[Music] thanks a lot good morning everybody sayings a lot Martin for this very kind introduction it's a great pleasure for me being here today thanks also to Philip for inviting me to present our research on history noises and the molecular repertoire of his terminus melodies as most of you might not be aware of the disease etc and the pathogen itself I will take you through a quick ride of the research periods how I call them and you will see why I call them research periods it all started in 1893 and this is the original description by Samuel Cushman of the disease he described the difficulties you had in turkey racing because turkeys were rooming over other lands but he also described the greatest obstacle however seems to be a disease that's carried of the young turkeys at certain age and that's the original description of blackhat disease later on in 1900 it was described in chickens as well by Chester and in Edie so in 1920 reclassified the pathogen as a flaky later system owners malarkey this a real landmark the work of a Tisa in coccidiosis research and also in his dominance research soon later it was discovered that one of the most important ways to transmit the parasite is via the intermediate vector at that time hit Iraqis Popolo so nowadays heterologous kalyanaraman and then the time period started to develop drugs because turkey production in the u.s. went down dramatically - to black hat disease how it's called as well and the first trucks developer arsenical trucks but then vilette's he made made a science paper and he published the efficacy of imidazoles flora solid own and then parama mice in an aminoglycoside antibiotic in 1962 by sick by Lindquist already published being effective as a prophylactic track not as a treatment immunological studies were carried out but the main conclusion was that you cannot prevail blackhead by vaccination at that time and genetic relationship between different chicken lines were investigated and after 1976 and it is nearly unbelievable there is no real scientific publication on histo Moniz history noses or blackhead research until 2003 and in 2003 I put up a paper of Larry MacDougall's group they showed that the transmission can also occur in an experimental setting between birds absent in the absence of the intermediate vector hierarchies and hetero sex so the situation which we face nowadays in the US and in the European is not resistance comparing to the talk in the morning and all a lot of other talks the problem we face is we have no trucks available anymore in the US with the withdrawal of histo start late in 2015 we have also no trucks anymore this is the situation on farms in Austria for example very complicated picture different modalities different intervention pyramids like Papa for this is an off-label use of parama mice in which people use this is a farm where we had blackhead several times on the same farm and for this farm it became reality the farmer has stopped turkey production due to the severe outbreaks there are no real figures on the importance of the disease in turkeys we know that in France it is of major importance in turkey production also for turkey breeders substantial outbreaks some latest figures are from the US in 2018 from Stephen Clark we have 127 out of 27 outbreaks in turkeys in Germany for example last year we had 1 million birds involved in outbreaks of which 150 thousands died or had to be killed had to be killed based on the statement of Samuel Cushman in 1893 this is again one phrase of his public and therefore he says therefore stamp out the disease when it first appears and that's something we do nowadays 130 years later again how could it look like this is a turkey barn the turkey toms are fifty three days old this is the day here and then you can see the increasing mortality you can have eight hundred nine hundred dead birds a day and at the end of the day you have ninety percent mortality but the mortality can also go like this it goes up to 20 percent and then it goes down again and these two situations more or less reflect an earlier study published from France where you see on the x-axis the mortality on the y-axis the frequency and you can see the major losses are in in in farms with mortality is below 10% so the scenario which you saw up here is probably the exception and it's not the rule now how does the lesions look like so if you miss everything in veterinary medicine and in poultry pathology you will never miss blackhead it's pretty obvious you have the substantial level lesions and you have to see collisions but it might look like this as well this is also blackhead these are turkeys and you won't find any liver lesions at all these are the livers and you can see the livers look pretty nice but if you look to the seeker then you can see the seeker I'm nearly erupting and full of seagull scores and this is the case which I showed you before where mortality went up to 20% and then it went down again what is the situation in chickens there's limited information in chickens to different groups our group in Austria and we L'Enfant group in the Netherlands did serology on onself develop the lysis and whereas we'll didn't found a difference between the housing systems we found a clear difference between the housing systems if you lose biosecurity in organic free-range in conventional to con eventually deep litter in comparison to conventional deep litter you see a substantial increase of outbreaks there are PCR papers from Germany for example and there is a paper from Poland showing that the type of production is P B and that's broiler breeder that it is an issue in broiler breeder and indeed it's an issue in chickens even so in the literature it might tell you that the chicken is just a reservoir and it's not a pathogen in chickens at all I think this statement can nowadays - and today be revised this is a picture I took about 15 years ago this is the same parrot inlay this is deliver and this is the seeker and that's something we do not see nowadays anymore what we see nowadays is something like this you do a post mortem and your first diagnosis as a veterinarian is calabasa Llosa's you see the peri hepatitis very severely and if you go back if you go further in your post-mortem you go to the seeker then you see the seagulls course and this is blackhead this is not coccidiosis and this is the same in all these cases which you see here you see a substantial thickening of the sequel war you see the seagulls course as for example here you see the coli bacillus is the Kohli parcel osis in all these cases and in clinical from a clinical perspective you are faced with a scenario that you have to treat then the Coley Coley infection of course and we see it in broiler in lair breeders as well and this scenario brought us up to picture where we thought could it be that histo Moniz forces the translocation of HIV shock orally from the gut to internal organs and what we did is we took three three isolates of HIV shakoora from the cecum from the heart and from the liver in case of kali barceló seized in connection with system analysis we did pfg analyzes and we found out of two hundred and fifty isolates one hundred and eighty completely were identical between the gut and internal organs so we do think this is a good proof clinical proof that mr. Moniz is a door opener for HIV Shakuni to translocate from the gut to internal organs if you in fact chickens SPF chickens experimentally and these are four week old birds then you can see you see substantial increase in seek relations and you see liver lesions but less substantial as in turkeys of course the chicken is less susceptible but you might see lesions as well but the seeker lesions are always present but of course you might conclude and this is shown also supported by this immunohistochemistry that mr. moehner system enosis is a gulf disease you see here turkeys you see the microscopic picture from turkeys you see the microscopic picture from chickens and this is the histology with immuno history histology and you see the parasites are innovating the cecal tissue this is the most glorious layer and you can see here the muscularis but in Turkey's it's much more substantial of course just to round up the epidemiology you have the parasite also in other bird species mainly in galliformes all galliformes species are susceptible for the parasite and for the disease peacocks for examples but also guinea fowls you can see here outbreaks which we just handle from France these are is a picture from Larry mcdougal these are quails and we did a substantial study also in the UK with colleagues on partridges on game birds so where does the problem start the problem starts here that's the isolation and propagation so this is one of our first cultures and I want to introduce you to the problem which we faced and show you how we try to solve this when you do culture from the seeker content of deceased or even maybe a healthy animal you see different protozoan parasites which are moving they are flagellates and you don't know if this is the same as this as this as this because they can change the morphology this is the first difficulty you have and you can see what I have put up here from blog and diamonds establish all these organisms in culture is far from a routine procedure growing them in the lab remains an art rather than a science so you can have a lot of inspiration and a lot of Howdy's how to grow them but this statement is not on his dominoes melodies this statement is on entamoeba histolytica which is a mobius in humans this statement is about Ballantine Koli in Carroll this statement is about diem and the entamoeba Fraggle is in pigs and humans so growing them is really a challenge what you do need is the rice starch as a carbohydrate source that's something they definitely need without carbohydrates you cannot grow them and you see they take up the carbohydrates I show you some better pictures later on now in order to solve this problem what do we have in the culture we came up with a very simple idea we remembered as veterinarians that in medicine we do in vitro fertilization on single cells and our idea was as you as those flagellates replicate grow by cell division we take a single cell under the microscope as you can see here and from this single cell we grow a whole population of cells and that means you have a clonal culture because the culture starts with a single cell and that's crucial for all later experiments which I will show you now so we did it for histo munis melaka this we did it for tetraplegia Moniz and these are already global cultures and you can see how they can change their morphology and this is plus the sisters is a protist formerly known as a funky quite important in disgusting humans as a as a sonic agent and what we did next is we assign those clones with the terminology of influenza viruses so we did this is system owners molecular this Turkey Austria this is the diagnostic number this is to clone six and this is the year 2004 so we can exactly with every single colony with every single clone clone we can go back to the outbreak and to the bird and based on this system having such clonal cultures available this made us brothers in fortune that we could develop various tools for example we develop diagnostic tools based on those clonal cultures we develop PC ours we developed Eliza in-house interact sandwich Eliza based on those cultures as well in the next step we raised antibodies this is a liver histology and you can see the liver cells and in here you see mr. Moniz Malacca this this is a bursa we developed in situ hybridization in the final stage the parasite invades disseminates in the whole organism and you find the parasite even in the brain in a turkey and this is a bursa you see the boards of four Lincoln Cent you see here inter follicular the coloring of the in situ hybridization and we use those cultures to test substances of course we tested a lot of substances herbal substances phyto biotics etc some of them showed an effect in veto but none of them showed an effect in vivo in chickens and in turkeys and we had a PhD student from Denmark with us for some time who was interested to test artemisinin Anwar artemisinin is a very important track in malaria therapy so approach of so and very important in humans of course and the idea was that artemisinin can be used to to cure to prevent a blackhead and you can see the in vitro data looks quite nice but we went when we did in vivo studies in chickens and in in turkeys we lost the birds and it didn't show any effect and then we went for vaccine development this was something we had in mind from the beginning I can say and we did a very simple approach again we used our clonal culture we passed our state 295 times every three days so you can see how long it takes takes about three years and then you are there probably or not and then we realized that the parasite is attenuated but it was not only attenuated which was questioned before quite substantially in 1967 by lund he wrote you can never attenuate mr. Moniz there might be attenuated strains around in the environment but you're not able to donate them but we were able to attend ate them because we start from a single cell and we did challenge studies different challenge that is even in chickens and this is a picture in laying chickens now and this is the non protected bird which stopped laying and this is the bird which was vaccinated so we did quite a lot of studies on efficacy but we also looked into safety what happens with our vaccine tentative vaccine and we realize when we do immunohistochemistry that our vaccine strain hardly evades from the seeker to internal organs it mainly resides in the cecum of turkeys and we did a study a very laborious study we did a reversion to virulence study which you have to do in licensing vaccines all kind of life vaccines we did five times back passages in chickens in turkeys and at the end of this we took the material and put it into 30 chickens again Turkey's again and we did a lesion scoring the ethanol aided strain in turkeys and in chickens versus the violent in Turkey's and in chickens and you can see that the attenuation is pretty substantial even after five back massages and bringing it back to 30 individuals again you have some seeker lesions in red but you have hardly liver lesions nearly zero and in chickens it's the same and we looked into immunology what is the immunological response on vaccinations we realized that interferon gamma even so it's extracellular protozoan parasite obviously that's our Syria at the moment plays a crucial role in the defense mechanism as well and just to show you how a vaccination experiment could look like these are the vaccinated Birds 15 days post challenge and these are the non vaccinated Birds three days earlier so at 15 days post post infection those birds are not alive anymore you can see the sulfurous diarrhea the birds are severely sick the maturity of birds has died off already and these are the vaccinated birds so the immunity which you can induce by a tentative vaccine is a pretty strong immunity as long as you get the birds vaccinated now leaving the field of the biology coming a bit to the molecular biology what do we know about the parasite and its molecular repertoire we do know that history Moniz malarkey this is a member of the treat Ramona day his closest relative is the under arm above Alice the entamoeba vocalist is present in pigs and is present in humans it's in a debate if it's a so notic agent or not it's isolated from healthy individuals but also from individuals with diarrhea it's very different difficult to culture the entamoeba fabulous as well so it's not another easy subject at all it's a flagellated protozoa of course system owners belonging to the class the Jamuna day as I said now based on 18s RNA typing some years ago we did a study together with colleagues in France and we detected that we might have two different genotypes of e.coli we find the 18s RNA typing still very reliable the maturity of isolates of types which we have from Europe are all genotype one genotype two is the exception and genotype two was exactly the field case which you saw before without any liver lesions so we think if you might come across such a performer of a logical picture it is it might be genotype 2 the separation into two different genotypes was first confirmed by using alpha acting in alpha actinin actin ins are very important proteins for this flagellated protozoa forest parasites because they need acting acting as for the cytoskeleton and to move forward and even if you use the RNA polymerase rpb1 you can see a clear discrimination between the two genotypes so we think this typing is very robust how can we use this typing go back to a field situation again occasionally at least in austria but also in France we have flocks where we have males and females together in a single shed they are just separated by this simple wire fence and occasionally there are reports there are reports in the literature that the male's all die heavy heavy mortality due to blackhead but the females are all healthy for a long time we asked ourselves the basic question are these females simply not infected and that's why they are not dying or are the females infected at all and for that in a recent study we did a lot of sampling we sampled us samples dust is still a good environmental sample which we established years back to at least raised the DNA of the pathogen and we sampled Sica from deceased males and we took cloaca swabs for males and from females and we did this on three different farms which faced the same scenario as you can see here and if you see this is from a this is from B and this is from C then you can see by 18s RNA typing that the positive samples from from B delivers the cloaca swabs the dust samples they all group together and they group together from farm C and from from a the rhesus we can confirm that obviously the females are infected but they don't die they even go to slaughterhouse at the end of the day we have no real explanation for that phenomenon what is known about the molecular data the molecular data heavily I rely on 18s RNA gene as I showed you for phylogenetic analysis there only a few studies reported on other sequences for example one of the first one was from a group in France they characterized three genes involved in hydro hydro some establishments hydrogenous ohms hydrogenous ohms our evolution of advice the precursors of mitochondria this primitive flatulates have no mixer mitochondria to produce energy as all our cells have they have what is called hydrogenous ohms and then we ourselves even a village together with Michael Abel we characterized alpha actinin and then the group of Robert Baxter did some work on cDNA libraries and better tubulin genes but the question when you are doing proteomics study when you are doing molecular studies there should be a video as well oh yeah now it's moving so this is histor Moniz you can even see the flagellum of the parasite here so we have a global culture that principle I explained you but we have a severe hindrance to perform detailed proteomic studies and the big hindrance is the fall what you see in the background and that's the wild-type bacterial flora because we still have the wild-type bacterial flora in the background of the eukaryotic cell so this parasite bacterial interaction is a very strange one we have zenyk are cultures which have undefined microbiota and this is what you see here we have a chronic absence of bacteria we cannot produce eggs anak pista Mona's cultures they are not published reproduced anywhere in the labs but we were aiming for monarchs and cultures and I will show you how we achieve that just to give you a picture how this relationship looks like between bacteria and protozoa this is the protozoan cell this is the nucleus this is the rice starch which you see here as well as as an important nutrient source this is the hydro china somes and what you see here inside by electron microscopy this is the same as here you see the bacteria this is the size dimension you see and the correlation you see so they photo so ties bacteria and this led to controversy on etiology for a long time people were not convinced that the Flakoll ate itself is causing the disease and it's unbelievable you have such a tremendous disease which I showed you on video and even in 1961 remember in 1917 six research stopped there were papers on the etiology of blackhead in 1966 another paper on the etiology of black heart disease and even Malcolm Reed made a paper in 1967 roading if the filterable virus theory the bacterial histor Moniz theory coughs postulate how can you fulfill them what is causing this disease is it really the parasite or not because you isolate different protozoan parasites as I showed you before and you have the wild-type bacterial flora so this shows you the parasite again here you see the flagellum much nicer well most likely after cell division and in the background you have the bacteria and if you take off the bacteria the protozoan parasite does not grow any more and it will die off but we achieved in a very substantial setting and very frustrating procedure at the beginning but quite luckily at the end to substitute the whole wild-type flora with a single bacterial isolate and the single bacterial isolate which you see here is in green this is a charcoal ETH valve alpha which we which we made genetically recombinant with which GFP so it's it's it's clean fluorescent protein and this is a cut through a single cell this is a single cell and you can see the history on our cell this is the nucleus and this is the ich aphasia Coley a chasing the mr. Munna cell and then you can see it's in the cell so this is the same cell so having this system available this is pretty smart because you can knock out the h valve alpha quite easily why are certain antibiotics and you can replace them with other bacterial isolates and then you can make monarchs any cultures which Clostridium perfringens with Enterococcus with salmonella with Pseudomonas whatsoever and you can investigate the impact of the bacteria on the protozoa which we are currently doing but you need those detailed cultures as well to do proteomic studies to study the proteome of your parasite because if you still have your wild-type bacterial flora in the background you do not know if a single protein comes from the bacteria or does it come from the parasite and that's why we developed assistance for the virulent and for the a virulent parasite going back to a single cell and both of them in the background of Eurasia call eth valve alpha as you can see here we did animal studies and we could show that the difference in virulence is mainly due to the parasite so even if you do a monoclinic virulent parasite in the background of the HVF alpha it kills the turkeys but with a certain delay of about one week whereas the a virulent still stays a virulent but what we do have available the biological system we have available is a defined system where all microorganisms in the culture are known we have only one defined microorganism this is the H of alpha plus system ona's instead of having histo moniz with hundreds of bacteria and with this setting we started to perform investigation on the transcriptome and the EXO proteome on the proteome and the EXO proteome of the parasite so this proteins which are released in the supernatant and which can act as possible virulence factors the strategy we did we did first of all transcriptome analyzes the noble sequencing of transcriptomes and we build up transcriptome database and then we did proteome analysis we did it on a child based methods we did 2d by Charles 2d died Charles and we did herself remasters what a mess technology and then we did bioinformatics and we did EXO proteome analysis we looked which kind of proteins are secreted from the a virulent from the virulent in comparison and we did it again by gel based methods and by child free methods and then we used the bioinformatics analysis and compared it with the transcriptome database which we established because there there are not many transcriptome databases on touch kind of regulated protozoan parasites there is of course information available for example on tissue Mona's vaginal is the most important sexually transmitted disease in humans and this is an outline of the transcriptome analyzes you can see we have certain context from the activated and from the virulent strain and the proteins identified are proteins which are involved in site or adherence for example but also proteins involved in destruction and in lysis of cells so we came up with a whole transcriptome map for the parasites and we went on to proteome analyzes proteome analyzes in 2d gel analyzes separating proteins by the isoelectric point and by the size of the proteins and we did it several times we purified the parasite get rid of bacteria as good as possible debrief them from serum which they also don't like and then we were running different gels in comparison to have to replicate and if you looked at those gels you might think they all look identical but that's not true if we go to the virulent parasite we have the majority of proteins on a low kilo daltons between 15 and 50 kilo daltons whereas on the attenuated one they are more beyond 50 kilo Dalton that might be due to the peptidase activity which we realize in the virulent strain in comparison to the attenuated strain which we repeat then also by selecting spots on the 2d gel electrophoresis and go on to direct on mullet of MS analysis and if you do this what you find is that you have proteins involved in carbohydrate metabolism you have peptidase active proteins and your plg plasma no gain binding proteins which are quite important plasma no gain binding proteins are important because they transfer plasminogen to plasmin and that's a important serine peptidase which is active in cells which need to migrate in the body like for example cancer cells like immune cells and pathogens use this as well and if we look to the a donated strain then we see most of the proteins are involved in metabolic processes in cell division but much less in peptidases and this correlates mr. morphological picture here you can see this is the very late form this is the Blair morphic attenuated form but if you put them under stress they look the same again and finally we look to the EXO proteome we characterize protein which the parasite secretes and in parasitology the proteins which are quite important are peptidases because as I said before they are needed to destruct extracellular matrices and you can easily control the secretion and the presence of peptidases and this is done in this kind of gels this is called cyma graphi you add shallot into your child's and then those places where the charity is digested you get white spots and you can see the comparison between the AVL and individual and the whiteness it's much more severe here and if you add TLC k which is a special inhibitor of cysteine and serine proteases in different concentrations and you can see you can inhibit quite well within the a virulent form but not in the virulent form so we think peptidases our important virulence factor just to summarize this transcriptome and EXO protein studies we found mainly system peptidases in the virulent form of which we sing our important virulence factors metabolism very important also especially in the attenuated form adapted to this environment different size categories of proteomes in proteomics on axial constitutions which started to characterize this peptidases now I took you through a ride through this disease and to the pathogen and I hope I could show you some of our investigations which we did since 2003 it's pretty obvious we have no syrup boutique no prophylactic drugs this is an economical issue but at the same time it's a very severe animal welfare issue if you see the turkeys suffering from blackhead this is a terrible disease for the animals so we sing defying cultures I try to explain you they could be the way to vaccine it would be the first life like elated vaccine developed in medicine ever to be honest and I showed you the advantage of molecular studies in a background of e.coli environment Sang's has to go to various corporation partners most substantial funding from Austrian organization like the Christian table organization like the Austrian Science Fund who did our basic funding on the broad term studies a lot of things has to go to the clinic especially to even a village here this is a picture from our last Christmas celebration and if your first are interested in the subject we have published quite a number of free reviews on this and as Martin said before we are hosting a parasite symposium together with the Royal Veterinary College in London and those who are first are interested in all kind of parasites infest aiding poultry you're cordially invited for that and we said I thank you for your attention thanks a lot [Applause] [Music]