so I would normally work on coccidiosis in terms of vaccine development but Martin Phillipe asked for some presentation on the role of I'm area in interactions with the microbiome so that's what are we going to do today I don't think I really need to explain to this audience what coccidiosis is so you'll be glad to know I haven't got life cycles and slides like that mmm I think the key point is that obviously coccidiosis is caused by AI Miria species parasites as Greg will say in his next talk coming up pretty much every chicken in the world is likely to be exposed to I'm area and in the absence of control many birds will become sick the way in which they become sick it would depend on where they're infected which parasites it is so it could be the small intestine could be in the seeker but important point to note is that we have quite a few drugs and live parasite vaccines and they are both very effective at controlling our Miria however neither a perfect so we know drug resistance develops very rapidly we know it's very widespread and there's increasing public and legislative pressure to reduce the drugs used in livestock production indeed we're gonna hear some more from Greg on this as well very soon as far as live vaccines go they're relatively expensive I mean on an individual basis they're not that expensive but if you compare them to the price of drugs prepared they are relatively expensive they're produced and they can only be produced using live chickens and so this gives an inherent limitation to the capacity to produce them but having said that billions of anti coccidia vaccine doses are sold every year historically that's been for layers and breeding birds but is very interesting especially in the u.s. seeing massive increases in the amount of vaccines sold for use in broilers and of course because of the interest caused by problems with drugs with vaccines there's a lot of effort now being made to look at host resistance so in which chickens may be resistant or susceptible to natural and meereen infection now historically from a scientific perspective we tend to compartmentalize so they've been studies focused on define strains of I'm area they might be strains that were isolated fifty years ago so you can question how relevant they are to what's in the field now we look at chickens but they're often specific pathogen free chickens there may be lines like light Sussex inbred lines again birds that are not necessarily directly relevant to what's in the field now there's a strong interest in hosts immune responses but of course if you're looking at SPF Birds and you haven't got the challenge in the field how relevant to those immune response is going to be as we've heard a lot of this meeting now there's a lot more interest lot more understanding in the microbiome and of course how this interacts with the chicken and particularly with a parasite I think if you're thinking about the bacteria and all the other components of the microbiome and the gut if you've got a relatively large pathogen they can punch big holes in the wall of the gut lead to appearance of mucus blurred lesions then inevitably it can have a profound impact on the microbiome and it's not just the host and the parasite obviously the environment is very important the environment of chicken is Reardon well impact to the microbiome as well it's food and within the microbiome there's components that we're particularly interested in so historically there were things like Campylobacter Salmonella Clostridium and as we move forward now especially with a more of an industry view we need to compare and look at all of these components in a combined system rather than individually so I'm going to split this talk into three sections in the first section I'll be looking at host resistance or susceptibility to I'm area so this is where we are looking at commercial islands to see if we can find the genetic basis of this resistance will then move towards bacterial issues microbiome so looks specifically at one bacterium that's Campylobacter before looking at the broader system that's the microbiome so why are we interested in natural host resistance - i'm Iria well so we're looking to select chickens that can pass handle the challenge better and can thrive potentially a dirtier environment and especially birds that could do that without drugs now we already know there's notable variation between chicken breeds there's some famous examples like the Egyptian for you me chickens which are very resistant to I'm area to nella and this could be in terms of colonization parasite replication and now there's other red lines such as the white McGowan chickens which tend to be much more susceptible to parasites like I Marianela again these aren't necessarily birds that you'd see on a farm how it's even more interestingly there's considerable variation between individuals and this is especially true when we're looking at some of the modern commercial hybrid lines where you can see individuals that are very resistant or very susceptible this is true for the cob for birds the Ross Birds pretty much all of these commercial islands so I'm talking about resistance to our Miri infection how do we define that well we've used three distinct phenotypes that we can use to quantify to assess resistance now the first you could say relates to severity of disease and this is a traditional lesion score so using the Johnson Reed system is very well known and we're looking primarily by Marianela and say hey we'll look for seagull lesions which will range from zero or as you can see up behind me apparently normal seeker all the way up to a lesion score of four where a bird may die you can see within the seeker that we have thick caseous plug lots of damage so we can say that's severity then we can also look at a consequence of infection a consequence of infection that we've looked at is variation in body weight gain so this is looking a variation perhaps within breeds or between different strains and lines or chickens and the example we have here it was published actually by NAT Bumstead and Ryan Millard where again they were looking at a range of inbred chicken lines as well as like Sussex and Rhode Island red and you can see by four days post are myriad another infection if you compare the body weight of chickens that were infected with the parasite or uninfected of the parasites there's an enormous difference and once this difference has happened it remains fairly stable even up to 12 13 days post-infection you can still see there's a big difference between these individuals the third measure we're looking at is a measure of disease response so how the chicken is responding to that infection and for this we're looking at serum interleukin 10 so this is a marker of an anti inflammatory immune response and in the example shown here as published last year there's a capture eliezer which is looking at aisle 10 in the serum and you can see in birds that have not been infected with our mirrior that they have very low levels of high or 10 in fact it's often undetectable with this assay but if you look in Birds 5 days after infection with I me return Ella you can see a considerable increase in serum I or 10 levels but what's very interesting if you look again behind me you can see there's an enormous variation between individual chickens both given high or a low parasite dose the very high levels of I or 10 or very low levels of I or 10 but by the time you move to 8 days post infection of course you know that the I me renew assists are pretty much left the chicken aura leaving the chicken to that point and the I or 10 levels are returning to normal so we've taken these three measures and we've employed them energy was so this is a genome-wide Association study now after this was supported by cops so we work with Cobb 500 to chickens where did this in three separate batches so we split our birds in two groups we gave some a high challenge of i merit another so it's 42 and a half a thousand new assists per bird and we chose this dose very carefully if you wanted to see a significant effect both on weight gain and lesions and then our control birds they would just mock infected with water nothing else we received birds from a commercial supplier so these are commercial cob 400 where 500 were three we raised them in commercial conditions up until the day 21 of age at which point we weighed them we expose them to my Marinella and then a few days later so four and a half days later we've taken blood samples and the two days after that we took another blood swab and religion scored for these birds and at that point we also collected DNA or tissue that we extracted DNA from so we could genotype each of these chickens what about the results well if we look at first at the range of sequel lesion scores you can see we had the full range now I said it was quite a severe parasite challenge and that's reflected here where you see the largest numbers of individuals had lesion scores three or four but you can see we did have nearly 80 individuals that had a lesion score of zero despite being exposed to such a large parasite dose we also saw that infected male birds had significantly fewer lesions than female birds which is not something you classically discover in the scientific literature but it's rare that you would study such a large group of birds forget the effects on body weight gain well regardless of infection males grew heavier than females that's no surprise you would absolutely expect that but if you look at the impact of infection and as I say once again we had quite a severe Amira infection and again you can see that the infected birds had a lower weight gain than the control birds all as you would expect and then we look at the serum il-10 levels once again the concentrations of the control birds of the birds who are not infected with I'm area had undetectable il-10 so there was no anti inflammatory immune response there but those that had been infected had a very variable level of aisle 10 so we saw significant differences between infected and uninfected birds but also between trials as I said we did it in three separate cohorts so you have to include trial as a variable in our analysis there wasn't a statistically significant difference between males and females you should see there's a fairly representation of male and female birds are both the high and the low levels of a or 10 but what is interesting if we repartition this data to look at aisle 10 levels against lesion score you can see that those birds with the highest lesions so leaders goes 3 or 4 also had the highest aisle 10 which if you think about it make so they've obviously had some dramatic injury in the gut and I alternate an anti-inflammatory so we took these data we put them into our genome-wide Association study so this is a statistical way in which we can take a phenotype or in this case the three phenotypes and actually match them to genetic data so that you can identify reach into the genome or better steal specific genes that will associate with whatever your phenotypes are now the key is from this large number Marcus there's 62,000 single nucleotide polymorphism markers the vast majority of them would not be linked to genes or parts of the genome that are under selection so they should be neutral but a small number those that are closely linked to genes that are under selection should be selected and you can see we've got an example here this is a secret lesion scores for all birds combined and there's two statistical cut-offs which is termed genome-wide suggestive and genome-wide significant and there were small numbers of these single nucleotide polymorphisms to fall into both of these groups so to give you a summary of the genome-wide Association for sequal lesion school we found five genetic markers three of which were segregating in both male and female birds - only in male birds and these were found to litigate on the Zed chromosome for body weight gain there were three snips even the males only again found on the z chromosome and for serum il-10 we found a single snip and this was only found in the female half of the data and that was located on chromosome 26 so it's quite interesting that so many of our traits and our snips linked chromosomes there's a sex chromosome we know there's many genes that encode interferons there thus genes which control chicken carcass traits so it certainly helps us to design in on what's likely to be behind these traits now perhaps more relevance to this meeting we could take as the same data and we can subject it what's called an eigen analysis so this is a statistical method to look at genetic structure within the data and I'll just show you the phenotypic matrix and that the bit that you could be interested in here is the correlations so you can see as you might expect body weight gain in Sikh religion score are correlated as leaves the score goes up what do you rate goes down not a surprise the same is the case for I or 10 and we see that Sikh religion score and I all ten have a significant correlation and I'll show you the data for that already oh it gets more interesting when we look at individual vectors within this group so you can look at the way chickens are responding in terms of our three measures and two groups that fall out entirely illogical we have chickens that we would consider to be susceptible so they have a low or negative trait loading for body weight gain and a high positive trait loading for sea collision score quite obvious for birds with high lesions don't very well they're susceptible we see the reverse in the second group these are birds which have good body weight gain have low lesions and they're considered to be resistant they see in both of these groups we have elevated I or 10 but what's really interesting is we have a third group which represents about a fifth of the birds were looking at in total and these birds had good body weight gain despite having good or high levels of lesions so these birds they have significant lesions there's damage to the gut but they grow anyway and so these are considered to be tolerant birds as we look at this in more detail the first group were they're susceptible chickens so you can assume that they're failing to thrive due to sequel damage they've got high interleukin-10 due to the damage that's been caused in the gut by the parasite but if we think about the role with interleukin 10 so interleukin turns an anti-inflammatory it'll be damping down things like interferon gamma and interferon gamma is essential to the immune response against iberia so it's a sort of no cause or effect it could well be that the interferon production here as a consequence they are 10 is leading to an immunopathology the second group they're these resistant Birds clearly these are the birds that we're most interested in they're potentially attractive to industry as breeding for these birds would allow you to look for those that are naturally resistant to I'm area to another infection that may be that they can overcome the disease they can become immune and until we return to a healthy state although we didn't wreck it out in these birds other groups it's very industry these tolerant birds so remember about a fifth of our chickens fit into this tolerant category an actual functional basis for this is not entirely clear but there's some important aspects there so if we're looking at birds that feed well grow well but are heavily parasitized they could become super shedders so these are the birds that could really drive contamination of the environment with feet with ear assists but another aspect we found is these birds are susceptible to sudden death so you could be monitoring them even hourly and you find if you look at them one hour they're happy they're walking around they're feeding they look normal and you come back and with an hour they could either have died will be very close to death so it really is a tightrope in these birds they can tolerate the parasite right up to the point when they can't and the last point or a decimate about this was the secret lesion scores so I said they were lower in males than females this agrees with some previous studies but it may well be to do with where these genes that associate where the traits are found as we know differences in inflammatory cytokine production or also occur between male and female birds as the males of course have two copies of the Zed chromosome and so practically if we are going to select birds in the future that are more resistant to our Miria it may be worth putting a greater selective pressure on the female lines than the male lines so you could accelerate the selection I guess that's host tolerance the way in which chicken is increasing able to live with the parasite we're now going to switch tack a bit and look at bacteria I'll start off with a specific bacterium can pilot back to judge annoyed so why were we interested in Campylobacter well traditionally it's been considered to be commensal in chickens although I think now it's widely recognized that's not the case especially in faster-growing chicken Lions the various reports associating it with Vibrio neck hepatitis diarrhea a range of other symptoms because the reason people were interested in it initially is because of course it's zoonotic it's a leading cause of foodborne infectious disease and in the UK in fact it's interesting now that Campylobacter outbreaks the most commonly associated with poultry liver pate or pathway that's been undercooked so in the past it used to be fecal contamination so Campylobacter being shed in the feces contaminating the surface of meat cause the problems but improvements in hygiene standards has actually reduced that as a primary cause of infection the UK so now we're looking at colonization of deeper tissues and chickens being more important so of course Campylobacter primarily colonizes the cecal lumen and chickens and if we think how do they get from the lumen to these deep tissues to the liver areas like that then clearly intestinal tegrity is very important and there's few pathogens that make bigger holes and make a bigger mess of the intestinal barrier than i Miria so we were speculating there could be a link between a Miria infection and systemic colonization of Campylobacter so we undertook a trial the co-infection trial so you can see at the bottom we had birds that were either left uninfected with Iberia they were given an attenuated vaccine strain of Jaime return Ella are they given a non attenuated strain and happen if you look at Oasis output we didn't find any air assists from the birds that were not infected with Iberia we saw some who assists from the attenuated parasite line and rather more from the non attenuated line that's what you expect but we then gave some of these birds are challenged with Campylobacter and some were left Campylobacter free and if you look at Campylobacter colonization so this is three days after the Campylobacter was administered you can see you get in our control group the group that had no can't be well they remain clear the group that had no Amiri infection have a high level of Campylobacter colonization in the seeker lumen but those that received I Marius are those that received the vaccine strain had significantly higher campy colonisation and those that receive the non attenuated strain had even higher levels because this is bad news we're worried about fecal shedding of Campylobacter and the surface contamination of meat if you've got a two or more log increase of Campylobacter then there's a significant problem but this is where the story gets a bit odd if you remember we were expecting if you had more intestinal damage you might see greater Campylobacter load outside of the gut and actually the reverse is true so if we look at Campylobacter in the liver again three days after can't be challenged we see around 3 log of campy but actually see about a one log reduction on average in birds that are been infected with Iberia and this is the same regardless of whether it was attenuated on non attenuated I Miriah of course this is essentially good news maybe that's why Martin looks so happy there he's celebrating if you like liver pate or pate and that then the fact that I'm eerie actually associates with reduced campi load could be good news I think these guys maybe they're celebrating as well there is red is he here he's talking later on so maybe they're celebrating the impact on Miriah has on Campylobacter or maybe not but the same is the case that what happened in the liver you looked at the spleen we see the same issue again you can see a fairly high level of colonization Campylobacter in the spleen it's reduced in the presence of attenuated on non attenuated I'm area now fair to say we were a bit dubious about these results to begin with it's quite striking but a little unsure so so we repeated it we actually on this several times now and you can see these two examples we were again looking at attenuated or non attenuated I'm area sampling three days after the can't be challenged and in each case we see an increase in Campylobacter load in the sequel lumen we see a decrease in the liver or the spleen the ones in brackets here were not statistically significant but they were a reduction because one question is asked if you speak to a bacteriologist is the three days post challenge a genuine colonization or as the can't be still transiting so we did the study again this time we sampled ten days post Campylobacter challenge and we're also interested to know whether the difference we see between attenuated and non attenuated Amir was because of the difference in parasite replication or the difference in the level of damage they actually caused in the gut so we in this occasion tried two different doses of the non attenuated I'm area and again essentially we saw the same thing so with the moderate dose so this is four thousand do assists per bird you see began an increase in Campylobacter in the sequel lumen you see a significant decrease in the liver non significant in the spleen on this occasion and it wasn't so apparent at a lower dose of just four hundred who assists and just to tie these two together we also looked at fecal shedding of Campylobacter a three days post infection and again you see that this increase in frequency of Campylobacter in these i'm area infected birds so what's the mechanism for this seems rather strange well obviously i'm erie is well known for stimulating some very potent immune responses so an example is interferon gamma this is quantitative pcr and you can see there compared to birds that receive no i'm area there's a very significant increase in transcription of interferon gamma it's about thirty to forty fold up regulated and this is the same for both attenuated and non attenuated parasite lines in action we looked at a whole raft of different interleukins who got interleukin-1 beta glucan six interferon gamma here aisle to aisle ten or some inducible nitric oxide synthase all of these are significantly increased by the presence of i mary infection but it doesn't differentiate whether your Campylobacter present or absent so it seems the immune response we're measuring is driven by the parasite rather than the bacterium the only difference we see is il-13 where this is generally reduced in a malaria infection but in a camp II only infection we see a modest upregulation so there is some potential here this could be different and it could have a functional effect I went on and if there's not any specific differences associated with an immune response what else could be important so we've looked Church the production of reasons so this is a mutant 5ac and again you can see in the presence of Iberia there's an increase in transcription both of the attenuated and the non attenuated the same is the case for me since 2013 and of course mutants and mucus are quite important especially the biology of Campylobacter as we know that chicken mucus is a place where Campylobacter can colonize where it can replicate so it could be if we're increasing the amount of mucus production you could be making an environment that's more amenable to the Campylobacter so we wanted to try and test this so we actually went to repeat our study but this time we included n-acetylcysteine in the diet as a mucus thinning agent now it's fair to say this is a fairly crude experiment because obviously if you're trying to thin the mucus it will have a profound effect on lots of bacteria not just Campylobacter but it was quite interesting results nonetheless so if we look in birds that haven't received NSO cystines they have a standard diet then we see what we now know is a reproducible trait in that if you add a Miria to a Campylobacter co-infection it increased the sequal lumen loader campi but when you add the n-acetylcysteine we see actually it seems to stop this increase so the Campylobacter still colonized but you didn't see the increase in numbers that you normally associate with the Ameri infection however when we look to systemic colonization looking in the liver we see in both case that there is still the reduction in the colonization of the Campbell about so while we're saying back here the an asset l-cysteine blocked the change you see in the gut itself in relation to the parasite it does not affect the change that's happening in the liver and the same is the case in the spleen so we've had to have two rather different mechanisms of work here so it's fair to say it's a fairly complex system we've got a working model and this was clearly an afternoon when I had too much time on my hands but you can see if we give a large Campylobacter challenge to chickens some of which will colonize so it may move into the mucus layer where it can replicate and of course some is flushed out we know transit time in the intestine is fast for chickens we're talking a couple of hours really now from here the Campylobacter can either stay in the gut or they can translocate out of the gut and we see they can colonize the liver the spleen or elsewhere of course we've added a parasite to the mix we can see that we increase mucus production some of which is collectively moved into the lumen of the gut and this is where we know campi can colonize and can replicate Dimity a proliferation of Campylobacter we also know that the parasite we're having a big impact in the gut so there could be blood there if you've got a high level challenge a considerable change in oxygen levels things like that and of course all of this again can favor Campylobacter make it a more nutritious environment for it but then the interesting part is of course we're reducing the Campylobacter to leave the gut now it could be that the increase in mucus is actually forming a physical barrier and is stopping them getting out so efficiently it could be that the immunity we're looking at and it may be these strong immune responses that are stimulated by i Miria maybe they're impacting on the Campylobacter as a bystander effect we can certainly say there's lots of interference sloshing around there's a strong th1 immune response is a big heterophile in flux maybe were just killing Campylobacter by accident well maybe there's some immune function going on outside of the gut we've done quite a bit of work looking at the impact of imary infection on a betta defensins for example but I'm not showing those real results today and actually goes the wrong way around so it's clean not to be too defensins it is worth remembering of course whilst these are mostly commercial birds in an experimental commercial setting it's not genuinely the field so we're now sampling chickens in the field looking for presence or absence of I'm area to see if this stands up as a genuine feature because you don't think well what about the rest of the microbiome that brings me to the last part of this talk so I think we've heard a lot about micro biomes at this meeting but I think I need to go into too much detail it's very clear that the microbiome impacts the production performance of a chicken it can impact on its immune status how it can control or withstand pathogen exposure it impacts the metabolism of that animal we could of course influence it with pre or probiotics with the choice of antibiotics we use if we have to use them because as we know there's no restrictions on their use and vaccines can also modify the micro floor in some way and we're interested particularly in what I Miriah does both direct infection of Iberia but also using I'm ear in a live vaccine so we come back to the study I mentioned right at the start in this large number of birds were using for the genome-wide Association we measured as I said lesion score for disease severity but we also had some birds that were left uninfected so we've got six groups of birds if you like here there's uninfected and this birds with the full range of lesion scores despite being infected with eye Marinella so we collected cecal lumen contents from each of these we extracted total genomic DNA we've got between 8 and 10 birds per lesion score category and these were then sequenced using alumina next generation sequencing we were looking at the v3 and v4 variable regions in the 16s right just in practical issues so we used flash to as a program to analyze and combine the reads we use chime to actually look at how we could filter the data assign ot use I think you came across this term already these are operational tax units the names we can give to divide our bacteria we then analyzed them using a series of tools available online so rarefaction curves which I'll show you in a minute we've looked at measures of alpha and beta diversity and when I say alpha diversity were looking at like the presence or absence of different Oh to use whereas in beecher diversity we're looking more at the levels of the odors that are present and we finished up by looking at differential abundance of bacteria using a program called dc2 so the first thing you do is we constructed rarefaction curves the idea here is as you look at more sequences you've generated of course you would expect to find more o to use but at a certain point you were saturate by them you the more sequences you look at you don't find any new Oh to use you found everything that's present so what you're looking for is for these curves to plateau because it shows you that you've found everything that's in that sample and so I say that most of these are approaching that plateau state so it's a reasonable data set now Rob showed you some slides like this at the beginning of the conference or be it from rather larger numbers of chickens and rather bigger pictures but you can see here the different phyla that are represented in this equal lumen microbiome because he firmicutes for example dominant in most of these birds there's a reasonable level of variation between individuals even within distinct lesion store groups and it's worth looking at the uninfected birds they're perhaps more different than the infected birds but there was no standard was statistically significant difference in foiler between the different lesion score groups we look at measures of alpha diversity and each of these is a different way of calculating alpha diversity so looking at observed shell 1a see Shannon Simpson and each one you can see if you're looking at uninfected and then lesion scores 0 1 2 3 or 4 there's quite a lot of diversity within that sample group but in each measure when you compare between the categories the averages are generally very similar and there was no statistical difference so this tells us the occurrence of tax at the occurrence of these ot use was not directly affected by I'm area another infection we don't see the loss the complete loss of any taxa we don't see any new ones appearing feature diversity was rather more interesting so this is a weighted Yuna frac plot just look at that plot it's rather difficult to tell we're talking about you've got different marks associated for uninfected birds and each of the lesion scores so what we can do is we can pull out some specific comparisons so in the top right you can see a comparison between uninfected birds which are the purple diamonds and those with lesion score four which are the blue triangles and you can see yes to some overlap but actually there is quite a significant clustering effect of these two on the bottom we're comparing infected but lesions caused zero birds with either lesion score three or lesions go four and again in both of these you can see there's quite strong elements of separation so there clearly are differences in the proportions of some of these bacteria so we can say the proportions of taxa are affected by only rat another infection so what's actually happening well if we put some numbers on this we start off by comparing lesion score zero Birds so these are birds that have been infected but have got no lesions that you can see when you compare them to birds with increasing severity of lesions so two the number of ODU's or species that vary increases and this is quite plausible because obviously we're getting a wider appearance in the phenotype that we're interested in but where it's really interesting is if you look at the uninfected birds as if you take an uninfected bird and you compare its microbiome with infected but lesion scored category birds while you might expect it to be most similar to the lesion score zero it's actually closest to lesion score two so the middle lesion score looks most like the uninfected birds and following on from Rob's comments at the beginning actual this is a trial that has been repeated and the data is being reanalyzed what probably not quite as we speak but certainly in a minute but where it gets really interesting you can see as you move up to loser scores three or four you've got again higher numbers of differentially abundant species or a to use but also as you move down the scale to lesion scores 1 or 0 and so we can plot this here this is looking at the number of different Oh to use versus those uninfected birds and so that's actually those in the middle that are most similar it's the outliers those that seemingly more resistant to the parasite or more susceptible that have also got the biggest change in the microbiome now actually look at what's going on within these micro biomes you can see comparing uninfected birds with infected you've got increases in ODU's associated with only you coli or Shigella we see decreased occurrence of streptococcus staphylococcus and many unidentified ot use perhaps was really interesting to look at lactobacilli and see the some lactobacilli are significantly increased in lesion score 0 birds some are significantly decreased in the lesion score 3 & 4 birds now if we look at just infected birds and compare these differences between 0 and the high Gleason scores we see an increase in facultative anaerobes in the high lesion score birds many entry back to EAC and again this is plausible because there's lots of blood there's lots of damage mucus so it's changing the environment in the in the seeker we see the Bacteroides that's almost disappeared not completely but almost disappeared in the lesion score 0 birds and again we have this comparison of lactobacillus so of course we could be looking at birds here you know as a cause or effect and these bacteria that are either doing well or not doing well as a consequence of iberia infection and the severity of that infection or are they actually contributing to either the severity or in this case the lack of severity so there's certainly some options for probiotics here so to summarize it's fair to say that modern chickens are very tolerant and we've selected them very intensively for performance and we can see now they can tolerate severe sequel so severe challenge with i Marinella so we have a mix between resistance and susceptibility but also these tolerant birds and we also see that we have a robust sequel microflora so even in the face of a severe eye Miriah challenge alpha diversity in the microbiome does not change and that's true until you push them too far when we look at these tolerant Birds in the face of the high-dose mortality can be very high and very very Swift so within an hour from Birds that seemingly healthy - clearly not we also see this variation in Campylobacter colonization and there's this really intriguing relationship between eye Miria and Campylobacter in the gut and the lumen of the gut and outside of the gutter in the liver or the spleen and this clearly impacts on contamination risks during processing and that's obviously food food safety and finally we can see that microbiome structure in terms of beta diversity does vary so just to finish up I'd like to acknowledge Sarah Williams McDonald who did the Campylobacter work and the microbiome work mat Nolan and K Bolton who did the G was genetics work and I particularly like to take a minute to remember Pete Kaiser and Steve Bishop who many of you may have come across both were working at Roslyn on the genetic mapping project and both unfortunately passed away during the project so I thank you for your attention if you have any questions I'll be very happy to answer them [Music]