiled by 16S rRNA gene sequencing, and the predicted metabolic capacities of gut microbiota were determined using genome-scale metabolic models. RESULTS: The inclusion of insect meals and fractions decreased abundance of Proteobacteria and increased abundance of Firmicutes in salmon gut. The diets that contained insect chitin, i.e., insect meals or exoskeleton diets, increased abundance of chitinolytic bacteria including lactic acid bacteria and Actinomyces in salmon gut, with fish fed full-fat meal diet showing the highest abundances. The diets that contained insect lipids, i.e., insect meals and oil diets enriched Bacillaceae in fish gut. The fish fed diets containing full-fat insect meal had a unique gut microbiota composition dominated by beneficial lactic acid bacteria and Actinomyces, and showed a predicted increase in mucin degradation compared to the other diets. CONCLUSIONS: The present results showed that the dietary inclusion of insect meals and fractions can differently modulate the composition and predicted metabolic capacity of gut microbiota in Atlantic salmon pre-smolts. The use of full-fat black soldier fly larvae meal in diets for salmon is more favorable for beneficial modulation of gut microbiota than larvae processed by separation of lipid or exoskeleton fractions.

The Spleen as a Target to Characterize Immunomodulatory Effects of Down-Stream Processed Cyberlindnera jadinii Yeasts in Atlantic Salmon Exposed to a Dietary Soybean Meal Challenge.

Aquaculture feeds have changed dramatically from being largely based on fishmeal (FM) towards increased use of plant protein sources, which could impact the fish's immune response. In order to characterize immunomodulatory properties of novel functional ingredients, this study used four diets, one based on FM, a challenging diet with 40% soybean meal (SBM), and two diets containing 40% SBM with 5% of Cyberlindnera jadinii yeast exposed to different down-stream processing conditions: heat-inactivated (ICJ) or autolysation (ACJ). The immunomodulatory effects of the diets were analyzed in the spleen of Atlantic salmon after 37 days of feeding, using a transcriptomic evaluation by RNA sequencing (RNA-seq) and the detection of specific immunological markers at the protein level through indirect Enzyme-linked Immunosorbent Assay (indirect ELISA). The results showed that SBM (compared to FM) induced a down-regulation of pathways related to ion binding and transport, along with an increase at the protein level of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). On the other hand, while ICJ (compared to FM-group) maintain the inflammatory response associated with SBM, with higher levels of TNFα and IFNγ, and with an upregulation of creatine kinase activity and phosphagen metabolic process, the inclusion of ACJ was able to modulate the response of Atlantic salmon compared to fish fed the SBM-diet by the activation of biological pathways related to endocytosis, Pattern recognition receptor (PPRs)-signal transduction and transporter activity. In addition, ACJ was also able to control the pro-inflammatory profile of SBM, increasing Interleukin 10 (IL-10) levels and decreasing TNFα production, triggering an immune response similar to that of fish fed an FM-based diet. Finally, we suggest that the spleen is a good candidate to characterize the immunomodulatory effects of functional ingredients in Atlantic salmon. Moreover, the inclusion of ACJ in fish diets, with the ability to control inflammatory processes, could be considered in the formulation of sustainable salmon feed.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Dietary Inclusion of Black Soldier Fly (Hermetia Illucens) Larvae Meal and Paste Improved Gut Health but Had Minor Effects on Skin Mucus Proteome and Immune Response in Atlantic Salmon (Salmo Salar).

The present study investigated effects of dietary inclusion of black soldier fly larvae (BSFL) (Hermetia illucens) meal and paste on gut health, plasma biochemical parameters, immune response and skin mucus proteome in pre-smolt Atlantic salmon (Salmo salar). The seven-week experiment consisted of seven experimental diets: a control diet based on fishmeal and plant protein (Control-1); three BSFL meal diets, substituting 6.25% (6.25IM), 12.5% (12.5IM) and 25% (25IM) of protein; two BSFL paste diets, substituting 3.7% (3.7IP) and 6.7% (6.7IP) of protein and an extra control diet with 0.88% of formic acid (Control-2). The 6.25IM diet reduced enterocyte steatosis in pyloric caeca, improved distal intestine histology, and reduced IgM in distal intestine. The fish fed 12.5IM diet reduced enterocyte steatosis in pyloric caeca, improved distal intestine histology, had a higher plasma lysozyme content compared to 6.25IM, and tend to increase phagocytic activity in head-kidney macrophages-like cells. On the other hand, 25IM diet improved distal intestine histology, but showed mild-moderate enterocyte steatosis in pyloric caeca, increased IFNγ and reduced IgM in distal intestine. In the case of BSFL paste diets, 3.7IP diet caused mild inflammatory changes in distal intestine, although it reduced enterocyte steatosis in pyloric caeca. The 6.7IP diet reduced enterocyte steatosis in pyloric caeca and improved distal intestine histology. Increasing level of BSFL meal in the diet linearly decreased plasma C-reactive protein, whereas increasing level of BSFL paste linearly increased plasma antioxidant capacity. Dietary inclusion of BSFL meal and paste had minor effects on the expression profile of proteins in skin mucus and no effects on immune markers in splenocytes. BSFL meal showed no negative effect on liver and muscle health as indicated by plasma alanine aminotranseferase, asparate aminotransferase and creatine kinase. The present study showed that replacing conventional protein sources with low to moderate levels of BSFL meal (6.25% and 12.5%) or paste (3.7% and 6.7%) reduced enterocyte steatosis in pyloric caeca, while replacing up to 25% with BSFL meal or 6.7% with paste improved distal intestine histology. Further, dietary inclusion of BSFL meal and paste had minor effects on skin mucus proteome and immune response in Atlantic salmon.

Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar).

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

The Use of Extracellular Membrane Vesicles for Immunization against Francisellosis in Nile Tilapia (Oreochromis niloticus) and Atlantic Cod (Gadus morhua L.).

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Comparison of Circulating Markers and Mucosal Immune Parameters from Skin and Distal Intestine of Atlantic Salmon in Two Models of Acute Stress.

Ensuring salmon health and welfare is crucial to maximize production in recirculation aquaculture systems. Healthy and robust mucosal surfaces of the skin and intestine are essential to achieve this goal because they are the first immunological defenses and are constantly exposed to multistressor conditions, such as infectious diseases, suboptimal nutrition, and environmental and handling stress. In this work, Atlantic salmon, split from a single cohort, were subjected to acute hypoxia stress or 15-min crowding stress and observed over a 24-h recovery period. Samples were collected from fish at 0, 1, 3, 6, 12 and 24 h post-stress to analyze plasma-circulating markers of endocrine function (cortisol), oxidative stress (glutathione peroxidase) and immune function (interleukin 10 (IL-10), annexin A1). In addition, mucosal barrier function parameters were measured in the skin mucus (Muc-like protein and lysozyme) and distal intestine (simple folds, goblet cell size and goblet cell area). The results showed that both acute stress models induced increases of circulating cortisol in plasma (1 h post-stress), which then returned to baseline values (initial control) at 24 h post-stress. Moreover, the hypoxia stress was mostly related to increased oxidative stress and IL-10 production, whereas the crowding stress was associated with a higher production of Muc-like protein and lysozyme in the skin mucus. Interestingly, in the distal intestine, smaller goblet cells were detected immediately and one hour after post-hypoxia stress, which could be related to rapid release of the cellular content to protect this organ. Finally, the correlation of different markers in the hypoxic stress model showed that the circulating levels of cortisol and IL-10 were directly proportional, while the availability of Muc-like proteins was inversely proportional to the size of the goblet cells. On the other hand, in the crowding stress model, a proportional relationship was established between plasma cortisol levels and skin mucus lysozyme. Our results suggest key differences in energy partitioning between the two acute stress models and support the need for further investigation into the interplay of multistressor conditions and strategies to modulate immunological aspects of mucosal surfaces.

Microbiota-directed fibre activates both targeted and secondary metabolic shifts in the distal gut.

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. Here, we test the accuracy of AcGGM within the complex endogenous gut microbiome of pigs, wherein we resolve 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially express AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifests a "butterfly effect", whereby numerous metabolic changes and interdependent cross-feeding pathways occur in neighboring non-mannanolytic populations that produce short-chain fatty acids. Our findings show how intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large.

Cyberlindnera jadinii Yeast as a Protein Source for Weaned Piglets-Impact on Immune Response and Gut Microbiota.

Supplying novel feed ingredients for pig production is crucial to enhance food security and decrease the environmental impact of meat production. Several studies have focused on evaluating the beneficial health effects of yeast in pigs. However, its use as a protein source has been partially addressed. Previously, we have shown that yeast at high inclusion levels maintains growth performance and digestibility, while nutrient digestibility, intestinal villi height and fecal consistency were improved. The present study combined microbiome, short-chain fatty acid, and immune parameter analysis to investigate the effect of high inclusion of yeast in diets for post-weaning piglets. Our results showed that yeast did not have a significant impact on the hematological or biochemical parameters in blood. The different immune cell subpopulations isolated from blood and distal jejunal lymph nodes (DJLN) were analyzed by flow cytometry and showed that yeast diet induced an increased number of the subtype of leukocytes CD45+/CD3-/CD8+, a special type of Natural Killer (NK) cells. Also, a very mild to moderate infiltration of neutrophilic granulocytes and lower IgA level were observed in the colon of yeast fed piglets. The microbiome profiling in different compartments of the gastrointestinal tract of piglets was performed using 16S rRNA metabarcoding. The results showed that 40% replacement of dietary protein had a statistically significant effect on the microbial communities in cecum and colon, while the microbial population in ileum and jejunum were not affected. Analysis of predicted microbial metabolic pathways analysis revealed significant upregulation of short-chain fatty acids, ether lipid metabolisms, secondary bile acids, and several other important biosynthesis pathways in cecum and colon of pigs fed yeast. In conclusion, the results showed that diet containing 40% of yeast protein positively shaped microbial community in the large intestine and increased the number of a specific subpopulation of NK cells in the DJLN. These results showed that yeast modulates the microbiome and decreases the secretion of IgA in the colon of post-weaning pigs.

Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota.

The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex ß-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex ß-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on ß-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders Clostridiales and Bacilli, respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order Enterobacteriales. The most abundant proteins detected in the ß-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community; furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source.

Candida utilis yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Local intestinal tissue and plasma proteome responses.

Microbial ingredients such as Candida utilis yeast are known to be functional protein sources with immunomodulating effects whereas soybean meal causes soybean meal-induced enteritis in the distal intestine of Atlantic salmon (Salmo salar L.). Inflammatory or immunomodulatory stimuli at the local level in the intestine may alter the plasma proteome profile of Atlantic salmon. These deviations can be helpful indicators for fish health and, therefore potential tools in the diagnosis of fish diseases. The present work aimed to identify local intestinal tissue responses and changes in plasma protein profiles of Atlantic salmon fed inactive dry Candida utilis yeast biomass, soybean meal, or combination of soybean meal based diet with various inclusion levels of Candida utilis. A fishmeal based diet was used as control diet. Inclusion of Candida utilis yeast to a fishmeal based diet did not alter the morphology, immune cell population or gene expression of the distal intestine. Lower levels of Candida utilis combined with soybean meal modulated immune cell populations in the distal intestine and reduced the severity of soybean meal-induced enteritis, while higher inclusion levels of Candida utilis were less effective. Changes in the plasma proteomic profile revealed differences between the diets but did not indicate any specific proteins that could be a marker for health or disease. The results suggest that Candida utilis does not alter intestinal morphology or induce major changes in plasma proteome, and thus could be a high-quality alternative protein source with potential functional properties in diets for Atlantic salmon.

CpGs Induce Differentiation of Atlantic Salmon Mononuclear Phagocytes Into Cells With Dendritic Morphology and a Proinflammatory Transcriptional Profile but an Exhausted Allostimulatory Activity.

Due to their ability to present foreign antigens and prime naïve T cells, macrophages, and dendritic cells (DCs) are referred to as professional antigen-presenting cells (APCs). Although activated macrophages may function as APCs, these cells are particularly effective at directly engaging pathogens through phagocytosis, and production of antimicrobial compounds. On the other hand, DCs possess superb antigen-presenting and costimulatory capacity and they are essential for commencement and regulation of adaptive immune responses. In in vitro models, development of mature mammalian DCs from monocytes requires sequential exposure to growth factors (including GM-CSF and IL-4) and proinflammatory stimuli such as toll-like receptor (TLR) ligands. Currently, except for IL-4/13, neither orthologs nor functional analogs of the growth factors which are essential for the differentiation of mammalian DCs (including GM-CSF and FLT3) have been identified in teleosts and data about differentiation of piscine APCs is scant. In the present study, primary salmon mononuclear phagocytes (MPs) stimulated in vitro for 5-7 days with a B-class CpG oligodeoxynucleotides (ODN 2006PS) underwent morphological differentiation and developed "dendritic" morphology, characterized by long, branching pseudopodia. Transcriptional profiling showed that these cells expressed high levels of proinflammatory mediators characteristic for M1 polarized MPs. However, the cells treated with CpGs for 7 days downregulated their surface MHCII molecules as well as their capacity to endocytose ovalbumin and exhibited attenuated allostimulatory activity. This concurred with transcriptional downregulation of costimulatory CD80/86 and upregulation of inhibitory CD274 (B7-H1) genes. Despite their exhausted allostimulatory activity, these cells were still responsive to re-stimulation with gardiquimod (a TLR7/8 ligand) and further upregulated a wide array of immune genes including proinflammatory mediators such as intereukin-1 beta and tumor necrosis factor. Overall, the presented data highlight the disparate effects TLR ligands may have on the proinflammatory status of APCs, on one side, and their antigen-presenting/costimulatory functions, on the other. These findings also indicate that despite the poor phylogenetic conservation of the growth factors involved in the differentiation of DCs, some of the processes that orchestrate the development and the differentiation of professional APCs are conserved between teleosts in mammals.

Rainbow Trout (Oncorhynchus Mykiss) Intestinal Epithelial Cells as a Model for Studying Gut Immune Function and Effects of Functional Feed Ingredients.

The objective of this study was to evaluate the suitability of the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immune function and effects of functional feed ingredients. Effects of lipopolysaccharide (LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides (MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventional culture plates and transwell membranes. Permeation of fluorescently-labeled albumin, transepithelial electrical resistance (TEER), and tight junction protein expression confirmed the barrier function of the cells. Brush border membrane enzyme activities [leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cells but activity levels were not modulated by any of the exposures. Immune related genes were expressed at comparable relative basal levels as these in rainbow trout distal intestine. LPS produced markedly elevated gene expression levels of the pro-inflammatory cytokines il1b, il6, il8, and tnfa but had no effect on ROS production. Immunostaining demonstrated increased F-actin contents after LPS exposure. Among the functional feed ingredients, MOS seemed to be the most potent modulator of RTgutGC immune and barrier function. MOS significantly increased albumin permeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production, cell proliferation and myd88 expression. Induced levels of il1b and il8 were also observed after treatment with nucleotides and beta-glucans. For barrier function related genes, all treatments up-regulated the expression of cldn3 and suppressed cdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, the present study has provided new information on how functional ingredients commonly applied in aquafeeds can affect intestinal epithelial function in fish. Our findings suggest that RTgutGC cells possess characteristic features of functional intestinal epithelial cells indicating a potential for use as an efficient in vitro model to evaluate effects of bioactive feed ingredients on gut immune and barrier functions and their underlying cellular mechanisms.

The Use of Chitosan-Coated Membrane Vesicles for Immunization Against Salmonid Rickettsial Septicemia in an Adult Zebrafish Model.

The introduction of fish vaccination has had a tremendous impact on the aquaculture industry by providing an important measurement in regard to disease control. Infectious diseases caused by intracellular pathogens do, however, remain an unsolved problem for the industry. This is in many cases directly connected to the inability of vaccines to evoke a cellular immunity needed for long-term protection. Thus, there is a need for new and improved vaccines and adjuvants able to induce a strong humoral and cellular immune response. We have previously shown that membrane vesicles (MVs) from the intracellular fish pathogen Piscirickettsia salmonis are able to induce a protective response in adult zebrafish, but the incorporation of an adjuvant has not been evaluated. In this study, we report the use of chitosan as an adjuvant in combination with the P. salmonis-derived MVs for improved immunization against P. salmonis. Both free chitosan and chitosan-coated MVs (cMVs) were injected into adult zebrafish and their efficacy evaluated. The cMVs provided a significant protection (p < 0.05), while a small but nonsignificant reduction in mortalities was registered for fish injected with free chitosan. Both free chitosan and the cMVs were shown to induce an increased immune gene expression of CD 4, CD 8, MHC I, Mpeg1.1, TNFα, IL-1ß, IL-10, and IL-6, but to a higher degree in the cMV group. Taken together, the results indicate a potential use of chitosan-coated MVs for vaccination, and that zebrafish is a promising model for aquaculture-relevant studies.

Isolation and Characterization of Serum Extracellular Vesicles (EVs) from Atlantic Salmon Infected with Piscirickettsia Salmonis.

Secretion of extracellular vesicles (EVs) is a common feature of both eukaryotic and prokaryotic cells. Isolated EVs have been shown to contain different types of molecules, including proteins and nucleic acids, and are reported to be key players in intercellular communication. Little is known, however, of EV secretion in fish, or the effect of infection on EV release and content. In the present study, EVs were isolated from the serum of healthy and Piscirickettsia salmonis infected Atlantic salmon in order to evaluate the effect of infection on EV secretion. P. salmonis is facultative intracellular bacterium that causes a systemic infection disease in farmed salmonids. EVs isolated from both infected and non-infected fish had an average diameter of 230-300 nm, as confirmed by transmission electron microscopy, nanoparticle tracking, and flow cytometry. Mass spectrometry identified 180 proteins in serum EVs from both groups of fish. Interestingly, 35 unique proteins were identified in serum EVs isolated from the fish infected with P. salmonis. These unique proteins included proteasomes subunits, granulins, and major histocompatibility class I and II. Our results suggest that EV release could be part of a mechanism in which host stimulatory molecules are released from infected cells to promote an immune response.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.