[Music] thank you mark for the introduction so what I will try to do is just give you the kind of a snapshot of a lot of things that are actually going on on the field of good health diagnostics and gotta love biomarkers is actually a quite complicated topic and you can use a lot of different technologies to actually identify good gut health biomarkers so the first thing I will just pave the way a bit for other speakers that will follow my talk for example one of the postdocs of our group is presenting really a practical solution in a few few presentations but also in this presentation I will just try to give you an overview of which potential biomarkers can be of value and which are the ones that are currently being developed so I'm quite confident that within the next few years some tools will be available to be used on forms of phlox to assess so to measure gut health first question of course is what is cutoff it's a very complicated thing there are a lot of different definitions and of course we first have to have an idea on what is actually what we mean with intestinal health before we can and of course try to identify and quantify this kind of markers now I'm not going to talk about humans but just there's a lot of interaction with a lot of knowledge present in in human medical science that we can actually use and we just want to give you two slides on the current diagnostic tools in humans to measure intestinal health or intestinal inflammation and of course you don't go to a medical doctor and give him a sample and ask him ok please what is my cattle status that's something you don't do so there's nothing available there of course to measure general intestinal health status of course when you have an infection then of course they take a sample they just plate it on to a Gerson they will tell you which pathogen you're infected with that's of course also the case for poultry but there's one well validated methods that they use in clinics to actually measure intestinal inflammation and that is based on one specific protein which is called protecting and calprotectin is actually a marker for intestinal inflammation in humans so what this protein is in fact it's a protein that is present within the granules the secretory granules of granular sites of neutrophils what you see here for example on the Left this is just intestinal fillers you see these immune cells these granamyr sites that really infiltrate the gut wall and there's some kind of a trigger then they translocate through the epithelium and end up in the intestinal lumen and also they did granulate what you see here they release granules they release proteins and these proteins they end up in the faeces okay and actually you can measure quantities of this specific protein in the feces and that's also something that would of course be of value for poultry and I will come back to this at the end stage of my talk because there are similar proteins and kind of as well be used in chickens what you see on the right side here is just a graph showing the quantification of this calprotectin in different human subsets on the left you see crohn's disease patients then you see that is also rate of colitis IBS is irritable bowel syndrome and then you have these healthy controls I hope okay so but what you can see here is that in cases of severe inflammation you have very high levels but in cases of well there's something not normal a bit of diarrhea maybe a bit of constipation just from some limited problem limited information you don't see any difference between this population and the control population so actually you need really severe inflammation to be able to measure it and this is of course kind of a problem and especially when you look into poultry you also have this on the one hand cases of severe inflammatory diseases necrotic enteritis is probably the most well known you have a massive inflammation of grandmas eyes there and also this kind of proteins will actually be released in intestinal lumen and you can measure these in the faeces but on the right side you see well this is just subtle problems I'd say in chicken terms in broiler terms that means a little bit of performance loss because of a little bit of intestinal inflammation some dysbiosis and so on and that's a complexity because for this kind of condition there's not well it's maybe not severe enough to find this kind of biomarkers that are used to measure severe inflammation I will come back to this in a few minutes because actually there are some possibilities there so this kind of problem just as a introduction this kind of wrong subtle intestinal inflammation or dysbiosis they can be evolved by a lot of different triggers can be micro toxins can be clostridial toxins can be the diet composition is very important and so on there are a lot of potential factors that influence this kind of condition screen so there are a lot of different potential triggers so we are not talking about measuring the triggers but measuring of course the outcome of this I will come back to this again now what we are really looking into is we want to try to identify some predictive biomarkers and that's a challenge okay what we try to do is like employers taking samples at about 2 weeks of age measuring some kind of a biomarker and then predict what is going to happen in the near future what the performance will be whether there will be health problems or not and so on so that would be the ideal biomarker something that is predictive for performance now oops I just told you I will give you some definitions this is something you already know of course microscopically when you have the animal has intestinal health problems you will just see it of course in cases of severe disease necrotic enteritis you see got necrosis but also there's a lot of other things you can see when you open the birds you look at the microscopy the macro capital appearance of the gut wall okay and there are a lot of people that have been using or implementing this gut health scoring systems looking at the gut and just giving a value there to be used of course this is kind of well it's not completely objective I would say there's a little bit subjective depends on the person that's doing that here's one of them just looking at different entities looking at thickness of the gut wall at the inflammation and I just feed particles and so on and this at least gives you an indication or gives you a score at the end about well intestinal health but as set it should be ideal to replace this kind of system with a very easy to take a fecal sample put it in a machine and get the value that's basically what we are looking for so now you can also look at it under the microscope of course and that gives you a lot of information of what is actually going on and if you look under the microscope of animals of which the veterinarians are saying this is here's a problem this is these biosis or what later or whatever then you will see that Phyllis length is decreased you have some intestinal inflammation you have a lot of immune cells that they filtrate the gut wall so this is something that can learn you a lot about what is actually going on and what you need to measure okay so just some pictures here this is on the left side necrotic and right it's okay this is the most severe case of these biosis which is actually got necrosis and you see that here the whole structure of the gut wall is completely disrupted this is normal on the right side normal gut wall appearance and also long slender villi and in the middle this is just some congested blood vessels and inflamed gut and what you can see here is the fillet articular a bit blunted they're shorter and all the brown dots these are immune cells okay in this case these are cd3 positive t-cells so these are dealing for sites so that actually means that in this middle case which the animals that don't have any symptom you don't see anything on the em on the animals the only thing that they will do is form a little bit less because they have to spend a lot of energy in this immune cell infiltration cost a lot of energy that the animals are not putting in muscle of course that's quite evident so actually just a little bit of a short molecular explanation of what is actually going on and if you have gut inflammation that basically means that there will be some disturbance in intestine integrity so this is actually the normal situation all these dots here these are bacteria different colors different genera different species different families whatever and actually what is happening is that in a normal individual also us here but also normal chicken that is perfectly healthy they just do not react to all these bacteria that are sitting in the gut lumen we have tolerance against these and actually when there's some kind of a breach here when you have epithelial cells that are dying due to toxins or micro toxins or even coccidiosis you will have some of these bacteria that go through these lesions and connect with receptors that are basically at the basolateral surface of these epithelial cells and that will be a trigger for gut inflammation that's what you can see here what you see here this is an increased permeability so some gut damage some damage to the tight junctions that's what you see here in the middle cells not connecting very closely and there is a breach and then you have these bacterial toxins maybe dietary antigens that leak through they connect with some receptors here and then of course you have cytokine expression and inflammation that's basically what we need to find some kind of biomarkers for if you look well here we also see a lot of these red bacteria and so you have also these B O's is quite often already mentioned by show well if you have inflammation you see a microbial shift and you see some bacterial populations going up others going down that's also something very specific now at the end you will also see that as I just mentioned granular sites go through to the intestinal lumen it doesn't really look like a granular site I try to draw it sorry and all these blue things here these are just also proteins from serum whatever that also are ending up in the faeces coming from the bloodstream from the intestinal mucosa and it just ends up in fecal material and actually what I want to point out is that if you look for bio markers if you want to identify biomarkers you can look at microbial populations microbial shifts microbial functionalities that are different you can look at these host molecules that are leaking true and end up in fecal material you can look at inflammation markers that sell that markers and so on okay so this is something that you can use a lot of different technologies for to try to identify these different proteins or molecules so I think this is an important one there's a lot of novel technologies being used for the moment - well talked about metagenomics yourself meta proteomics meta metabolomics and so on there's a huge set of novel techniques that you can use and really use some experimental models inflammation models and try to identify all these different potential biomarkers and that's actually what we are trying to do and you can learn a lot from that and especially you can identify potential markers so first of all microbial shifts if you think about microbial shifts you think about 16s sequences this is differences in families genera species you also think about different functionality meaning functional genes in this microbiome will be changing you also think about metabolites bacterial metabolites bacteria produce a lot of different molecules and metabolites and also when you have some kind of dysbiosis different microbial composition actually the metabolic profile of this microbiota is changing this is also something you can measure okay question is of course which metabolites are linked with dysbiosis and inflammation and which ones we should actually measure now I will come back to that and give a reply to this in a few slides so just a third definition of gut health okay and this is the microbiological definition saying that if mike the microbiological diversity is high this really is related to intestinal health and of course it should be a microbial composition that produces metabolites that basically and the inflammatory and not pro-inflammatory that's a very vague definition I will come back to this in a few slides in fact already she well talked a lot about it cos when you look at the human datasets you can see which bacteria are doing what you have to butyrate producers this is anti-inflammatory you have some others that are pro-inflammatory and of course also these you can try to use as biomarkers this is already mentioned as well we have also been using this kind of model it's a different experiment that to identify 16s sequences that are actually either or not associated with performance and weight with inflammation what you see here is just it's just a model okay it's a little bit unpractical but what we wanted to do here is induce intestinal inflammation induce Phylis length decreases induce performance losses induce some kind of pathology between brackets that is more or less identical as what you sometimes see in birds that do not form well so actually what we have been doing is using only digestible diet no enzymes we depleted microbial composition using antibiotics then in fact bacterial cocktail was given of some opportunistic pathogens equalised some non-pathogenic lost Rydia even some lactobacilli and then also we gave a coccidiosis challenge okay and then we just sampled and looked at 16s sequencing so basically the question is first of all is difficult model secondly what type of microbial populations are changing okay these are some data has already been mentioned but what you see here on the left that of course and the challenged animals you have a body way to decrease you have a feed conversion increase that's evidence so the animals are clearly performing less you see some coccidiosis lesions on the right side so each dot here is one animal and these are the animals at day 26 so more or less three to four weeks of age this is at this beauty score this is just a microscopical gut wall appearance score the ones I just showed you some pictures off okay this is also increased so you see some gut wall abnormalities if you open the animal and look at the intestine and you also see performance losses and if you look to the histology also syphilis length decreases and inflammation just showing you this data to tell you that this is this is a model that you can use to identify potential biomarkers that's basically what I'm saying here okay you know what you see here on the left this is the finished length decrease it goes from over 2000 to 2500 micrometer in the duodenum which is quite strong it's a 25% reduction in villus length villi also thicker and as you can see here right below this is a picture of the challenged animals meaning there's a massive immune cell infiltration these are dealing for sites again I also see that on the graph about 15% of the whole gut mucosa is full of it's filled with this brown staining and these brown staining or brown cells and these are T lymphocytes okay now if you do 16s sequence thing you immediately see that the challenged animals they have a lower diversity okay that's one thing but importantly as well is that if you compare control animals with challenged animals I will look at the observed taxonomical units so numbers of bacterial taxa if you if you want in the control population it's it's evidence while animals that waken a little bit less or more they all have the same diversity but in challenged animals you see this very clear correlation meaning that animals under challenge and this has already been mentioned by Joelle in the previous talk animals under challenge with the high diversity they have a higher wait they have less problems of coping with the challenge okay animals with the lower bacterial diversity seem to well be the ones that react very strong to the challenge this is also linked to it immune cell infiltration and so on you can clearly link that with bacterial diversity and this is something you can measure it's still a bit of complex to measure it but anyway this is in principle a tool to look at responses to challenges so going much more into detail in fact we have a full list of all sequences that are going up and down and so on but what always comes back in this kind of models and also in other inflammation models is that quite a lot of bacteria that are depleted in challenge conditions these are Beauty rate formers okay what you can see here these are the blue families of room Monocacy a lactose bitter CI which are broad families that contain a lot of butyric acid producers and in a lot of different models also in mice even in pigs but also in chickens under different conditions if you challenge the animals or sweet coccidiosis also with some other pathogens you see a depletion in these specific families and there are a lot of other names there I just highlighted two of them as well fickle a bacterium burrows niche sorry the one that you all mentioned as being very very strongly connected with intestinal health in humans we also find it here as like thousandfold depleted in challenged animals patristic caucus is another one this is one that we have been isolating from chickens before and as if you administer this one to the chickens actually they improve their intestinal health and also here you see this huge depletion what you see here on this graph in green that is the well what is more abundant in control animals but actually it's not more abundant in control animals its depleted when you challenge the animals that's basically what it says so potentially if you just quantify these populations you have something to work with okay and quantifying these population is fairly easy you can just use quantitative PCR methods apply it to fecal samples or to intestinal samples actually measure abundances of these populations which is fairly easy to do okay just one step back to humans just as I just mentioned this is something that looks very similar to human intestinal inflammation what you see here is just a study comparing human healthy individuals with people with inflammatory bowel disease and actually there's in people with inflammatory bowel disease you see a depletion of butyrate producers l'hospital's their family identical as what i'm just showing an increase in Proteobacteria so this contains a lot of enterobacter I see a lot of e.coli and opportunistic pathogenic organisms you also see an increase in cost in poor engines for example so there's like a balance okay there's a on the one hand you have the strictly anaerobic butyrate producers and all the other ones that are connected with it and on the other hand you have these enterobacter CI crostini perfringens and some others and it's really about balance between these two populations it's also easy to quantify again what they have been found in this paper also is that battery Secaucus for example is actually depleted quite severely in oxidative colitis patients and actually here coli of course is increased in abundance in this disease entity so it's very similar and it seems to be that if you look at microbial composition over different animal species also humans there's a lot of similarities in the way microbes respond to inflammation to challenge and the way that the host is reacting to microbial disturbances okay just to show you that well in this paper we have been working together with people from human medical departments in the University of Leuven in fact people that develop severe inflammation after resection of parts of the gut in inventory bowel disease patients the outcome of what happens there is really depending your microbial composition and people with high concentrations of costly perfringens they will develop severe inflammation leading to an additional surgery so costing for engines really linked to severe pathology here also some papers showing the entry back to see I am Proteobacteria that are very important here and also many other papers showing that beauty date forms are very important also human gut inflammation and this is one that we have been publishing in few years ago in gut in human science paper so showing that in fact a strain or genus that we have been isolating from chickens it's actually also found in humans and this is also very important there in gut information so the less abundant this bacterium is the more chance that people get got health problems okay and you can do exactly the same with the crowd can to write this uses similar shifts so it's all a bit related now of course butyrate formers are important I'm not going to focus too much on it but that means of course well it's booted production and butyrate is a very strong anti-inflammatory compound it strengthens epithelial cell layer integrity it increases expression of tight junctions it's causing cell proliferation and differentiation so it's a very important compound so also here measuring beauty rate concentrations could actually be of relevance especially when you also start measuring other fatty acid profiles and that's also something that can potentially be done now one of the postdocs in our laboratory has looked at all these 16s sequences and then just try to map functional pathways just we're just wondering in that moment what is really happening in a functional way and if you do that it's a little bit of a complex picture but actually what I want to point out here is that challenged animals chickens under a gut inflammation models they actually have depletion in butyrate production pathways and they have an increase in ammonia and hydrogen sulfate reducing bacteria communities and this is quite important as well because in fact this means that you can look at let's say quantifying genes that encode functional pathways just using quantitative PCR and that would also gives you some gotta love biomarkers I will come back to this in a few minutes it's about if you would be able to measure the potential of the bacteria to produce Beauty rate the potential to produce hydrogen sulfide to produce ammonia and try to bring this in a quantitative way you actually have a good idea of microbial health or intestinal health but of course by measuring functional activities of the microbial population just to explain this this is the normal situation a lot of diversity anaerobic conditions of course cause you're speaking about the guts the intestine high microbial diversity and a lot of butyrate producers this beauty rate is taken up this it's oxidized in the cells oxygen is used and this is used as an energy source on the other end you can have this well microbial population shift lots of diversity depletion of butyrate producers in an expansion of this gram-negative Proteobacteria which contain all ecoli like organisms and actually what then happens is that there are some problems in butyrate production butyrate is not taking up any more by epithelial cells you even have glucose being used by a Patil yourselves lactate being produced and it all becomes a bit more aerobic because what you get is immune cell infiltration these are granulocytes you get reactive oxygen species you get more or less while conditions that are not strictly anaerobic anymore and this type of condition also means that you are killing off stupidly anaerobic bacteria even more so it's like a vicious circle you're also killing of these tricky anaerobic butyrate producers so this depletion in this kind of bacteria is even further in expansion so again your self production of nitrates by immune cells you will have a reduction and production of Armagnac you will have sulfate-reducing bacteria just in fact oxygen but also nitrates sulfates used as electron acceptors by bacteria and production of different components that are toxic okay that's basically what it is saying healthy conditions let's cherish conditions that's what is happening I think this is now all quite well documented in different animals pieces also in chickens this seems to cure and in fact that means that as I just mentioned you can start quantifying this functional pathways if you make a ratio between this enzyme this gene bacterial gene that encodes this enzyme and you make a ratio between this one that is evolved in butyrate production and the other ones then actually also have a value that gives you a hint on intestinal microbial health okay this is giving you a hint on okay is the microbial community in the gut positive beneficial or potentially toxic that's basically what this is meaning so just well just to explain a bit more on on the mechanistic about mechanistically what actually this means for an epithelial cell why is this so important well this is an epithelial cell you have some transporters these are transporters for butyrate this is a mono carboxylic acid transporter another transporter you also have receptors these are receptors for Beauty rates amongst some other small molecules and actually what is happening is that these compounds such as ammonia for example produced under challenge conditions they block expression of these transporters hydrogen sulfide it's blocking better oxidation so again there's this balance you have butyrate then you have a lot of other metabolites that are harmful so there's always this balance that you have and you can actually quantify one or the other and make these kind of ratios luckily you also have some other compounds that bind to these beauty receptors and the difference is here that these receptors actually they promote anti-inflammatory activity okay so maybe a nice thing but that's not about Diagnostics maybe about feed additives is to try to figure out how we can activate these kind of receptors that will stimulate and if matauri properties okay but that's something is not of relevance in this specific talk I've talked a lot about 16s sequencing functional pathways of course you can also to metabolomics or meta metabolomics even looking at what type of metabolites are present in the intestinal lumen or if fecal material tell what you see here just just one specific example we have a huge data set of all different metabolites that are going up and down when you challenge chickens with coccidiosis Orchestra Liam or any dietary challenge for example and what you often see is that protein proteolytic activity is going up meaning you have some products here that are actually a result of protein degradation or protein fermentation and that's something that's very specific also for a lot of lactic acid bacteria will also enterobacter CI so this points to this shift I was just explaining cadaverine and putrescine these are poly amines these are well known in humans because they are really associated with colorectal cancer there it's completely irrelevant for chickens of course but still actually these are also compounds that are produced by the same populations so lactic acid bacteria but also a lot of enterobacter are CI so this is just an example if you can measure this kind of compounds you actually also have some kind of markers for intestinal health so these are all potential targets so I think using all these meta technologies you can just identify a lot of potential targets that you can quantify and then of course it's the major question will be these all works and experimental models but you have to apply that to field conditions okay and tests in the field and this is something we are currently working on so final part of the talk is about host responses so what we now had in this first 25 minutes says microbial biomarkers and actually there are a lot that you can actually use and this is something that is currently being developed so I hope that in a few years there will be something available but also a host site you can have a lot of things because if you look at the hosts you can look at go inflammation markers you can look at epithelial cell markers because if you have a Patil yourself that it will anyway end up in the feces okay all these molecules that cell's proteins coming from these death cells and also of course some proteins that leak out from plasma to the intestinal lumen and this you can also find in fecal material but only of course if they are not degraded and that's a major challenge you have a lot of proteins that are present host proteins that are like produced and end up in fecal material and that are much more abundant in challenged first as control animals but of course there are lot of protease is as well a lot of proteases that break down all these different proteins so you need to find stable proteins that are not broken down by proteases that are found in fecal material and then you haven't had an ideal biomarker that's what calprotectin is calprotectin is an infra matauri marker in humans that is very stable and you can actually find that in stool samples of course in chickens is a bit more complicated because stool sample is easy to give in chickens you want you can take fresh samples but ideally you should use something or later so there's one additional talk later on about this because these are three different models I'm showing here a coccidiosis model necrotic and to write this model and model I was just explaining in a few minutes AV will explain you about a biomarker that is found to be increased in abundance in all three types of conditions I will leave that for her but actually what I will do is just describe you a few biomarkers that you can find in this model okay and one of them we did some proteomic analysis some meta proteomic analysis trying to find proteins that you find in intestinal content in colonic content and a fecal material that are more abundant in challenged animals and actually one of them is this this is mild with protein one and this is in fact found in chicken granulocytes it's exactly the same type of molecule as calprotectin in humans but this is brought in that you find in granules or petrol filler granule sites or the chicken counterpart of the human neutrophil and you find that increased in colonic content of animals under Jana's conditions highly increased so it means that this is a stable compound it's a stable protein that is present when the animals have some kind of gut damage and gut inflammation because this is really pointing towards granular site infiltration in the gut wall so this is clearly a nice biomarker and of course this kind of molecules you can detect using Eliza and further on you can quantify them so just about in developing antibodies and setting up a test so another one I just give you a few examples we have a whole list of different proteins that you can actually find to be increased in abundance either in Jana's conditions but you also have some that tend to be well present at high concentrations in control animals and are depleted in challenged animals okay so you have like different variety of different proteins actually being present this is one it's called amino peptidase Eve why it's just involved in protein digestion okay and if you have food proteins of course they are first of all converted to two polypeptides in the stomach with them later on smaller peptides they are further broken down to amino acids and then of course taken up by epithelial cells and these amino peptide I see why this is just a brush border enzymes produced by epithelial cells it's present in the brush border to digest proteins actually what it means is that the more epithelial cells you have the more of this kind of protease or peptidase is being produced if you have shorter Feli or if you have less epithelial cells you will have less production that's one thing or maybe it's just less produced anyway but actually in this kind of inflammation models we have been using you see a serious depletion or decrease in allele and colonic content and also a fecal content material actually there's a depression or a decrease in concentration of these kind of proteins Conte enterin cherished animals so it's very some kind of an epithelial cell abundance marker have two other examples before I close this is fibronectin its effect compounds or a molecule that is found in a basal membrane in extracellular matrix and it also makes sense because you find it much more abundant in colonic contents of cherished animals and of course that's because you have some kind of gut damage okay if you have gut damage this compound will be released in the intestinal lumen because actually it's part of the basal membrane to which all the epithelial cells are connecting okay if epithelial cells are dying this extracellular matrix components and also basal membrane components will just be released in the intestinal content and actually you can also measure this now there are quite some other proteins that of all having to do with inflammation gut damaged even epithelial layer integrity and so on that you can actually find there and I think this is very promising okay because it's quantifiable in an easy way and the first thing of course is developing antibodies developing a lysis this is something we are currently doing and we are currently also just using field samples and using this kind of lysis systems on the fields amps check whether abundance of these proteins actually connected with animal performance if you have that you have something beautiful of course and especially when you would measure it like that see at two or three weeks of age and then predict what is going to happen at later stages so what next well we know that a lot of these technologies that currently being used will still generates much more data than I have been presenting is just a snapshot but ideally this is the way to go this is practical product in Si it's a bit of pregnancy stick but you can just dilute a fecal sample put it here it's like leather flow system and it can contain different markers if you want okay if you for example put three or four different protein markers on such a stick you have quantification of different ones and above a certain tres hold this will be positive or negative and gives you an indication and does not have to be used to for example evaluate feet additives to be used to evaluate intestinal health of a flock and so on I think this is something that can be very important for the future just the one but final slide I just looked into the literature about easy systems to measure this kind of but do this kind of quantification and actually have quite some companies that are already now having systems available but you just put this kind of dipstick in the smartphone actually read it okay just take a smartphone either you take a picture of the color or you just put a stick in the smartphone actually you have the value this is things very promising and there's quite a lot of papers in this specific journal biosensors and bio electronics I didn't know it at all but they describe this smartphone applications is biosensors so this is something I think this is clearly what we should do next final slide I want to thank a lot of people once all the people in our group and especially also of course Vanessa doing a lot of work in the animal modeling also AV doing a lot of work on bioinformatics and fin also doing a lot of work in the animal models itself and also the proteomics and metabolomics group at our University and of course also the multiple industrial collaborators we have of which some of them are on the sponsor list here and also the people from that work sport forum because actually if we do animal models and animal trials we often discuss it with them and also for analysis of field samples this is together with Martin and his company so that's basically what I wanted to tell you thank you [Applause] [Music]