[Music] I'm very happy that we started talking about biomarkers because this is something we have been working on quite a lot recently but my talk will not be on biomarkers effective it's a bit it's a bit of a pity if I would have known that few days ago I would change my topic definitely but it's not the case now one of the guys of the second group there that asked more questions than answers I asked a lot of questions basically because I hope they come to me because I knew the answer so that's basically why I did this so I'm from Kent University and in fact we are part of the Pointer we are also part of different consortium one of them is also working on intestinal integrity and inflammation in humans and different animal species are also part of two different Federation consortium meaning that we try to develop vaccines and feed additives and food additives for of course but also for humans we try to bring these to the market so this is my topic as you can see what we basically do in our laboratory is trying to steal microbial composition using substrates fit additives or feet or foods on itself to try to make sure that these microbes produce metabolites that affect host responses inflammation metabolites that affect intestine integrity but also metabolites that affect the behavior of pathogens and this is very convenient because if you ask me questions about nutrition I can say ok I don't know another nutritionist the same here if you ask me questions about immunology I have no idea I'm not a monologist and also not really a bacteriologist so it's fairly convenient in fact I'm a molecular biologist so we try to link all these things with each other which is a very nice topic this is what we have seen already beforehand this is how the normal gut looks like in small intestine at least very long villi and also of course very thin villi so a lot of absorptive surface no inflammation which is basically how the gut should look like in optimal conditions now they're quite a lot of microbes in the gut so one of the questions is of course why don't we react to all these different bacteria that are in quite close connection with these epithelial surface and basically I think there are two different answers to it first of all when animals or we are born we present a lot of antigens coming from bacteria to our immune system and we just tell our immune system that we do not have to react against these antigens that's called oral tolerance and the second reason basically is that if you look at the epithelial layer you have a lot of receptors there that are basically responding to microbial components these are called toll-like receptors and these are basically found at the basolateral surface so not at the apical compartment meaning that once you have a breach in this epithelial layer when you have intestinal integrity increases when you have permeability increases then this bacteria can just go through the epithelial lining connect with these receptors and that's really a trigger for inflammation okay and that's of course what many pathogens do and if you think about poultry the poultry situation you have quite a lot of triggers that induce this kind of inflammation this kind of permeability increases here I just listed a few of them viruses of course enterovirus s3 or viruses whatever but definitely also coccidia do that also some pathogens clostridial toxins even mycotoxins make sure that there's a breach in this epithelial layer by giving epithelial cells of course bacteria can just penetrate and connect with these receptors and cause inflammation and inflammation means that there's quite a lot of immune cells that infiltrate in the gut mucosa and inflammation definitely also means that there's a lot of energy that the animal has to spend to attract these immune cells though there's a loss in performance that basically what this is saying now if you look at intestinal sections of chickens of broilers if you look at it using histology you always see inflammation the gut is more or less never normal so there's always something wrong some minor damage some subtle damage but you always see something so it's never perfectly normal so as I just said well this is the histological slide I've already seen this is how a section of the same intestinal segment looks like that is inflamed that means shorter feel i blunted feel I see curved villi and all these brown dots here these are immune cells that infiltrate the intestinal wall that's of course quite energy costly and that's some other stuff whatever doesn't matter so much but this is a consequence of intestinal integrity losses now I'm just going to talk a bit about bacteria especially mucosa associated bacteria this term has been touched upon already by a few people these are of course bacteria that are very close interaction with epithelial cells and these are the ones that produce a lot of metabolites that are sensed by these epithelial cells and we talked about blue Krait in the working group that's one of them that's a very important one that is very well but it's often produced very close to the epithelial cells and also used by epithelial cells I will come to this back in a few minutes if you look at the microbial composition in different parts of the intestinal tract you will see quite some differences of course and the most diverse microbial composition you have in the Sica this is the place this is the fermentation chamber where a lot of bacteria exists that actually use undigestible stuff use saccharides also to ferment and to produce short chain fatty-acids acetate propionate butyrate whatever there's a lot of different anaerobes a lot of different families species genera present in the Sica in the small intestine you only have a few families present and quite a lot of lactobacilli so if you sequence microbial composition if you look at microbial composition the small interesting it's full of lactic acids producing bacteria which is completely different in the psycho compartment as I just said well you have bacteria present in the lumen quite a lot and these are the ones that degrades a lot of polysaccharides and other substrates they produce other compounds and these are used by other bacteria it's called cross feeding bacteria break down certain components but use intermediates other bacteria use these intermediates to produce other intermediates or ant metabolites and especially important and this is what I'm going to focus on today is or these bacteria that really are in very close interaction with the epithelial cells and there's not a lot known about this especially not for poultry but there are quite a lot of studies being performed in mice rats and also humans using biopsies and also some studies in poultry is just one of them we have done some others just using scrapings of the intestinal wall and looking at microbial composition in this new Kosar very close to the epithelial lining and if you do that you see that you have well only a few well specific bacterial groups being present and that means in the small intestine you have some lactic acid bacteria that are very close and interaction with the epithelial layer huh and you have also bacterium with a very weird name this is Canada to serve a gala it's renamed in the meanwhile doesn't matter so much this is a bacterium that is only very recently being cultured but is very difficult to culture but it seems to be very important I come back to this later in the seeker you have butyrate producers that attach very closely to well at least some general species of butyrate producers that attach very closely to the epithelium this is a family of black conspiracy a family Freeman a coca CI and as microbiologists do you give very fancy and weird names to make sure that people cannot pronounce it this is a competitive advantage of a microbiologist okay but these ones are really important because they are found very closely to the epithelium they produce boutique acids so they are very important I will give you some examples why these bacteria are so important in a few slides on the other end you also find if you sequence bacteria closely associated with the epithelial cells so in the mucus layer you also have other bacteria to offer find here and these are the more pathogenic ones Clostridium perfringens ecoli and also in this Sica a lot of entra back to the CI that also contain a coli and as you can imagine there's some kind of a balance okay to be very simple I would say that if you look at mucosa associated bacteria and you find a lot of these ones that means if there's some kind of dysbiosis unfavorable conditions if you have a lot of these ones that's favorable and I will tell you in a few slides that if you do that and you look at these species there you will see that it's always like connected to it gut health parameters such as phyllis length okay if you have a lot of these ones you have a high phyllis length if you have a lot of these events you have lowly dose length and that of obviously also is connected with animal performance but i will give you some data a few minutes actually now so this is the question we have been asking how is microbiota composition changing when you have an affected intestinal integrity we have used a lot of different models to try to reduce animal performance and infuse intestinal inflammation and then measure microbial composition okay I just giving to give you one example of such a model this is one of the models we have been using basically what we do is we use poorly digestible diet we use coccygeal challenge we use bacterial challenges and we just a few days later is sample and measured microbial composition why we use this kind of model well just basically we want to have an intestinal tract that is inflamed as poor intestinal integrity short fillers lengths etc so that's ideal of course using these triggers these are just some data just to show you that these kind of models is actually having the effect we want to see the challenged animals of course have a lower body weight this is day 26 and these are just some macroscopic scoring systems either for coccidiosis this is just an sum of all the different coccidiosis scores and also these Bo z-scores which take into account different parameters inflamed gut ballooning of the gut thickness of the intestinal wall etc doesn't matter so much I just want to show you here that using this model we see some kind of performance losses and we also see that the intestinal wall looks abnormal it doesn't look normal it looks inflamed thin and it also has a lack of donors so you just do something that is affecting integrity now again if you look at histology you see that there's the Veals length is decreased and that you have this inflamed gut wall all these cells or cd3 positive T cells these are T lymphocytes that infiltrate the gut wall and you see that the percentage of the mucosa of the wall and of the intestinal wall that is having these immune cells is going up quite honestly using this challenge and village length is decreased so this is the situation we wanted to create and then we wanted to look at microbial composition shifts okay so basically this that's what we were aiming for huh we were looking for bacterial taxa families genera species that had a good association with Phillips lines meaning the lower the village length the higher or the lower a certain bacterial group is present in the gut that's basically what we have been doing and as you can imagine well a lot of bacteria that are potentially located very close to the epithelial cells of phones I will give you the exact date in a few minutes this one seems to be very important the high of the village length the more of this type of bacteria you find in the small intestine it has to do it well because it attaches to the epithelial cells of the gut it is more abundant in the ileum and here you have better weight producers so just giving you an idea and I just go to the real data this is one of these betrayed producers in challenged conditions it's depleted and also you see that the higher of the fields lengths the more of these bacteria you find so this is what you can consider as a microbus as a bacterial genus that is positively associated with intestinal health okay also on the other hand the more inflammation you have the less abundant this population is okay so this is fickle a bacterium pros nits i which has been described also in humans to be very important for intestinal health but also here in this chicken model and also in other chicken models using necrotic enteritis or coccidia whatever you see that this is positively associated with different guitars parameters so the more this bacterium is present the higher the finished length the more it is present the less inflamed the intestinal wall is so it's very important and this is actually one of the first biomarkers if you wish that you can look at that because if you quantitatively analyze the presence of these bacteria in the intestinal in the fecal material you actually have some kind of a first test that's what you could use potentially this is the second one I can give you a lot of this information but this is one that we have been isolating and doing some experiments with and also here you see a positive association with phyllis length and a negative association with t cell infiltration in the intestinal wall so this is what we can consider as beneficial well microbes that are linked to cartels just one slide I added this ten minutes ago just because all of you were talking about biomarkers at least I flicked one of the slides in just to tell you that we have been looking quite a lot of last year on host biomarkers for gut health and actually we just have patented a set of 9 different proteins that can be used in analyzer on fecal material just to measure gut health and this is just one of them I just give one example because you really find some proteins in fecal material of chickens that have a disturbed intestinal integrity this is an example of protein that is found in at a Philly granular site so in immune cells that go to intestinal wall but also translocate to the lumen and then release a lot of proteins okay one of these proteins as this main my lead protein one and if you look at it in fecal material of animals with disturbed dirty integrity so with inflammation in the gut you also see that the lower the villa's length well the higher the villus length I'm sorry the lower the amount of this protein or of course if you have a damaged gut you find a lot of these proteins in the fecal material of the animal the opposite way if you have an inflamed gut you find a lot of this protein so this is something that can potentially be used as biomarker okay so we have been identifying quite a lot of these proteins and I'm quite confident that some of these will be available in the future on analyze our dipstick whatever to be used on fecal material so if you can just go to a flock and measure this kind of proteins and say okay here in this flock you have intestinal inflammation so we have to do some intervention or not so this is really markers this is a host biomarker for intestinal inflammation you also have leakage biomarkers you also have even epithelial damage biomarkers you have all set of these that will become available back to the story epithelia integrity this is something that seemed to be very important today in fact there are a few different ways how intestinal integrity can be maintained how we can repair damage one of them is of course increased cell proliferation if you have some damage in a village some epithelial damage of course cells have to renew and double much more tight junctions we have been talking about tight junctions already today has strengthening of tight junctions is important and also of course anti-inflammatory effects just because inflammation on itself is also triggering epithelial cell that and loss of tight junctions now as you can imagine butyrate is having all these to be effect i'm not going to give you all the details but there are a lot of studies showing that butyric acid has these three effects on these to be different important and the mechanisms for repair of damage and of course well looking at these two microbial groups or microbial markers for cut off these are beauty read produces and as you remember these were positively associated with villus lengths with animal performance and negatively associated with inflammation so it seems to be that these bacteria that closely are located close to the epithelial cells especially this one is very key in maintaining intestinal health because they practice beauty rate which is sensed by the epithelial cells going a bit faster now here in this we have well this is a very complex light i think nobody can read it but anyway we have been insulated a lot of these beauty raid producers tweekly anaerobic strains that's a disadvantage they're all strictly anaerobic we have been isolating a lot of these from a chicken intestinal tract including these patristic cookers and this fickle a bacterium frozen inside so we have a lot of chicken isolates and of course then we can wonder if you deliver these strains to the animals what is happening so this is basically a question that we have been asking ourselves we have been using the karate can try these models you see some data here this is a study this is the protocol it doesn't matter so much you just induce lesions using Castilian perfringens challenges so when use necrotic enteritis and if you give this strain here as a probiotic meaning that we just administer it in freeze-dried form in the feet okay then you actually see that in four different independent trials you reduce the percentage of animals that develop necrotic lesions and so again telling you that this kind of bacteria are very efficient in counteracting gut damage okay this is just what happens with normal beauty rate at different levels this is also of course quite efficient just going to well this is just showing you that this strain is also having an effect in different dysbiosis models but I'm not going to focus too much on it I just want to tell you that if you use models the traditional models in which you give poor digestible feed to animals in using some performance losses that also administration of these bacteria stimulate performance and again that makes sense if you I've listened to the whole story because these bacteria per gives beauty rate and also repair subtool get damaged and actually what this is saying is these are just data from a 16s sequencing study showing that if you administered this kind of bacteria it really affects the whole microbial composition so what you don't have is I mean you give this bacterium to the animals it's not only about increasing abundance of this bacterium but you also increase lactic acid bacteria you know and you read use Enterobacter ACI which contain also e.coli and opportunistic pathogenic strains that's just what this slide is telling you okay just a small side story on butyrate and pathogens we have also been doing quite a lot of studies using blue t rate and salmonella and basically if you use protected butyrate and I think I'm replying to some of the the questions already if your ad protected Beauty rate you actually are able to induce increased butyrate concentrations also in the seeker and you can measure that using HPLC depends a bit on the type of compound you use with some of the compounds you can even measure it with a lot of other compounds you count maybe because it's not released or because it's gradually released we don't know but if you do that you actually are able to reduce colonization with someone aligned in the seeker and someone Ella for example as a pathogen it's really colonizing the sickle content and basically this is because Beauty rate is a signal for this pathogen to reduce is its Fitness gene expression so this is something that we have been doing a lot of studies on and I've been publishing in multiple papers so it's also very important and Salmonella compounds and that also means that the more of these bacteria producing bacteria you have the less Enterobacter Asiya and also someone else one of these enterobacter CIA so that's always some kind of a balance you have either the other I did the other one so it's one or the other that is most colonizing just very briefly butyrate production in the gut can be stimulated using a variety of different methods and basically it is just a general scheme showing you that if you have any polysaccharides it is converted by some bacteria to smaller components for example oligosaccharides that are used by other strains to produce the short chain fatty acids including butyrate and there are a lot of pathetic acid producers that consume lactate in other words if you have a lot of lactic acid bacteria lactic acid on itself it's in fact toxic it's not really to beneficial but it's consumed by a lot of betrayed producers to produce but your rate that's why a lot of these lactate producing bacteria lactobacilli are very important because they feed you today it produces to produce butyrate and as you can see in this graph you can supplement some oligosaccharides to the feet these are feed additives Seiler oligosaccharides fructooligosaccharides inulin whatever and this is also beautiful genic because it really steers this pathway okay not going to give you too much of specific data are just some examples this is just a study we have been doing with showing that enzymes have even a beauty or genic effect what we have been doing here was just taking wheat adding enzymes in a fermenter system and then just use what is coming out from this digestion to add as a feed additive for broilers what you can see is that it increases levels of glutamate producing bacteria in the seeker okay just showing you that if you have enzymes okay you also make sure that you release some components out of polysaccharides that of silence of course Arabic silence that are muted or genic that's just nothing more than that but that's of course also an effect that is important it's not only about viscosity reduction etc it also stimulates you today it produces just addition of enzymes and here of course this is what enzymes do they be great polysaccharides and one of the components coming out of course when you add xylene ages to the feet or arabic xylene oligosaccharides or maybe if you also remove the arabinose then you have silo oligosaccharides and just to show you that these components also affect butyric acid production we did some trials this just one of the trials we did quite a lot of trials with with oligosaccharides as substrates this is just trial we have been doing with supplementation of Kiley Oregon saccharides to the diet of broilers using a bottle with poorly digestible substrates without enzymes okay just making sure that the animals perform less this is a study of a few years ago actually showing that in these dysbiosis model because that's basically what it is we steel microbial composition to an unfavorable bond showing that this tile oligosaccharides increase row led to an increase in body weight decrease in feed conversion and also stimulate fillers line okay but basically what is important is that these substrates these oligosaccharides they induce butyrate production in the gut of the animals that's what you see here is the quantification of a bacterial gene gene copies of a pack T of this bacterial gene that encodes for this enzyme again it has a difficult name but this is the final gene this is the gene that encodes the final step in the butyric acid production pathway in bacteria okay so this is a quantitative PCR showing you that if you add sila oligosaccharides to diet that the number of gene copies of this very important gene involved in the butyric acid pathway is increased so you have this is a measure in fact a marker for the body rate producing capacity of a microbiota of the gut okay so this is a test we often use to show butyl genic activities so it's an induction of butyric acid production in the gut of the animals it's a bit of an alternative for measuring butyric acid just using the pathway just some data on the microbes we did we always why we often do some 16s sequencing studies looking at the global microbial profile of the gut of these animals if you add Seiler oligosaccharides you have an increase in certain lactobacilli this is just one of them like the bacillus Chris photos this is in the hindgut you also have an increase in some butyrate producers and actually what happens here you can read all of it in this this paper of saline the mascot but what happens is that these guys here they consume cider oligosaccharides and produce lactate and the lactate is consumed by this bacterium to produce butyrate so this is a very specific example of this cross feeding mechanism and shows you that lactate producers are important because they feed beauty rate producers to produce beauty rate and that's of course where feed additives can play quite a role you can steer this whole interaction you can steer production of these beneficial metabolites and then of course as the observed health effects just one final story just a few minutes just because some people were interested in this it was not in the first slides but if you think about Beauty rate and epithelial cells there are different things on the left side you have transporters and it means that if you have butyrate in the gut the epithelial cells can transport it in an active way inside the epithelial cells you have different transporters this is for example a very important one mono carboxylate transporter one but also you have some others you also have transporters that pump it further to the blood okay all this is not why they don't find it in high concentrations in the bloodstream Beauty rate can actually feed the TCA cycle so it can lead to announced proliferation of epithelial cells but it can also have an effect while an anti-inflammatory action because it goes into the nucleus and is involved in in histone deacetylation without too many details on the other end you have quite some receptors and you have two important receptors for beauty rate this one here and this one and these are also involved in anti-inflammatory actions of this molecule okay but I just want to point out here is that you have Beauty rate okay but you also have many other metabolites and Beauty rate is a well-known one but if you look at all the metabolites that bacteria produce often these metabolites have effects on one of these transporters or one of these receptors that's that's that's amazingly interesting because for example if you have a lot of proteolytic degradation of 30 minutes this is the first time I do it I didn't realize it made such a noise otherwise I would have done that but just one minute ago so if you have proteolytic degradation sore too many protein degradation for example in the in the gut you see that you have a lot of Armagnac and this is really blocking this transporter so if you have too much proteolytic degradation there you have less butyric acid uptake in epithelial cells so that's why for example this ammonia in the gut is quite harmful same it hydrogen sulfide also here too much proteolytic degradation for example sulfur-containing amino acids it blocks TCA cycle and you can give you much more examples lactate it can fuel as I just said it can fuel butyrate production by other bacteria but it can also bind to some receptors present on immune cells and what you see here this is like the receptors that respond to beauty rate they actually drive the immune system towards a lot of regulatory t-cells okay this is also an anti inflammatory cascade producing interleukin 10 okay and basically you can have a lot of different other compounds propionate is also affecting this butyrate receptor we have vitamin b3 it's binding to this receptor that is also anti-inflammatory and so on and so on so all metabolites produced by bacteria or at least a lot of these metabolites produced by bacteria they one or another have an effect on one of the receptors transporters or systems that are responsive to butyric acid so it's very complex but I think butyrate is really a main metabolite and all the others that are either produced by fermentation of amino acids or proteins or sugars they actually play a role somewhere in this beauty rate pathway okay and lucky you this is the final one as I said beauty rate is important you can have a lot of strategies that enforce butyrate production but it's much more complicated than that and I think future will tell us how complicated this really is that's the final one thank you thank you for the exciting presentation and I think there's a lot to think about thirteen questions so we start with number one and finish with number thirteen or I deliberately went over time to not reply to it but it seems yeah no chance yeah whatever maybe I take this one I think well transcriptomics I wouldn't really focus on cuz it needs RNA of course and that's unstable but any but I get the idea ideally you shouldn't just look at species of genera or whatever but you should look at genes that encode for microbial pathways I think this is the future doing metagenomic studies check which pathways of the bacteria are important and then quantify this I think that's that's that's basically very important because for example this bacterium maybe other bacteria have exactly the same function can produce exactly same metabolites and that's why probably we don't need functional markers instead of just markers based on taxa or phylogeny that's just one one is enough for should I do more one more okay okay I don't think it's it's wrong to look at the macro behind the lumen because well I think that the end aim is of course to look at microbiota in in fecal material or colonic content because that's the easy way to do for the field so I don't think it's wrong and I also think that if you look at luminal content this is something what we are obviously always do quite often you also see patterns that look very similar as what you see in mitosis yet community it's different but in terms of ratios between populations is actually you can often see similarities also when you look at fecal material which is obviously what we should do for field testing [Music]