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Diet composition, ingredient and nutrients, are important to consider for maintaining intestinal functions. Studies on both positive (using feed additives) and negative effects (using high inclusion of plant ingredients) of fish feeds are numerous, however, between studies results are often highly variable, both in type of response and in significance. The central hypothesis of this study was that adverse environmental conditions may aggravate negative effects of plant ingredients on the intestinal functions to the extent that mild effects become severe and perceptible. To do so, dietary factors and environmental conditions were evaluated and the interaction between diet composition and environmental conditions were studied in Nile tilapia. In Chapter 2, six common raw materials including hydrolysed feather meal (HFM), soybean meal (SBM), rice bran (RB), rapeseed meal (RM), sunflower meal (SFM) and dried distiller grains with solubles (DDGS) were chosen to determine the effect of nutrient digestibility, nitrogen/energy balance and changes in intestinal morphology. The study demonstrated that feed ingredients do have an impact on the alteration in intestinal parameters, but also on the nutrient digestibility and the nitrogen/energy balance. Although being well digested, soybean meal caused the most obvious alteration in the intestinal morphology. These alterations were not related to the nutrient digestibility nor to nitrogen/energy balance parameters. Soybean meal, causing the most alterations in the intestinal morphology, was further used in all subsequent chapters of this thesis. Chapter 3 and 4 described the interaction between diet composition and environmental conditions on the intestinal functions. This was studied with two different environmental conditions, dissolved oxygen (Chapter 3) and salinity (Chapter 4). These two chapters evaluated whether suboptimal environmental conditions (low dissolved oxygen or elevated salinity in water) may interact with a soybean meal based diet in nutrient digestion and intestinal morphology of tilapia. The study demonstrated that environmental stressors can aggravate/reveal the negative intestinal morphology changes induced by a soybean meal based diet. However, these effects of adverse environmental conditions on the intestinal functions were not homogenously dispersed over the whole intestinal length. The effect of salinity on the intestinal morphology occurred predominantly in the distal intestine, whereas the effect of low oxygen concentration was more visible at the proximal intestine. Alterations in the intestinal morphology, as found in this study, have wider effects on the performance of the affected fish. In Chapter 3, the protein digestibility decreased under hypoxic conditions at week 8, which parallels with the time related alteration in intestinal morphology. Chapter 4 showed that when fish were raised at 15 ‰ salinity, nutrient digestibility increased; however, this positive effect decreased when the intestinal morphology changed. The study also found that the combined effect of a soybean meal based diet and hypoxia was stronger compared to the combination with elevated salinity. Therefore, the combination with hypoxia was further used in the next study of this thesis. In Chapter 5, the combination of hypoxic condition and a soybean meal based diet was chosen to test the hypothesis that only under stressful conditions, the effects of feed additives can be noticed. The impact of two dietary organic acids, formic acid and butyric acid, on nutrient digestibility and intestinal morphology was determined under optimal (normoxia) and suboptimal conditions (hypoxia). The results showed that although organic acids did not significantly improve growth performance and nutrient digestibility under normoxic condition, they did so under hypoxic conditions. Fish fed organic acid supplemented diets all showed improvements in the morphology of intestine under normoxic conditions, and these effects were more enhanced under hypoxic conditions. This indicates that environmental conditions can alter the effect of organic acid on nutrient digestibility and intestinal morphology in tilapia. Finally, Chapter 6 provided a synthesis of the main findings, and a reflection on the methodologies used in Chapters 2-5 as well as a discussion on the relevance of this study to aquaculture. It is concluded that although being well digested, soybean meal caused the most obvious alteration in intestinal morphology. The adverse environmental conditions aggravated negative effects of soybean meal based diets on the intestinal functions to the extent that mild effects become severe and visible. The negative effect on intestinal morphology of soybean meal in the diet is enhanced at low oxygen level and at elevated salinity. The effect of salinity on the intestinal morphology occurs predominantly in the distal intestine, whereas the effect of low oxygen concentration is more visible at the proximal intestine. The thesis indicated that the impact of organic acids on intestinal functions is dependent on environmental conditions, being more pronounced under challenging conditions (e.g. hypoxia). Therefore, studies on both positive (using feed additives) and negative effects (using high inclusion of plant ingredients) of dietary factors should be done under suboptimal conditions.

The alarming increase in obesity rates creates an urgent need for effective prevention and treatment strategies. The most effective treatment for obesity today is bariatric surgery. Bariatric surgery comprises a number of different procedures having in common that they induce weight loss and alter gut hormone release. Gut hormones are well known for their effects on food intake behavior and their role in weight loss after bariatric surgery is undeniable. In addition, the therapeutic use of GLP-1 (Glucagon-Like Peptide-1) analogues including liraglutide in type II diabetes and obesity is on the rise. This underlines why gut hormones are considered promising targets for the development of new treatment strategies against obesity and its comorbidities. The secretion of gut hormones, among which GLP-1, is influenced by nutrient ingestion. The interactions of dietary components or their breakdown products with receptors and transporters located on the enteroendocrine cells of the intestinal tract can induce their release, a process called intestinal nutrient sensing. In this thesis, we aimed to further elucidate intestinal nutrient sensing mechanisms on a cellular level. First, the regional expression of several gut nutrient sensing related genes along the intestinal tract was assessed in three commonly studied species, namely mouse, pig and man. Gene expression of receptors, transporters and peptides involved in nutrient sensing shows a distinctive distribution pattern along the small intestine, which is in the distal small intestine highly similar between the species. Subsequently, we sought to investigate if this expression was changed after a weight loss inducing bariatric procedure. By whole transcriptome analysis, we showed that upper gastrointestinal tissue expression of genes associated with nutrient sensing was hardly changed. In contrast, a considerable reduction in inflammatory pathways was observed. Next, we sought to investigate the effects of the non-caloric sweetener rebaudioside A. This Stevia rebaudiana-derived compound was approved on the European market in 2011. As there is still some controversy about the effects of sweeteners in general on GLP-1 release, we investigated the effects of this specific sweetener. Because of the short half-life of GLP-1, the effect of nutrient stimulation was mainly studied in ex vivo and in vitro models in which local intestinal hormone release could be determined. A two dimensional gut model using intestinal organoids derived from murine intestinal crypts was developed to study location-specific hormone secretion. Rebaudioside A was found to induce GLP-1 and PYY release ex vivo from porcine intestinal tissue and in two dimensional organoids. This induction of the release was specific for the intestinal location, with the ileum being most potently stimulated by rebaudioside A. Moreover, prolonged exposure to rebaudioside A increased enteroendocrine cell numbers in two dimensional organoids. When studying the underlying mechanism in enteroendocrine STC-1 cells, we concluded that rebaudioside A-induced GLP-1 release was independent of the sweet taste receptor. The studies presented in this thesis add to our understanding the role of receptors and other molecular structures that are likely to be involved in nutrient sensing and the modulation of gut hormone release. What we know now is that several factors play a role in gut hormone release. This includes not only the nature and dose of the active compound(s), but also the location and timing of its (their) interactions with receptors and other targets along the gastrointestinal tract. We have shown that rebaudioside A may be a potential compound to induce gut hormone release in vivo, especially when applied to the distal small intestine. Therefore, rebaudioside A may be a promising compound to influence food intake, possibly most potent when delivered in the ileum.

The alarming increase in obesity rates creates an urgent need for effective prevention and treatment strategies. The most effective treatment for obesity today is bariatric surgery. Bariatric surgery comprises a number of different procedures having in common that they induce weight loss and alter gut hormone release. Gut hormones are well known for their effects on food intake behavior and their role in weight loss after bariatric surgery is undeniable. In addition, the therapeutic use of GLP-1 (Glucagon-Like Peptide-1) analogues including liraglutide in type II diabetes and obesity is on the rise. This underlines why gut hormones are considered promising targets for the development of new treatment strategies against obesity and its comorbidities. The secretion of gut hormones, among which GLP-1, is influenced by nutrient ingestion. The interactions of dietary components or their breakdown products with receptors and transporters located on the enteroendocrine cells of the intestinal tract can induce their release, a process called intestinal nutrient sensing. In this thesis, we aimed to further elucidate intestinal nutrient sensing mechanisms on a cellular level. First, the regional expression of several gut nutrient sensing related genes along the intestinal tract was assessed in three commonly studied species, namely mouse, pig and man. Gene expression of receptors, transporters and peptides involved in nutrient sensing shows a distinctive distribution pattern along the small intestine, which is in the distal small intestine highly similar between the species. Subsequently, we sought to investigate if this expression was changed after a weight loss inducing bariatric procedure. By whole transcriptome analysis, we showed that upper gastrointestinal tissue expression of genes associated with nutrient sensing was hardly changed. In contrast, a considerable reduction in inflammatory pathways was observed. Next, we sought to investigate the effects of the non-caloric sweetener rebaudioside A. This Stevia rebaudiana-derived compound was approved on the European market in 2011. As there is still some controversy about the effects of sweeteners in general on GLP-1 release, we investigated the effects of this specific sweetener. Because of the short half-life of GLP-1, the effect of nutrient stimulation was mainly studied in ex vivo and in vitro models in which local intestinal hormone release could be determined. A two dimensional gut model using intestinal organoids derived from murine intestinal crypts was developed to study location-specific hormone secretion. Rebaudioside A was found to induce GLP-1 and PYY release ex vivo from porcine intestinal tissue and in two dimensional organoids. This induction of the release was specific for the intestinal location, with the ileum being most potently stimulated by rebaudioside A. Moreover, prolonged exposure to rebaudioside A increased enteroendocrine cell numbers in two dimensional organoids. When studying the underlying mechanism in enteroendocrine STC-1 cells, we concluded that rebaudioside A-induced GLP-1 release was independent of the sweet taste receptor. The studies presented in this thesis add to our understanding the role of receptors and other molecular structures that are likely to be involved in nutrient sensing and the modulation of gut hormone release. What we know now is that several factors play a role in gut hormone release. This includes not only the nature and dose of the active compound(s), but also the location and timing of its (their) interactions with receptors and other targets along the gastrointestinal tract. We have shown that rebaudioside A may be a potential compound to induce gut hormone release in vivo, especially when applied to the distal small intestine. Therefore, rebaudioside A may be a promising compound to influence food intake, possibly most potent when delivered in the ileum.

The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth. Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.

The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth. Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.

Aquaculture has realized considerable growth over the past years while the world demand on seafood has been increasing. As aquaculture intensifies, the production sector needs to tackle major bottlenecks such as suboptimal growth and high and unpredictable mortality, especially in larval cultures. Fish-microbe interactions are closely related to overall fish health. To obtain a healthy and resilient microbial community (MC), it is important to understand the underlying mechanisms of microbial colonization in the fish gut. The goal of this thesis was to investigate the role of water and feed microbial communities on shaping gut communities during early development of Nile tilapia. To determine the contribution of stochasticity to overall variation, we first characterized the spatio-temporal variation in MC composition between individuals reared within the same or in replicate recirculating or active suspension systems (RAS vs. AS). Highly similar MCs developed in the gut when larvae shared the same water and diet. Rearing larvae in replicate production systems resulted in significantly different gut communities indicating that compositional replication of the MCs of an ecosystem is not fully predictable. We found that mainly water MCs, and to a lesser degree feed MCs, were associated with changes in MCs. Thus, we could conclude that steering gut MCs can be possible through water MC management tailored on the specifications of the rearing system in use. Next, the possibility of early life steering of gut communities via microbial manipulations of feed MCs was explored. We hypothesized that gut microbial composition is strongly shaped by selective pressures in the gut and by the MCs present in the water. Thus similar MCs should develop between treatments regardless of the dietary treatments. Fish larvae were fed either a control feed or the control feed containing MCs derived from aerobic, methanogenic or denitrifying sludge reactors. We found that gut microbiota shared a much higher number of operational taxonomic units (OTUs) with microbiota in sludge-based feeds than with water, resulting in distinct gut MCs between treatments. Our findings suggest that Nile tilapia gut MC has a certain plasticity, which makes it amenable to interventions through proper feed microbial management. Subsequently, we tested the imprinting effect of early exposure to the probiotic Bacillus subtilis on shaping gut MC composition even after the administration of the probiotic discontinues. For this, we constrained the initial contact with microbes from the environment by producing axenic tilapia larvae, which were then exposed to normal husbandry conditions. Early life probiotic exposure affected gut MC composition during B. subtilis administration but also within the first two weeks after its administration stopped, thus indicating that early exposure to the probiotic strain via the water had a sustained impact on gut MC composition. Finally, overall conclusions and practical implications of our results for aquaculture production were presented. A meta-analysis was also performed to examine (1) the phylogenetic similarity among gut MCs of the same and different fish species reared in different habitats, fed different diets and at different developmental stages and (2) the factors primarily shaping gut MCs. We showed that the selective pressure responsible in shaping gut MC composition highly depends on the host as gut communities clustered primarily together by host and to a lesser extent reflected differences in habitat and diet. The phylogenetic analysis of gut communities revealed a clear clustering by study thus indicating that manipulation of gut communities is conceivable. Study-to-study variation could be attributed to the methodology used for MC analysis highlighting also the importance of methodological uniformity when comparisons between studies are made. Overall, this thesis provided fundamental knowledge on MC composition and development in aquaculture rearing systems. Although the insights generated by this thesis are still premature to fully explain, predict or steer MC composition, and though additional studies are needed, we believe that, in the long run, this approach will facilitate the development of safe and effective methods for manipulating gut microbial composition to promote fish health in aquaculture rearing systems.

Aquaculture has realized considerable growth over the past years while the world demand on seafood has been increasing. As aquaculture intensifies, the production sector needs to tackle major bottlenecks such as suboptimal growth and high and unpredictable mortality, especially in larval cultures. Fish-microbe interactions are closely related to overall fish health. To obtain a healthy and resilient microbial community (MC), it is important to understand the underlying mechanisms of microbial colonization in the fish gut. The goal of this thesis was to investigate the role of water and feed microbial communities on shaping gut communities during early development of Nile tilapia. To determine the contribution of stochasticity to overall variation, we first characterized the spatio-temporal variation in MC composition between individuals reared within the same or in replicate recirculating or active suspension systems (RAS vs. AS). Highly similar MCs developed in the gut when larvae shared the same water and diet. Rearing larvae in replicate production systems resulted in significantly different gut communities indicating that compositional replication of the MCs of an ecosystem is not fully predictable. We found that mainly water MCs, and to a lesser degree feed MCs, were associated with changes in MCs. Thus, we could conclude that steering gut MCs can be possible through water MC management tailored on the specifications of the rearing system in use. Next, the possibility of early life steering of gut communities via microbial manipulations of feed MCs was explored. We hypothesized that gut microbial composition is strongly shaped by selective pressures in the gut and by the MCs present in the water. Thus similar MCs should develop between treatments regardless of the dietary treatments. Fish larvae were fed either a control feed or the control feed containing MCs derived from aerobic, methanogenic or denitrifying sludge reactors. We found that gut microbiota shared a much higher number of operational taxonomic units (OTUs) with microbiota in sludge-based feeds than with water, resulting in distinct gut MCs between treatments. Our findings suggest that Nile tilapia gut MC has a certain plasticity, which makes it amenable to interventions through proper feed microbial management. Subsequently, we tested the imprinting effect of early exposure to the probiotic Bacillus subtilis on shaping gut MC composition even after the administration of the probiotic discontinues. For this, we constrained the initial contact with microbes from the environment by producing axenic tilapia larvae, which were then exposed to normal husbandry conditions. Early life probiotic exposure affected gut MC composition during B. subtilis administration but also within the first two weeks after its administration stopped, thus indicating that early exposure to the probiotic strain via the water had a sustained impact on gut MC composition. Finally, overall conclusions and practical implications of our results for aquaculture production were presented. A meta-analysis was also performed to examine (1) the phylogenetic similarity among gut MCs of the same and different fish species reared in different habitats, fed different diets and at different developmental stages and (2) the factors primarily shaping gut MCs. We showed that the selective pressure responsible in shaping gut MC composition highly depends on the host as gut communities clustered primarily together by host and to a lesser extent reflected differences in habitat and diet. The phylogenetic analysis of gut communities revealed a clear clustering by study thus indicating that manipulation of gut communities is conceivable. Study-to-study variation could be attributed to the methodology used for MC analysis highlighting also the importance of methodological uniformity when comparisons between studies are made. Overall, this thesis provided fundamental knowledge on MC composition and development in aquaculture rearing systems. Although the insights generated by this thesis are still premature to fully explain, predict or steer MC composition, and though additional studies are needed, we believe that, in the long run, this approach will facilitate the development of safe and effective methods for manipulating gut microbial composition to promote fish health in aquaculture rearing systems.

Het onderzoeksprogramma Feed4Foodure werkt aan een meetlat om te voorspellen wat het vermogen is van een dier om adequaat te reageren als het wordt blootgesteld aan ziekteverwekkers. Dit wordt de immuuncompetentie van het dier genoemd. Deze immuuncompetentie is te beïnvloeden door onder andere diervoeding en voederadditieven, vertelt onderzoeker Mari Smits van Wageningen UR.

This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos. Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier. We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements. The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.

Gut microbial colonization and immune competence development are affected by early-life environmental and dietary interventions. The interplay between microbiota in the intestinal tract and the gut mucosal surfaces of the host is critical for the development of an accurate immune competence. In the present study we intervened during early life of suckling piglets by a daily oral administration of fructooligosaccharides (FOS solution) from day 2 – 14 and investigated the effects on intestinal microbiota composition (by 16S rDNA sequencing) and biological processes of the intestinal mucosal tissue (by genome-wide intestinal gene expression analysis) during the suckling phase.

'We zijn nog niet zo ver dat we nu al kunnen zeggen wat iemand met een bepaald microbiootprofiel van de darmen het beste kan eten, maar dit zal in de toekomst wel mogelijk zijn', zegt prof. dr. Willem de Vos. Hij doet samen met prof. dr. Ellen Blaak onderzoek om duidelijkheid te krijgen over de ideale samenstelling van microbiota zodat iemand gezond en slank wordt.

Summary Silver nanoparticles (Ag NPs) are among the most promising groups of NPs (particles with all dimensions below 100 nm) for application in numerous consumer products due to their broad spectrum antimicrobial activities. Examples are incorporation in textiles and plastics, personal care products, water filters, food supplements etc. The extensive application and use together with the not yet fully understood properties of Ag NPs as well as the toxicity of Ag itself has raised concerns on potential impact of Ag NPs on human and environmental health. The research conducted within this thesis aimed at the evaluation of potential hazards of Ag NPs and identification of some key factors that determine the toxicity of Ag NPs. A tiered approach was employed using a battery of standard bioassays with model aquatic organisms, followed by the determination of sub-lethal concentrations for mechanistic endpoints, the identification of target tissues and organisms for Ag NP exposure and uptake and the integration of a proteomic tool to identify subtle changes. One of the main uptake routes for Ag NPs is through ingestion making the gastrointestinal epithelium one of the first ports of potential NP uptake and cell-particle interactions. An in vitro co-culture model incorporating a mucus layer mimicking the gastrointestinal epithelium was established for a more realistic evaluation of Ag NP potential toxicity than using intestinal epithelial cells alone. Indeed, the absence of mucus resulted in an overestimation of Ag NP toxicity. To be able to elucidate subtle changes in cellular functions and identification of particle specific effects and NP modes of action, a proteomic approach was employed. Differences and commonalities were observed between the cellular responses induced Ag NPs of different sizes and AgNO3 as a source of free Ag ions. As the Ag NPs are expected to reach the aquatic environment, a combination of adapted standard ecotoxicity assays with organisms of different trophic levels were used to evaluate the toxic effects Ag NPs. Synthetically produced Ag NPs of different sizes (Ag 20 and 200 nm) as well as Ag NPs synthesized by a biological method (using plant leaf extracts of Ocinum sanctum and Azadirachta indica, Ag 23 and 27 nm, respectively) were used as model particles in order to elucidate the relation between size, synthesis method, NP surface properties, ion dissolution and toxicity. Based on earlier indications of interference of another type of NPs with the multi xenobiotic resistance mechanism (MXR), a first line of defense against xenobiotics, the effects of Ag NPs on the MXR mechanism were studied as well. The MXR mechanism is present in all animals, including humans and aquatic organisms. MXR can be compromised by chemical agents that are structurally and chemically unrelated, and interference with MXR could be the basis for enhanced toxicity by contaminant mixtures. A fast in vitro cellular efflux pump inhibition assay (CEPIA) was established evaluating first the effects of contaminants commonly found in the environment. Next, an in vivo CEPIA assay was established using the juvenile D. magna model aquatic organism and the potential of the MXR modulation by Ag NPs and ionic Ag was quantified in vitro and in vivo. This integrated approach revealed that the size and the synthesis method are the factors affecting most the uptake and toxicity in both cells in vitro as well as in vivo in freshwater crustaceans (daphnids) and dissolution in the different media with the biologically synthesized Ag NPs being more potent compared to the conventional Ag NPs. The gastrointestinal tract is expected to be a target site for Ag NPs exposure and a co-culture of Caco-2-TC7 and HT29-MTX cells optimized and employed in the current study represents a more realistic model compared to Caco-2 monocultures. The mucus layer provides an additional protective barrier and its absence can lead to overestimation of effects in in vitro studies. Ag was detected both in the cells in co-culture, in the gut of daphnids’ as well as specific areas, seemingly developing oocytes, indicating a potential translocation of Ag NPs that could have consequences for fecundity. MXR efflux transporters were found to be modulated by Ag at low concentrations (0.18 µg/L), that are slightly lower compared to the predicted environmental concentrations. The extent to which the Ag ions contribute to the effects of Ag NPs depends on the size and surface properties of the Ag NPs. For the conventional, uncoated Ag NPs, the Ag release is minimal and the size is the determining factor while for biologically synthesized particles the biomolecules present due to the synthesis method and Ag release affect most the uptake and effects. The simultaneous presence of Ag ions and NPs releasing ions can lead to an exacerbation of the effects. The proteomic approach was successfully applied and it proved to be a useful technique in discerning subtle cellular changes in response to Ag NP exposure that would otherwise be unnoticed. Ag NPs 20 nm regulated different sets of proteins with a distinct pattern of cellular responses compared to Ag 200 nm and AgNO3, suggesting a different mode of action with effects being particle- and size-dependent. These results obtained during this thesis are promising for future toxicity testing of new materials using invertebrate organisms and more realistic in vitro models leading to more meaningful results and more accurate assessment of Ag NP hazards.

Summary Silver nanoparticles (Ag NPs) are among the most promising groups of NPs (particles with all dimensions below 100 nm) for application in numerous consumer products due to their broad spectrum antimicrobial activities. Examples are incorporation in textiles and plastics, personal care products, water filters, food supplements etc. The extensive application and use together with the not yet fully understood properties of Ag NPs as well as the toxicity of Ag itself has raised concerns on potential impact of Ag NPs on human and environmental health. The research conducted within this thesis aimed at the evaluation of potential hazards of Ag NPs and identification of some key factors that determine the toxicity of Ag NPs. A tiered approach was employed using a battery of standard bioassays with model aquatic organisms, followed by the determination of sub-lethal concentrations for mechanistic endpoints, the identification of target tissues and organisms for Ag NP exposure and uptake and the integration of a proteomic tool to identify subtle changes. One of the main uptake routes for Ag NPs is through ingestion making the gastrointestinal epithelium one of the first ports of potential NP uptake and cell-particle interactions. An in vitro co-culture model incorporating a mucus layer mimicking the gastrointestinal epithelium was established for a more realistic evaluation of Ag NP potential toxicity than using intestinal epithelial cells alone. Indeed, the absence of mucus resulted in an overestimation of Ag NP toxicity. To be able to elucidate subtle changes in cellular functions and identification of particle specific effects and NP modes of action, a proteomic approach was employed. Differences and commonalities were observed between the cellular responses induced Ag NPs of different sizes and AgNO3 as a source of free Ag ions. As the Ag NPs are expected to reach the aquatic environment, a combination of adapted standard ecotoxicity assays with organisms of different trophic levels were used to evaluate the toxic effects Ag NPs. Synthetically produced Ag NPs of different sizes (Ag 20 and 200 nm) as well as Ag NPs synthesized by a biological method (using plant leaf extracts of Ocinum sanctum and Azadirachta indica, Ag 23 and 27 nm, respectively) were used as model particles in order to elucidate the relation between size, synthesis method, NP surface properties, ion dissolution and toxicity. Based on earlier indications of interference of another type of NPs with the multi xenobiotic resistance mechanism (MXR), a first line of defense against xenobiotics, the effects of Ag NPs on the MXR mechanism were studied as well. The MXR mechanism is present in all animals, including humans and aquatic organisms. MXR can be compromised by chemical agents that are structurally and chemically unrelated, and interference with MXR could be the basis for enhanced toxicity by contaminant mixtures. A fast in vitro cellular efflux pump inhibition assay (CEPIA) was established evaluating first the effects of contaminants commonly found in the environment. Next, an in vivo CEPIA assay was established using the juvenile D. magna model aquatic organism and the potential of the MXR modulation by Ag NPs and ionic Ag was quantified in vitro and in vivo. This integrated approach revealed that the size and the synthesis method are the factors affecting most the uptake and toxicity in both cells in vitro as well as in vivo in freshwater crustaceans (daphnids) and dissolution in the different media with the biologically synthesized Ag NPs being more potent compared to the conventional Ag NPs. The gastrointestinal tract is expected to be a target site for Ag NPs exposure and a co-culture of Caco-2-TC7 and HT29-MTX cells optimized and employed in the current study represents a more realistic model compared to Caco-2 monocultures. The mucus layer provides an additional protective barrier and its absence can lead to overestimation of effects in in vitro studies. Ag was detected both in the cells in co-culture, in the gut of daphnids’ as well as specific areas, seemingly developing oocytes, indicating a potential translocation of Ag NPs that could have consequences for fecundity. MXR efflux transporters were found to be modulated by Ag at low concentrations (0.18 µg/L), that are slightly lower compared to the predicted environmental concentrations. The extent to which the Ag ions contribute to the effects of Ag NPs depends on the size and surface properties of the Ag NPs. For the conventional, uncoated Ag NPs, the Ag release is minimal and the size is the determining factor while for biologically synthesized particles the biomolecules present due to the synthesis method and Ag release affect most the uptake and effects. The simultaneous presence of Ag ions and NPs releasing ions can lead to an exacerbation of the effects. The proteomic approach was successfully applied and it proved to be a useful technique in discerning subtle cellular changes in response to Ag NP exposure that would otherwise be unnoticed. Ag NPs 20 nm regulated different sets of proteins with a distinct pattern of cellular responses compared to Ag 200 nm and AgNO3, suggesting a different mode of action with effects being particle- and size-dependent. These results obtained during this thesis are promising for future toxicity testing of new materials using invertebrate organisms and more realistic in vitro models leading to more meaningful results and more accurate assessment of Ag NP hazards.

This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos. Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier. We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements. The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.

'We zijn nog niet zo ver dat we nu al kunnen zeggen wat iemand met een bepaald microbiootprofiel van de darmen het beste kan eten, maar dit zal in de toekomst wel mogelijk zijn', zegt prof. dr. Willem de Vos. Hij doet samen met prof. dr. Ellen Blaak onderzoek om duidelijkheid te krijgen over de ideale samenstelling van microbiota zodat iemand gezond en slank wordt.

Gut microbial colonization and immune competence development are affected by early-life environmental and dietary interventions. The interplay between microbiota in the intestinal tract and the gut mucosal surfaces of the host is critical for the development of an accurate immune competence. In the present study we intervened during early life of suckling piglets by a daily oral administration of fructooligosaccharides (FOS solution) from day 2 – 14 and investigated the effects on intestinal microbiota composition (by 16S rDNA sequencing) and biological processes of the intestinal mucosal tissue (by genome-wide intestinal gene expression analysis) during the suckling phase.

In dit artikel wordt de relatie weergegeven tussen voedingsfactoren en diverse stoornissen in het darmstelsel. Het betreft een bewerking van een artikel over Nutrition and gut health of turkeys van Dr. Ferket.

Mammalian RegIII proteins are expressed in the intestine and in the pancreas in response to inflammation or infection. In the mouse intestine, expression of RegIIIβ and RegIIIγ is increased by microbial colonization, inflammation and infection. At the outset of this thesis human PAP and mouse RegIIIγ were reported to be bactericidal for Gram-positive bacteria. Additionally, human PAP had been shown to attenuate NF-κbsignallingin human monocytes and epithelial cells and administration of anti-PAP antibodies increased inflammation in an experimental rat model of acute pancreatitis. The overarching goals of this thesis were to find out more about the protective role of mouse RegIIIβ and RegIIIγ in the intestine and explore their protective role in colitis and bacterial infection. In Chapter 2 we investigated expression of RegIIIβ and RegIIIγ in intestine of Muc2 knockout (-/-) mice, which develop colitis after about 4 weeks, due to the absence of a secreted mucus layer in the small intestine or colon. RegIII proteins were expressed in Paneth cells, enterocytes and goblet cells pointing to a new function for goblet cells in innate immunity. Ang4 expression was confined to Paneth cells and goblet cells. Absence of Muc2 increased expression levels of RegIIIβ, RegIIIγ, and Ang4 and colitis appeared first in the distal colon where the RegIII expression is lowest. In Chapter 3 we investigated the distinct phases of colitis development in Muc2-/- mice from before weaning to 4 and 8 weeks of age, also taking into account the effect that mucin deficiency has in the ileum. Gene set enrichment approaches showed increased expression of innate and adaptive immune pathways associated with colitis over time, whereas in the ileum many immune signalling pathways were down-regulated. Nevertheless, RegIIIβ and RegIIIγ were significantly upregulated, suggesting their proposed antimicrobial and/or anti-inflammatory activities might be related to the suppression of immune pathways and avoidance of immune-mediated damage. Furthermore, we showed that RegIIIβ could specifically bind to mucin and fucosylated glycans in vitro, which may serve to inhibit bacterial binding to membrane bound mucins on the epithelium, and also enable RegIIIβ to be retained in the secreted mucin. An in vitro approach was used in Chapter 4, where we investigated the activities of RegIIIγ and RegIIIβby expressing and purifying recombinant proteins. Both proteins were insoluble when expressed in E. coli but RegIIIβ could be expressed and secreted in baculovirus as a soluble protein. As previous work reported that RegIII proteins were bactericidal even when produced as inclusion bodies in E. coli and refolded, we followed similar procedures to obtain soluble RegIII proteins. In our hands both the E. coli and baculovirus produced proteins bound strongly to both Gram-positive and Gram-negative bacteria after processing of an N-terminal pro-peptide by trypsin, but lacked any appreciable bactericidal activity. Furthermore these proteins did not influence the growth of Salmonella enteritidis andListeria monocytogenes. Attempts to crystallize the proteins were unsuccessful but structural models of the protein were generated based on the crystal structure of human PAP. These models were used to dock known ligands of RegIIIγ or RegIIIβ. Only one ligand is known for RegIIIγ, which is peptidoglycan, but for RegIIIβ the ligands include peptidoglycan, lipid A and the fucose-containing glycans identified in chapter 3. RegIIIβ was predicted to have two different binding sites which would allow it to bind to mucins and bacteria simultaneously, thereby preventing penetrating of the mucus. In Chapter 5 a RegIIIβ-/- mouse was used to study the role of the protein during infection with Gram-negative Salmonella enteritidis or Gram-positive Listeria monocytogenes. Whereas recovery of S. enteritidis orL. monocytogenes from faeces was similar in RegIIIβ-/- and wild type (WT) mice, significantly higher numbers of viable S. enteritidis, but not L. monocytogenes, were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the RegIIIβ-/- than the WT mice. The results suggest that mouse RegIIIβ plays a protective role against intestinal translocation of the Gram-negative bacterium S. enteritidis but not against the Gram-positive bacterium L. monocytogenes. In Chapter 6, the generation of a RegIIIγ-/- mouse is described. One of the main phenotypic differences between the RegIIIγ-/- and WT was an altered distribution of the ileal mucus and increased bacterial contact with the epithelium. Additionally, measurement of innate immune markers in the mucosa suggested heightened inflammation in the RegIIIγ-/- mice. Compared to WT mice, RegIIIγ-/- mice infected with S. enteritidis and L. monocytogenes showed an increase of mucosal inflammatory markers indicating protective, anti-microbial roles of RegIIIγ in defense against both Gram-positive and Gram-negative bacteria. Chapter 7summarizes and discusses the key results of the thesis in the context of the wider literature and possible directions for future research

Mammalian RegIII proteins are expressed in the intestine and in the pancreas in response to inflammation or infection. In the mouse intestine, expression of RegIIIβ and RegIIIγ is increased by microbial colonization, inflammation and infection. At the outset of this thesis human PAP and mouse RegIIIγ were reported to be bactericidal for Gram-positive bacteria. Additionally, human PAP had been shown to attenuate NF-κbsignallingin human monocytes and epithelial cells and administration of anti-PAP antibodies increased inflammation in an experimental rat model of acute pancreatitis. The overarching goals of this thesis were to find out more about the protective role of mouse RegIIIβ and RegIIIγ in the intestine and explore their protective role in colitis and bacterial infection. In Chapter 2 we investigated expression of RegIIIβ and RegIIIγ in intestine of Muc2 knockout (-/-) mice, which develop colitis after about 4 weeks, due to the absence of a secreted mucus layer in the small intestine or colon. RegIII proteins were expressed in Paneth cells, enterocytes and goblet cells pointing to a new function for goblet cells in innate immunity. Ang4 expression was confined to Paneth cells and goblet cells. Absence of Muc2 increased expression levels of RegIIIβ, RegIIIγ, and Ang4 and colitis appeared first in the distal colon where the RegIII expression is lowest. In Chapter 3 we investigated the distinct phases of colitis development in Muc2-/- mice from before weaning to 4 and 8 weeks of age, also taking into account the effect that mucin deficiency has in the ileum. Gene set enrichment approaches showed increased expression of innate and adaptive immune pathways associated with colitis over time, whereas in the ileum many immune signalling pathways were down-regulated. Nevertheless, RegIIIβ and RegIIIγ were significantly upregulated, suggesting their proposed antimicrobial and/or anti-inflammatory activities might be related to the suppression of immune pathways and avoidance of immune-mediated damage. Furthermore, we showed that RegIIIβ could specifically bind to mucin and fucosylated glycans in vitro, which may serve to inhibit bacterial binding to membrane bound mucins on the epithelium, and also enable RegIIIβ to be retained in the secreted mucin. An in vitro approach was used in Chapter 4, where we investigated the activities of RegIIIγ and RegIIIβby expressing and purifying recombinant proteins. Both proteins were insoluble when expressed in E. coli but RegIIIβ could be expressed and secreted in baculovirus as a soluble protein. As previous work reported that RegIII proteins were bactericidal even when produced as inclusion bodies in E. coli and refolded, we followed similar procedures to obtain soluble RegIII proteins. In our hands both the E. coli and baculovirus produced proteins bound strongly to both Gram-positive and Gram-negative bacteria after processing of an N-terminal pro-peptide by trypsin, but lacked any appreciable bactericidal activity. Furthermore these proteins did not influence the growth of Salmonella enteritidis andListeria monocytogenes. Attempts to crystallize the proteins were unsuccessful but structural models of the protein were generated based on the crystal structure of human PAP. These models were used to dock known ligands of RegIIIγ or RegIIIβ. Only one ligand is known for RegIIIγ, which is peptidoglycan, but for RegIIIβ the ligands include peptidoglycan, lipid A and the fucose-containing glycans identified in chapter 3. RegIIIβ was predicted to have two different binding sites which would allow it to bind to mucins and bacteria simultaneously, thereby preventing penetrating of the mucus. In Chapter 5 a RegIIIβ-/- mouse was used to study the role of the protein during infection with Gram-negative Salmonella enteritidis or Gram-positive Listeria monocytogenes. Whereas recovery of S. enteritidis orL. monocytogenes from faeces was similar in RegIIIβ-/- and wild type (WT) mice, significantly higher numbers of viable S. enteritidis, but not L. monocytogenes, were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the RegIIIβ-/- than the WT mice. The results suggest that mouse RegIIIβ plays a protective role against intestinal translocation of the Gram-negative bacterium S. enteritidis but not against the Gram-positive bacterium L. monocytogenes. In Chapter 6, the generation of a RegIIIγ-/- mouse is described. One of the main phenotypic differences between the RegIIIγ-/- and WT was an altered distribution of the ileal mucus and increased bacterial contact with the epithelium. Additionally, measurement of innate immune markers in the mucosa suggested heightened inflammation in the RegIIIγ-/- mice. Compared to WT mice, RegIIIγ-/- mice infected with S. enteritidis and L. monocytogenes showed an increase of mucosal inflammatory markers indicating protective, anti-microbial roles of RegIIIγ in defense against both Gram-positive and Gram-negative bacteria. Chapter 7summarizes and discusses the key results of the thesis in the context of the wider literature and possible directions for future research

Intestinal health is an important condition for sustainable animal production. Since it is known that there is significant variation in intestinal health and functionality, there is much to gain in this respect. However, to fully exploit the biological potential of the animal’s gastro-intestinal tract, the mechanism and regulation of major intestinal processes need to be unraveled first. In addition, identification of key components and processes involved in intestinal adaptation mechanisms may help to identify internal and external factors that influence the health and functioning of the gut. Improved knowledge in this area may contribute in defining rational strategies to improve sustainable animal production. Traditionally research used reductionist approaches and focused on specific components or isolated processes related to intestinal functioning. However, the recent developments in the areas of genomics and computational sciences provide tools and methods that allow studying the system of the gut as a whole. In this thesis we have set first steps in the use of such Systems Biology approaches towards the identification of the key components and processes involved in intestinal functioning and health. We investigated molecular processes associated with gut development in chickens under two extreme contrasting conditions. We used an infection with Salmonella immediately after hatch and control animals to create the two contrasting phenotypic conditions. We used microarray-based genome-wide mRNA profiling to identify patterns of gene expression and cellular processes associated with each conditions. Comparisons between the two conditions and the application of modeling approaches revealed genes, groups of genes, molecular pathways, gene networks, and high level regulators of system behavior. We also used a mathematical modeling approach to describe the dynamics of cellular components of the immune system and their corresponding interactions under the same two contrasting conditions. We identified different temporal gene expression profiles associated with morphological, functional and immunological processes. Several of these processes differed between the two contrasting conditions, whereas others were not affected be the experimental treatments. By inferring gene association networks, we observed that an infection with Salmonella considerably changes the behavior of intestinal tissue as well as the regulation of the underlying molecular processes. For each contrasting condition, we identified a specific set of potential high-level regulator genes (hubs). We hypothesize that these hubs are steering systems behavior. Bioinformatic analysis of the hubs suggested that the disturbance with Salmonella is associated with a shift from transcriptional regulation in the non-disturbed tissue to cell-cell communication in the disturbed tissue. Furthermore, the generated mathematical model describes the dynamics of the cellular components of the immune system as well as the dynamics of the invading pathogen well. The model was able to predict the cellular immune response of the host against an invading pathogen. We developed basic knowledge of (molecular) processes that are associated with different physiological conditions of intestinal tissue and we acquired global views on adaptation mechanisms of the intestine, including the regulation thereof. This information can be used to formulate new hypotheses about behavioral aspects of the gut, for the discovery of new biological mechanisms, and ultimately for the development of tools and rational strategies to improve intestinal functionality and health, either via diet and/or the host genotype. Such developments are urgently required to diminish the incidence and impact of intestinal diseases in farm animal species and to reduce the use of antibiotics in animal husbandry.

Intestinal health is of vital importance for health and welfare of laying hens and nutrition may have a significant contribution. Insoluble non-starch polysaccharides (NSP) and feed additives such as herbs, phytogenic material, probiotics and prebiotics, organic acids and enzymes may have a beneficial effect on intestinal health.

Een overschot aan cholesterol in het bloed (hypercholesterolemie), een belangrijke risicofactor voor hart- en vaatziekten, komt veelvuldig voor. Dit komt door te hoge dagelijkse inname en doordat cholesterol zeer moeilijk afbreekbaar is. Het lichaam kan cholesterol slechts kwijtraken door het ofwel direct uit te scheiden in de ontlasting, ofwel door het eerst in de lever om te zetten in galzouten. Galzouten mediëren cholesteroluitscheiding, maar zijn ook nodig om efficiënt cholesterol in de darm op te kunnen nemen. Galzouten worden echter door darmbacteriën omgezet in toenemend lipofiele (‘vetminnende’) vormen, die darmcelwanden kunnen beschadigen en in verband gebracht zijn met (voorstadia van) darmkanker. Recent werd duidelijk dat transport van cholesterol via de bloedbaan naar de darmholte, “TransIntestinale Cholesterol Excretie” (TICE) genaamd, een grote rol kan spelen in cholesteroluitscheiding. Stimulatie van TICE zou een aantrekkelijk therapeutisch doel kunnen worden. Wij hebben de invloed van verschillende interventies in de darm op cholesterol- en galzouthuishouding getest. Onze studies laten allereerst zien dat versnelling van de darmpassage met het veelgebruikte laxeermiddel polyethyleen glycol (PEG) in ratten geen invloed had op cholesterolopname en -uitscheiding. Daarentegen verminderde PEG de omzetting van galzouten in de darm. In muizen bleek dat een hoogvet dieet met zeer weinig meervoudig onverzadigde vetten TICE stimuleerde en de cholesteroluitscheiding verdubbelde in vergelijking met een standaard hoogvet dieet. Tenslotte vonden we dat bepaalde muizen, die spontaan extreem veel hydrofiele (‘waterminnende’) galzouten produceren, nauwelijks cholesterol opnamen en zeer veel cholesterol uitscheidden in de ontlasting via TICE. Samenvattend kunnen eenvoudige interventies gezondheidsbevorderende effecten hebben op de cholesterol- en galzouthuishouding. Vervolgstudies kunnen bij mensen het effect van inname van bijvoorbeeld niet-absorbeerbare vetten (om negatieve effecten van verzadigde vetten te voorkomen) en hydrofiele galzouten op TICE vaststellen, en het mogelijk beschermende effect van PEG op galzoutomzetting uitwijzen.

Intestinal health is of vital importance for health and welfare of laying hens and nutrition may have a significant contribution. Insoluble non-starch polysaccharides (NSP) and feed additives such as herbs, phytogenic material, probiotics and prebiotics, organic acids and enzymes may have a beneficial effect on intestinal health. Darmgezondheid is essentieel voor een goede gezondheid en welzijn van leghennen en voedingsmaatregelen kunnen hieraan een belangrijke bijdrage leveren. Niet-wateroplosbare vezels en diverse voederadditieven zoals kruidenpreparaten, fytogene stoffen, probiotica en prebiotica, organische zuren en enzymen kunnen de darmgezondheid positief beïnvloeden.

Een overschot aan cholesterol in het bloed (hypercholesterolemie), een belangrijke risicofactor voor hart- en vaatziekten, komt veelvuldig voor. Dit komt door te hoge dagelijkse inname en doordat cholesterol zeer moeilijk afbreekbaar is. Het lichaam kan cholesterol slechts kwijtraken door het ofwel direct uit te scheiden in de ontlasting, ofwel door het eerst in de lever om te zetten in galzouten. Galzouten mediëren cholesteroluitscheiding, maar zijn ook nodig om efficiënt cholesterol in de darm op te kunnen nemen. Galzouten worden echter door darmbacteriën omgezet in toenemend lipofiele (‘vetminnende’) vormen, die darmcelwanden kunnen beschadigen en in verband gebracht zijn met (voorstadia van) darmkanker. Recent werd duidelijk dat transport van cholesterol via de bloedbaan naar de darmholte, “TransIntestinale Cholesterol Excretie” (TICE) genaamd, een grote rol kan spelen in cholesteroluitscheiding. Stimulatie van TICE zou een aantrekkelijk therapeutisch doel kunnen worden. Wij hebben de invloed van verschillende interventies in de darm op cholesterol- en galzouthuishouding getest. Onze studies laten allereerst zien dat versnelling van de darmpassage met het veelgebruikte laxeermiddel polyethyleen glycol (PEG) in ratten geen invloed had op cholesterolopname en -uitscheiding. Daarentegen verminderde PEG de omzetting van galzouten in de darm. In muizen bleek dat een hoogvet dieet met zeer weinig meervoudig onverzadigde vetten TICE stimuleerde en de cholesteroluitscheiding verdubbelde in vergelijking met een standaard hoogvet dieet. Tenslotte vonden we dat bepaalde muizen, die spontaan extreem veel hydrofiele (‘waterminnende’) galzouten produceren, nauwelijks cholesterol opnamen en zeer veel cholesterol uitscheidden in de ontlasting via TICE. Samenvattend kunnen eenvoudige interventies gezondheidsbevorderende effecten hebben op de cholesterol- en galzouthuishouding. Vervolgstudies kunnen bij mensen het effect van inname van bijvoorbeeld niet-absorbeerbare vetten (om negatieve effecten van verzadigde vetten te voorkomen) en hydrofiele galzouten op TICE vaststellen, en het mogelijk beschermende effect van PEG op galzoutomzetting uitwijzen.

Een overschot aan cholesterol in het bloed (hypercholesterolemie), een belangrijke risicofactor voor hart- en vaatziekten, komt veelvuldig voor. Dit komt door te hoge dagelijkse inname en doordat cholesterol zeer moeilijk afbreekbaar is. Het lichaam kan cholesterol slechts kwijtraken door het ofwel direct uit te scheiden in de ontlasting, ofwel door het eerst in de lever om te zetten in galzouten. Galzouten mediëren cholesteroluitscheiding, maar zijn ook nodig om efficiënt cholesterol in de darm op te kunnen nemen. Galzouten worden echter door darmbacteriën omgezet in toenemend lipofiele (‘vetminnende’) vormen, die darmcelwanden kunnen beschadigen en in verband gebracht zijn met (voorstadia van) darmkanker. Recent werd duidelijk dat transport van cholesterol via de bloedbaan naar de darmholte, “TransIntestinale Cholesterol Excretie” (TICE) genaamd, een grote rol kan spelen in cholesteroluitscheiding. Stimulatie van TICE zou een aantrekkelijk therapeutisch doel kunnen worden. Wij hebben de invloed van verschillende interventies in de darm op cholesterol- en galzouthuishouding getest. Onze studies laten allereerst zien dat versnelling van de darmpassage met het veelgebruikte laxeermiddel polyethyleen glycol (PEG) in ratten geen invloed had op cholesterolopname en -uitscheiding. Daarentegen verminderde PEG de omzetting van galzouten in de darm. In muizen bleek dat een hoogvet dieet met zeer weinig meervoudig onverzadigde vetten TICE stimuleerde en de cholesteroluitscheiding verdubbelde in vergelijking met een standaard hoogvet dieet. Tenslotte vonden we dat bepaalde muizen, die spontaan extreem veel hydrofiele (‘waterminnende’) galzouten produceren, nauwelijks cholesterol opnamen en zeer veel cholesterol uitscheidden in de ontlasting via TICE. Samenvattend kunnen eenvoudige interventies gezondheidsbevorderende effecten hebben op de cholesterol- en galzouthuishouding. Vervolgstudies kunnen bij mensen het effect van inname van bijvoorbeeld niet-absorbeerbare vetten (om negatieve effecten van verzadigde vetten te voorkomen) en hydrofiele galzouten op TICE vaststellen, en het mogelijk beschermende effect van PEG op galzoutomzetting uitwijzen.

Het onderzoeksprogramma Feed4Foodure werkt aan een meetlat om te voorspellen wat het vermogen is van een dier om adequaat te reageren als het wordt blootgesteld aan ziekteverwekkers. Dit wordt de immuuncompetentie van het dier genoemd. Deze immuuncompetentie is te beïnvloeden door onder andere diervoeding en voederadditieven, vertelt onderzoeker Mari Smits van Wageningen UR.

Dit proefschrift beschrijft de gevolgen van antibiotica en voeding op de darmbacteriën die ons beschermen: de intestinale microbiota. Op de intensive care (IC) werden de effecten van het toedienen van antibiotica op het voorkómen van infecties door schadelijke darmbacteriën onderzocht: de Selectieve Darm Decontaminatie (SDD). Daarnaast werd getest of het alleen onderdukken van de bacteriën in de mond en keel helpt: de Selectieve Ororfaryngeale Decontaminatie (SOD). SDD en SOD bleken beide de sterfte te verminderen. In dit promotieonderzoek werd aangetoond dat bij SDD in tegenstelling tot SOD bepaalde groepen gunstige bacteriën – vooral F. prausnitzii – sterk werden onderdrukt. F. prausnitzii produceren boterzuur dat essentieel is voor een gezond darmslijmvlies. Op de IC krijgen patiënten niet alleen antibiotica maar ook vloeibare voeding via een maagslang. Daarom werd ook het effect van sondevoeding met of zonder voedingsvezels op deze intestinale microbiota onderzocht. De toevoeging van bepaalde voedingsvezels bleek een groot effect op deze gezonde bacteriën te hebben. Door een antibiotische kuur kan diarree ontstaan. Dit proefschrift verzamelt argumenten voor een nog weinig bestudeerd mechanisme: onderdrukking van boterzuur producerende bacteriën. F. prausnitzii – zo blijkt uit dit proefschrift – is gevoelig voor veel soorten antibiotica. SDD doet een bepaalde groep bacteriën in de darm toenemen: de enterokokken. Meestal worden deze bacteriën als betrekkelijk onschuldig beschouwd. Ernstige enterokokkeninfecties blijken toch regelmatig in het UMCG te worden ontdekt, blijkend uit een verkennend onderzoek waarbij alleen de bloedbaaninfecties werden meegerekend. Er was geen invloed merkbaar van het SDD onderzoek op het voorkomen van deze infecties.

Dit proefschrift beschrijft de gevolgen van antibiotica en voeding op de darmbacteriën die ons beschermen: de intestinale microbiota. Op de intensive care (IC) werden de effecten van het toedienen van antibiotica op het voorkómen van infecties door schadelijke darmbacteriën onderzocht: de Selectieve Darm Decontaminatie (SDD). Daarnaast werd getest of het alleen onderdukken van de bacteriën in de mond en keel helpt: de Selectieve Ororfaryngeale Decontaminatie (SOD). SDD en SOD bleken beide de sterfte te verminderen. In dit promotieonderzoek werd aangetoond dat bij SDD in tegenstelling tot SOD bepaalde groepen gunstige bacteriën – vooral F. prausnitzii – sterk werden onderdrukt. F. prausnitzii produceren boterzuur dat essentieel is voor een gezond darmslijmvlies. Op de IC krijgen patiënten niet alleen antibiotica maar ook vloeibare voeding via een maagslang. Daarom werd ook het effect van sondevoeding met of zonder voedingsvezels op deze intestinale microbiota onderzocht. De toevoeging van bepaalde voedingsvezels bleek een groot effect op deze gezonde bacteriën te hebben. Door een antibiotische kuur kan diarree ontstaan. Dit proefschrift verzamelt argumenten voor een nog weinig bestudeerd mechanisme: onderdrukking van boterzuur producerende bacteriën. F. prausnitzii – zo blijkt uit dit proefschrift – is gevoelig voor veel soorten antibiotica. SDD doet een bepaalde groep bacteriën in de darm toenemen: de enterokokken. Meestal worden deze bacteriën als betrekkelijk onschuldig beschouwd. Ernstige enterokokkeninfecties blijken toch regelmatig in het UMCG te worden ontdekt, blijkend uit een verkennend onderzoek waarbij alleen de bloedbaaninfecties werden meegerekend. Er was geen invloed merkbaar van het SDD onderzoek op het voorkomen van deze infecties.

Dit proefschrift beschrijft de gevolgen van antibiotica en voeding op de darmbacteriën die ons beschermen: de intestinale microbiota. Op de intensive care (IC) werden de effecten van het toedienen van antibiotica op het voorkómen van infecties door schadelijke darmbacteriën onderzocht: de Selectieve Darm Decontaminatie (SDD). Daarnaast werd getest of het alleen onderdukken van de bacteriën in de mond en keel helpt: de Selectieve Ororfaryngeale Decontaminatie (SOD). SDD en SOD bleken beide de sterfte te verminderen. In dit promotieonderzoek werd aangetoond dat bij SDD in tegenstelling tot SOD bepaalde groepen gunstige bacteriën – vooral F. prausnitzii – sterk werden onderdrukt. F. prausnitzii produceren boterzuur dat essentieel is voor een gezond darmslijmvlies. Op de IC krijgen patiënten niet alleen antibiotica maar ook vloeibare voeding via een maagslang. Daarom werd ook het effect van sondevoeding met of zonder voedingsvezels op deze intestinale microbiota onderzocht. De toevoeging van bepaalde voedingsvezels bleek een groot effect op deze gezonde bacteriën te hebben. Door een antibiotische kuur kan diarree ontstaan. Dit proefschrift verzamelt argumenten voor een nog weinig bestudeerd mechanisme: onderdrukking van boterzuur producerende bacteriën. F. prausnitzii – zo blijkt uit dit proefschrift – is gevoelig voor veel soorten antibiotica. SDD doet een bepaalde groep bacteriën in de darm toenemen: de enterokokken. Meestal worden deze bacteriën als betrekkelijk onschuldig beschouwd. Ernstige enterokokkeninfecties blijken toch regelmatig in het UMCG te worden ontdekt, blijkend uit een verkennend onderzoek waarbij alleen de bloedbaaninfecties werden meegerekend. Er was geen invloed merkbaar van het SDD onderzoek op het voorkomen van deze infecties.

De pluimveehouder kan zelf maag-darmproblemen bij de dieren beperken door oog te hebben voor kipsignalen. Onderzoekers Monique Bestman en Marko Ruis (auteurs van het boek "kipsignalen") weten hoe dat moet.