Good evening and welcome to tonight's farm webinar on ruminant immunology. First of all, the housekeeping. I'm sure many of you joined us previously, but for those of you who haven't, we do have the opportunity for you to post questions throughout the presentation.

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Alex Corley is a farm animal vet, currently working as a lecturer at the University of Edinburgh. He's worked in clinical practise in Northamptonshire, Cheshire, Midlothian, and he completed his PhD in cattle immunology and vaccine development at the Roselynn and Morden Research Institutes in 2015. He now divides his time between teaching vet students, clinical consultancy and research, and his main research interests include antibiotic use and resistance, immunology and clinical veterinary medicine.

So over to yourself, Alex. Thank you very much. So this webinar really came from a vet record article about 2 years ago where they interviewed a number of vets regarding undergraduate teaching and CPD on vaccinology specifically, but also thinking about the new immuno stimulant products that have come to the market and will be coming to the market.

And that really identified that there's sort of a lack of teaching and resources available on sort of basic immune function and, and vaccine use in farm animals. So to try and address this, and given that we're under increasing pressure to reduce the antibiotics, that we're using and therefore, to think more about immune function and vaccine use, we've put together this webinar, which if you can bear with me for the 1st 20 minutes, where we'll just sort of recap the basics of immunology and some of the specific things we need to consider with ruminants. Can then go on to consider how vaccines work, and then some common questions that I get asked regarding how we can use vaccines and, and how they interact with immune function.

And then finally, to spend a little bit of time thinking about the immunostimulants that are now coming to market. So thinking about the very basics of immunology, we can steal some of the vocabulary of Donald Rumsfeld at the time of invading Iraq. And the immune system's really evolved to deal with, you know, a variety of different pathogens, those that are known knowns, and that's our innate immunity, which are things like our monocytes and macrophages.

And our NK cells where we've evolved to recognise specific pathogen features like flagellin or LPS. We've then got things that we don't know about, and I've split those into the known unknowns, and that's one arm of the adaptive immunity, and I'll, I'll justify why I've put T cells in this known unknowns category. And then we've got the real unknown unknowns where our immune system's got no idea what it may come across.

And it needs to be able to adapt to these new challenges and threats. And in that category, I've put the B cells, antibody, and plasma cells. So what about the known knows?

So, these, I use pattern recognition receptors to identify what are called pathogen-associated molecular patterns or tissue damage. And there are many different types of these PRRs and not wanting to get into lots of detail, but those called tolite receptors are probably the most studied. They're mainly present on epithelial cells and, and leukocytes.

They're also soluble PRRs. So for example, things like C-reactive protein and other acute phase proteins in the blood. So this schematic here shows the different TLR combinations and the sorts of things they recognise.

So for example, TLR4 is recognising LPS TLR 5, flagellin, we've got recognition from viruses and bacteria. And whilst this may seem like a rather non-clinical level of detail to look at these in, when we start thinking about the immunostimulants, we'll see that some of these are specifically targeting these aspects of innate immunity. Once these TLR receptors bind to a pathogen feature, that will then activate a relatively non-specific inflammatory cascade, and that will immune cells to the tissue.

It might induce an antiviral state, which is important when we consider how we might use live vaccines, sequentially, which I shall talk about later. And also, in some instances, they may actually be anti-inflammatory, but that won't be a focus of today's webinar. So think about vaccines.

Think about what we've put in a vaccine. And actually, many of the bits of pathogen that go into a vaccine are in themselves PAPs, so they're activating aspects of innate immunity, whilst also the adjuvants that we use in vaccines, may be PAMs in themselves, or may emulate, pAP features, so they may directly stimulate some of these receptors. So when we get activation of the innate immune response, we get a number of generic responses, which include things like increased cell turnover, increased mucus or antimicrobial peptide production, blocking of viral replication, and then activation of things like fibrocytosis and, and immune cell degranulation.

But we also result in activation of our specific re responses. So we'll process antigen and present that and activate BMT cells. And this taken from the common immunology textbooks is just a reminder that those innate immune responses we see really start being activated within minutes to hours and tail off within days, whilst their acquired immunity will take days to weeks to reach its full effect.

And again, when we come back to think about immuno stimulants, this feature in the short duration of activity of innate activation is really important in how we consider the use of these products in the field. So think about this time course whenever we're thinking about use of different vaccines and products. So antigen presentation is really how we link innate and adaptive immunity together.

And antigens or peptides, when they've been cut up, are presented on these molecules called MHC, so major histocompatibility complex molecules through two routes. So there's the endogenous pathway, which is proteins from inside the cell that chewed up. They're loaded onto these molecules and they're presented on the surface, and in fact most cells that are doing that all the time, both to their own proteins and with pathogen proteins.

And then there's the exogenous pathway, which goes on to class 2 molecules. This is, antigens from outside the cell which are processed, loaded onto MHC and then presented on the surface. And that's really done by the professional antigen presenting cells.

So, antigen presenting cells, you find them all the way through the body. They include things like dendritic cells, macrophages, and also B cells that are producing antibody. They don't just present antigen.

They're also providing co-stimulation and activation signals and cytokines to the T cells, and they set up the type of response that you see. And that will really govern whether a vaccine, for example, is going to push more towards let's say an antibody or a cellular immune mediated response or a combination of the two. So think about what cells are seeing your vaccine, what route the vaccines being delivered by, and therefore how that vaccine is being processed and what responses you'll be seeing, and over what sort of time course.

So moving on to the known unknowns of adapt to immunity. This is where I've classified the peptides that are presented on MHC, and they're recognised by the T cell receptor that's on T cells. And the T cell receptor is restricted, depending on the type of co-receptor that's on the T cells.

So we have CD4 T cells, which only recognised peptides presented on class 2 molecules, and you've got CD8 T cells, which are our killer T cells, which only recognise antigens presented on class 1 molecules. And here we can see the antigen processing cells providing co-stimulation to those T cells. And these cells have got a different function.

And once they're activated, they'll divide, and they'll differentiate into different affected cells. So activated T cells, they are responsible for killing. They're cytotoxic.

You can get these responses, at the site of infection relatively quickly after, say, vaccination. And they're also part of, anti-tumor activity. The CD4 T cells are the helper cells, and they're the ones that release all these cytokines to sort of promote and shape the response that you get down.

And broadly speaking, these are the cells that give you either cellular or antibody-based immunity. And it's how this interaction and this co-stimulation is driven, that will actually determine the ultimate response you see to your vaccine. Now, don't bother too much worrying about this slide, but it's just to say that classic antibody and cellular immunity that we were all taught as undergraduates is hopelessly simplistic, and there are actually lots of other, sort of skews the T cell response you can get, so you can get regulatory responses and, and a sort of a TH17 response.

But for the purposes of this webinar, we can really focus on antibody versus cellular immunity. So why call them known unknowns? Well, the T cell receptor diversity occurs in the thymus, and it's generated by somatic recombination of different gene segments, and you can get around 10 to 16 different TCR combinations.

Now, as those cells develop in the thymus, you get positive selections, only T cell receptors that actually recognise MHC on an antigen presenting cell are retained. And then you get negative selection, where self-peptides that are presented by stromal cells in the thymus are presented. And if the T cell recognises self-peptides, it's deleted, and therefore, only T cells leave the thymus that are known to recognise MHC but not self-peptides leave.

And that therefore allows us to avoid auto reactivity. But for that reason, the T cell repertoire we have is fixed. This T cell cannot become any better at recognising antigens.

It's fixed, it is what it is, and therefore, it's a known unknown. We don't know what it's recognising, but it, it can't change. That's in big contrast to antibodies, which, are the different harm of the adaptive immune system.

And because we can't predict every potential pathogen, and our T cell receptors only recognise linear fragmented proteins, we need those antibodies to recognise native antigens in their folded form, and they're produced by B cells, and initially, they have the same diversity as a T cell receptor. But during the time course of infection, those antibodies can actually increase their affinity. And that's why we have multiple doses with the vaccine.

It's one of the reasons why we have it, so that when we represent that antigen, that B cell and therefore the antibody response can become better and more powerful. This happens in the germinal centres of the lymph nodes, and we get mutation within the genes that encode the antibodies. If that results in a poorer antibody, that mutant is deleted, but if it results in a better antibody, we get amplification, and this process by which we can edit that receptor and end up with a higher affinity antibody.

We can then switch classes from early stage IHDM to a much higher affinity antibodies. And again, not trying to get bogged down in this table, but there are a range of different isotypes of antibodies that are produced, and early on, we produce lots of IGM which has lots of binding sites, but doesn't have particularly high affinity for our pathogens and our antigens. And then later on, so after a 2nd or a 3rd dose of a vaccine or later during infection, we get IgG responses, which are the main antibody of our secondary response.

It's worth reminding ourselves that in ruminants, IGG is a major mucosal antibody. So whereas in things like humans and mice and pigs, we'll be thinking about IGA in vaccine responses at mucosal sites. In cattle, there is a lot of IGG secreted, and that's relevant with the way that we design these vaccines.

So this process of somatic hyper mutation is potentially very dangerous. You could end up getting antibodies that are auto-reactive. And that's why we have T cells to actually regulate and control, B cells so that we can avoid autoimmunity by tightly regulating how auto-reactive these B cells can become.

B cells also need some TLR stimulation, so things like LPS or flagellin to maintain the sustained responses. So we need to think about that in terms of the adjuvants and, and PAs that might exist in our vaccine preparations. Just very briefly, you don't always need a T cell, to have an antibody response.

So B cells can actually respond directly to antigens, if they bind TLRs directly or if there's an antigen that cross-links a lot of B cell receptors on the surface of the B cell. These responses tend to be quite weak and transient, so vaccines that, that rely on these tend not to give a very sustained response, and we won't get any somatic hyper mutation if there's no T cell help. So there then needs to be a process where this links back to an 8 immunity, and the cytokines from these CD4 T cells, that are interacting with antigen don't deactivate these cells, depending on the SKU that that CD4 T cell sets up will impact on things like monocytes, or osinophils, epithelial cells, NK cells, and gamma delta T cells.

So what are these NK cells and gamma DL to T cells and why should we care about them? The NK cells are sort of the hit squad of the totalitarian state. They go round the cells in the body, making sure that they are presenting antigens on, on MHC class one.

And if they are down regulating that presentation, potentially because they're tumorous or infected with a bacteria or virus, then they'll kill those cells. They can also recognise specific pumps, but they don't require, T cell help, so they're rapid responders early on an infection. But we're learning more about NK cells, and it looks like that they possess some memory type functions.

And that's really relevant when we look at things like leptospirosis vaccines, where NK cells have been shown to be a really important part of immunity following the use of, of both, leptospirosis vaccines that are on the market. Likewise, gamma-delta T cells, they're a major T cell population of ruminants. So, unlike in humans and, and pigs, for example, they, they really are quite predominant, source of T cells.

They can recognise, antigens independently of MHC so they can look at things like, lipids and, and non-peptide antigens. And they are also a bit of a mix between both innate and adaptive immunity, and can have anti-inflammatory roles themselves. Just like the NK cells, they're really important in responses to leptospirosis vaccines.

So we can see here at 16 weeks after vaccination, the gamma delta T cells in vaccinated, animals are responding significantly compared to unvaccinated animals. Just finally, it's worth thinking about comp compartmentalization during, a response to a vaccine. So we may give, a vaccine, mucosally or experience an infection mucosally, that will go back to the lymph node and there'll be crosstalk between the lymph node and the, mucosa.

There'll be also crosstalk to other sites. So vaccination at one mucosal site can give you immune responses at the other mucosal sites, but they won't be as strong as that original site. You'll then get cells travelling to the bone marrow where you'll set up a central memory and then these can also redistribute around places like the thymus or lymph tissue in the intestinal tract.

What we're trying to achieve with the vaccine is a good strong effector memory response at the mucosa surface. It's all well and good having a, a nice memory in the central pool in the bone marrow, but if it takes a long time to get these memory reactivated, you might find that actually the infection has moved faster than the immune system, and therefore, the protection of the vaccine isn't going to be sufficient. So think about, you know, where is antigen being processed and experimentally, people are looking at actually delivering vaccines straight into lymph nodes, for example, rather than letting mucosal site, or thinking about intranasal versus oral or even rectal and vaginal administration of, of novel vaccines.

So, I've already mentioned, think about memory, we've got, Bs and T cells that will divide to give us this pool of memory cells, and they may be at that, affected site, so at the mucosal sites, or they may be centrally in the bone marrow. And it's worth looking at where antibodies produced following vaccinations. So, after a primary immune response to an infection, the spleen produce lots of antibody, but then the secondary response to the bone marrow becomes much more predominant.

And also, even places like the lung and, and the liver. But when giving a, an oil-based adjuvanted vaccine, that lump that you see on the side of the animal after giving the vaccine, that granuloma is potentially in many products, one of the major sites of antibody production, following vaccination. So again, it's worth bearing in mind we've given a vaccine under the skin or intramuscularly.

It, it may be that that granuloma that's formed is an important, source of antibody production subsequently. So moving on to some of the specifics now about ruminant immunology, the first thing that often comes up is things like pregnancy and immune function. Your pregnancy is a, is particularly part tuition, is a major time of production stress, and therefore, there's a lot of attention looking at, at how that impacts on immune function and therefore, how we could potentially change management or think about the use of vaccines and immuno stimulants.

So there's lots of misunderstanding around this. So, of course, the dam doesn't want to reject the foetus, and for that reason, the fetotrophoblast, so the placenta doesn't express MHD but it therefore does need to express some NK cell inhibitors to stop those totalitarian policemen from killing, the, the foetus. There are things that faecal glycoproteins and phospholipids and interferons that are modulating the maternal immune response.

And from all of the undergraduate teaching we have, we know there's a really strong TH2 humeral skew. Early in pregnancy. But it's worth noting that this happens early on in pregnancy.

And the changes that we see in immune function in late pregnancy are not due to this TH2SK. That's already occurred really early on during placenation. And it's therefore very difficult in late pregnancy and, and even early lactogenesis to really disentangle, the sort of modifications in immune function for metabolic demands, and immunoglobulin transfer in lustrum, for example, versus any impact of, being pregnant or the, the hormonal changes during pregnancy.

It's worth noting that, of course, infections that drive that strong TH1 or cellular type immune response will threaten pregnancy. So that's where, things like toxoplasma and the ospor becomes so harmful. But it's also worth recognising that a healthy, normal immune response is really important in both placental development and placental detachment, at the time of lambing or calving, for example.

And just to illustrate this, there's been a lot of interest in, immune cell function in dairy cows during the periparuric period. And we'll talk about immunostimulants, in a minute that, that target that. But what we're looking at here are the neutrophil counts of dairy cows, around the time of tuition on the left, and neutrophil function on the right.

And in the coloured in black line, we have mastectomized cows, and in the dotted line, we've got normal cows. And actually, in the run-up to calving, you see a profound neutrophilia in dairy cows. So not a neutropenia, as has been commonly cited by, by many authors.

And that neutrophilia is really important in terms of placental detachment and, and sort of the normal process of, of pipe tuition. What we do see though, and what's different between, our mastectomized and our, entire cows, is that the mastectomized cows actually don't have a reduction in immune function that lasts after calving. So in the run-up to calving, we get a reduction in neutrophil functions, so they become less able to, phagocytose or or produce, myelop peroxidase.

And then in the period after calving, if they've been mastectomized, that neutral function returns to normal, whereas if they've been left intact, then that immune function remains suppressed. And the implication here is that this really isn't related to being pregnant per se. It's more to do with the metabolic demands of late pregnancy and then the ongoing metabolic demands of early lactation.

We see similar things when we look at other cell subsets. So, we see here again, total leukocyte counts going up in cows around calving, that's mainly driven by the neutrophils. But we can see here a reduction in mononuclear cells, in entire cows versus mastectomized cows.

And that's really due to a big reduction in things like gamma-delta T cells and CDH cytotoxic T cells around the time of calving. So there are some big changes in, in numbers and in function, but in terms of neutrophilia, for example, there isn't a reduction in neutrophil number. There's also a lot of potential looking at nutrition and immune function, and the peripaturan rise in worm egg output in sheep is, is well reported and has been known about for a number of decades.

But what's interesting is that there are clear genetic effects here. So what we're looking at in this study is the nematode egg excretion of, of ewes of two different breeds. So we have mules in the white, and we've got Scottish black faces in the black.

And they're both nursing twins, and we've got those on low protein diets, which are, here and those on, the high protein diets, which are down here. So we can see with, views on a low protein diet, we see this increase in nematode worm egg output due to relaxation of immunity, but we don't see that in the Scottish black faces on the low protein diet, and we don't see it on the mules on the high protein diet. So there's an interaction of both genetics and .

Nutrition in terms of immune function in the periparent period. There's also an impact on nutrition on passage transfer, for example. So in this study in Ireland, they fed multiparus cows, silage, they fed them straw, or they fed heifers silage.

And they looked at the yield of colostrum, and unsurprisingly, heifers produced less colostrum than cows. But what was interesting is they also looked at the amount of immunoglobulin that was produced and cows fed straw produced less antibodies than cows fed silage and heifers produced less antibodies than both. And they followed this through to the calves and found that it was quite a significant impact on average blood IgG concentrations in the post-cholesterol calves.

So cows-fed straw had carbs with lower blood IgG levels. So we've therefore got a number of examples during the peripartuan period when nutrition is impacting on immune function and metabolic demands are impacting on immune function and an interaction with genetics. So just to look at this in terms of a case study that we dealt with up here in Edinburgh, we have a local 250 cow, spring calving herd, mixed laying, Angus and limousines.

They finished their bulls at 12 to 15 months, and the heifers finished around 18 months. Herds BVD free, but it still vaccinates for BVD and uses IBR and lepto vaccines as well as road to vet corona. It's a relatively high risk.

Yoni's herd. It's got no history of salmonella, and they use two rounds of synchronised AI followed by sweepables. So they carve very, very quickly, and intensely, and they started carving on the 15th of March 2015.

So not long after the start of carving, 23rd of March 1 of our vets got called out to look at a couple of carbs that weren't right. And one had onalittis and the other septic arthritis. But then later that afternoon, Stockman called us back out again, because they sort of identified 5 carbs that really weren't right.

It's 3 to 4 days old, we dropped tears. 4 had a mild nasal discharge. So nothing really particularly outrageous on, in terms of rectal temperatures, but there was one animal approaching 40 degrees.

And at that time, with this sort of increased concern over neonatal calf health, we were potentially concerned about failure of plastic transfer, but we were also considering differentials like DVD, salmonella and hisophillus and mycoplasma. So we did some, calf GGT and ZST testing, just on a random selection of calves, so not particularly ill calves. ZST isn't a particularly specific test for, failure of plaster transfer, but it's a commonly used one that, that many vets will be familiar with.

And we can see that just in a random selection of cars, you've got 123456789, 10 out of 12 calves with a ZST result below 20. And we could argue about the cutoff, but if you went for a cutoff of 12, which will have a higher specificity, although a low sensitivity, we've still got 1234567 animals with significantly, poor passive transfer in this herd. In a herd that hadn't really had any major changes from previous years, and no real history of failure of passive transfer problems in the past.

Just a little note about GGT, Closterol GDT is taken up by calves and it decays very quickly in the first week of life. So there's a conflict in the literature, and it is often poorly correlated with blood IgG concentration, but it can be used in carbs that sort of a couple of days old as an indication of cholesterol consumption. And various cutoffs that are proposed and different labs use different cutoffs, but it's sort of one day, we're looking at around 200 and 4 days, we're looking for around 100 units per mL as our cutoff.

And we can see here that there are a number of cards in this early category where actually the GGT levels are really quite low, suggesting that actually they probably haven't really drunk very much lostrum at all. So it's not just in a matter of necessarily poor lostrum quality, although that might have been a feature of this outbreak, but potentially also poor lom yields in these calves, as well. So, as this problem continued, we looked at the calf records in the first month of the calving block, and we plotted them out by the day the calf was born in the calving period, and whether that calf required antibiotic treatment or not.

We also plotted it by the age of the dam, just to see whether there was an effect of being a heifer versus an older cow. And we also did the same for mortality. So there were 6% of the calves actually died in the 1st 30 days of the calving block, and a third ended up being treated with antibiotics.

So there's a major issue with, with calf health in this herd. So we looked at the ration, thinking, you know, why, why have we got such problems with failure of passive transfer? And not an untypical ration for around here.

These cows were on 2 kg of reasonable average quality grass silage, 5 kg of whole crop, 2 kg of kilos of distillers grains, and then they're allowed to eat to appetite, for the remainder of their dry matter intake, straw. And we metabolic profile of the herd, and we looked at butyrates and Nephers and saw that really they were generally within normal limits. There's one cow here with a mildly elevated Neer, but no obvious signs of an energy problem in the cows in this herd.

But actually, nearly all the cows coming out with low urea nitrogen results indicating that the ration, was, was pretty marginal with respect to effective room degradable protein. Therefore, the implication in our view being that poor EIDP supplying this herd potentially resulting in a poor immunoglobulin production by the cows and therefore, poor passive transfer and health in the calves. So in this herd, we recommended that they supplement the cow ration with a high ERDP protein source.

We would probably have preferred that they went for something a bit more cost effective and, and rich in ERDP like rape meal, but they decided to go in with some soya. We suggested they sourced lostrum from a dairy of the same health status. And to really focus on, supplementing the at-risk carbs and, you know, identifying and treating sick carbs very quickly.

And as mentioned on the previous slides, about 32% of the calves born in the first few weeks of the carving block required antibiotic treatment, and in total, 10 died. After the diet change, we was, we noted a significant improvement in calf health, and actually, only 11% of the calves born subsequently required any antibiotic treatment and, and none died. So whilst this is still quite anecdotal and something that we're actually pursuing in further depth with, with HDB at the moment, in this herd, at least, it did appear that supplementing these cows in the last month of gestation with additional protein did improve that immune function.

In terms of antibody, transfer to the cars. Now, we're certainly not advocating that all suckler herds are feeding 1 kg of soya in the last month of gestation. But there appear to be instances where, clusterm production and antibody transfer, is potentially dependent on that protein supply to the cow in the run-up to calving.

Just because I get asked it quite commonly, you know, what sort of cluster replaces do we recommend? There's very little data out there, but Murray, Cork and Cambridge did publish a few years ago, a comparison of IGG, supplied by the recommended dosage of the different clustering places on the on the market. And you can see that the, the best product that he's tested subsequently to this presentation was Euol Platinum, which gave about 80 grammes of IgG per sachet.

You can see that significantly below even the sort of 400 grammes that heifers are producing and, you know, nowhere near the 800 grammes that, cows being fed silage are being produced. So really in terms of clustering replaces, clustering from the dam or from a cow is by far superior. So changing tack a little bit and now moving on to specific considerations of, of veterinary vaccines that we use in, in ruminants.

So it's worth reminding ourselves that most veterinary vaccines were developed empirically. Nearly all of them can be fitted into sort of three categories, either live attenuated, they're killed whole organisms or, or they're axo. And the method of attenuation of the vaccines we use is really often poorly understood.

So attenuation may only be very modest, and it may depend on the dose or on the, route of administration. It's often been attenuated through serial passage in vitro, but there are more modern vaccines. So, for example, Bevila has been attenuated by design through the deletion of the ER and the Erns genes.

And that's really the big comparison between, say, something like a live endotic abortion or or vaccine, which has really got minimal attenuation and is really relying on being administered at a time or through a route through which we don't see a production limiting disease problem. Versus a vaccine that's been made safe through a very almost surgical approach to to spine. There's also a few odd ones out there.

For example, Husqva, which has been attenuated by radiation, therefore preventing the L3 developing into reproducing adult, parasites. And of course, the major factors that really underpin, the, the way vaccines are being developed for veterinary use are really price and convenience of administration, rather than necessarily what might be an ideal vaccine formulation or vaccination course. So it's worth just, reminding what toxoids really are.

So toxoid is a modified toxin from a pathogen, it's no longer toxic, but still elicits a neutralising antibody response. And that can be done physically through treatment with chemicals like formaldehyde or glu aldehyde or heat treatment. And most of the, toxoid vaccines that we're using for, ruminants would be, physically inactivated through glu aldehyde treatment.

And then also by rational design, and, and we don't have any of these ruminants, but the sticks2E vaccine that's been developed for pigs. There's a specific mutation that's been made at the, Active site of the toxin, so that the toxin still has its normal confirmation, you're still getting an antibody response, but the molecule itself isn't actually harmful and therefore can be included in the vaccine. Certain vaccines also depend on specific growth conditions.

So if you think about how, Manheimia vaccines, they own both, Australia and sheep in Mannheim and cattle. They've been developed through, culturing bacteria in certain conditions. So we have here, A, a gel produced in the lab, looking at the proteins produced by bacteria that have been grown in the animal, and then bacteria that have been grown in iron restricted conditions.

And we can see when you compare that to being grown in normal lab media, we get these additional proteins being produced in iron restricted conditions. So quite often the way the vaccine is grown, the way it's produced, will determine what, proteins it expresses and therefore how useful it is as a vaccine. And vaccines based on being grown in normal conditions are not really very protective, whereas vaccines grown in unrestricted conditions because of these additional proteins are protective.

So what sort of challenges do we have going ahead with, with vaccines? You, how do we get a better response, at a slight distance to the route of administration? So if you give an injectable vaccine, intramuscular or subcutaneous, how can we make sure that's directed to a mucosal site?

How can we target different, antigens between strains? So do we need to be looking for better conserved antigens or, bugs that are escaping the vaccines? We need to care for what we do if there's no protective immunity.

So Barber vax, which is the Hemonuss vaccine developed by Mordon and used in Australia, needs to be delivered every few weeks to continue boosting antibody levels because there's no natural, antibody production. We need to think more about how we ensure vaccinated animals maintain an intact immune system, so that might be through better, nutrition and periprour management, for example, or it may be through, genetics, and we're seeing that with certain semen companies, launching branded products from, semen products from animals have been selected for the better immune function. We also need to make sure we can maintain these immune responses at the site of infection so that we can get a good response, at that site for especially for infections that may move faster than an antibody recall response.

So things like pastoralosis. And then we also need to think about how we get the right type of response. So we're looking for antibody or we're looking for a cellular response, and how do we design our, our antigens and, and our adjuvants to, to achieve that?

And then finally, we need to think more, much, much more about the factors that are affecting field performance of vaccines. And you know, true vaccine failures are very, difficult to firstly demonstrate and then actually investigate depth. There are often many holes simply in the way a vaccine is handled and, and administered, before considering whether the vaccine itself, was efficacious or not.

So some common questions that we're often asked about vaccines, that I thought would be useful to go through today are what are the differences in the immune response to sort of live versus killed IVR vaccines? Are there reasons why they can't get multiple vaccines concurrently? Why does MDA not protect very well against BRD pathogens, but yet it interferes with vaccination.

And then also, why is it that some vaccines have a single dose claim? What is it about those that, that make a single dose appropriate? So the first one on, on sort of live versus killed IBR vaccines is, is relatively straightforward to answer.

If we look here at antibody responses, to IBR vaccine administration, we can see here in the triangles a live intranasal IBR vaccine, where there really is no antibody response at all. However, if you give that live vaccine systemically, then we do see an antibody response in the blood. But it's not as strong and it's not as sustained than if we give two doses of the killed vaccine systemically and we see that antibody response lasting a lot longer.

Actually, in terms of viral challenge, it's not a big difference in terms of, viral shedding. But there is a big difference in cellular immunity. So the live vaccine, whether it was given intranasally or intramuscularly, gave a really good interferon gamma, so it's cellular immune response, following challenge of cells taken out of the animals, whereas the killed vaccines didn't really give us that response at all.

So the implication really between live and killed IVR vaccines is we're seeing much more cellular immunity with the live vaccines. We will be getting mucosally targeted responses as well if they're given intranasally. But for a really strong and sustained antibody response, then actually the killed vaccines are important there.

And that really is what underpins the current IBR vaccine protocols of starting with a live vaccines given systemically and then followed up by a kills vaccine given systemically. So, the next question about giving multiple vaccines concurrently, are there any reasons why we can't do this? It's commonly done in the field, but there are, relatively few products that are licenced to be given concurrently.

And there may be reasons why this is not a, a good idea, but have we really done the work to look at it? And quite often I'd advocate that it's probably more important that animals are protected, from a vaccine at the appropriate age. And prior to the expected period of risk.

And if that involves making an informed decision with your clients about giving unlicensed vaccine combinations, then that may be what's necessary within that system to provide the protection in time. So in humans, there's been a lot of work done, and actually, both serial conversion and adverse events following vaccination are similar, where the vaccines are given concurrently or sequentially. So that would imply that it's generally a, a safe thing to do.

And that sort of makes sense when you consider the immune system has evolved to cope with multiple pathogens at the same time. There is, however, some evidence in humans that giving two live vaccines sequentially can result in an increased failure rate of the second live vaccine. The reason being for this, that there's potentially an anti viral response that's triggered from the first vaccine, and if that anti-viral response is still active when you give the 2nd live vaccine, it may actually prevent that second live vaccine from replicating and therefore interfere with the immune response.

So giving multiple live vaccines concurrently as you see with sort of RSV and PI3 intranasal preparations where they're both live and they're both in the same, product, seems to make sense, but giving them, shortly after one another sequentially could actually impact on the performance of the second vaccine. So sort of general advice, you know, we really do need to give different sites because we do want to avoid any interaction between different antigen adjuvant, combinations. I suppose giving different sites may, reduce that theoretical risk of immunodominant, antigens in one vaccine, potentially interfering with responses in the second.

And theoretically, it may actually ensure that that T-helper cell skew is not affected, . But, really, the, the advice to give at those different sites is really trying to avoid interaction between the constituents within the products. So next question in terms of why is MDA not protect very well against, BID pathogens, if it interferes with vaccination.

Really sort of, it's worth looking at these sorts of, graphs where you can see the maternal antibody in the calf dropping off over the first few weeks of life. And the tides are required for protection is usually relatively high. So you need a reasonable amount of antibody and it may only be sort of 2 or 3 weeks or even sooner when that maternal antibody drops below that tights required for protection.

But actually the tier of maternal antibody that inhibits the kind of response is often quite low, and there's there this gap between the waning of maternal immunity and the time at which maternal antibodies is not interfering with vaccines anymore. It's worth noting that not all protection is antibody mediated. There is a, this important cellular component.

And also that maternal antibody levels against BRD pathogens may not be particularly high. So, you know, the, the, there's good reasons why just the antibody in the clusterm alone may not be that protective against respiratory pathogens. So antibodies in a maternal antibodies can inhibit B cell function, how they may directly bind to B cells, and that's something that's well established in patients with myeloma, for example, where high antibody levels inhibit, inhibit B cells.

But also, they may just be simply binding with the, antigen, and therefore masking it, in the vaccine. So, yeah, MDA may also inhibit the replication of, of live vaccines as well. So there's a whole host of reasons as to why MDA may interfere, with, with a vaccine response.

So what sort of advice have we got? And there was a nice, review by Amelia Wolrems, in the bariatric Congress a few years ago, and, the sort of general principles we can use when considering MDA. The calves, they generally don't serro convert if they've got high levels of MDA following vaccination.

But cards, vaccinating the presence of MMDA do often demonstrate an animastic response when they're then challenged with the pathogens. So they are developing an immune response, it's just not necessarily a very, good antibody response. And the most reliable responses we can see, in calves with MDA are when we use modified live vaccines.

If we give a couple of doses 3 or 4 weeks apart, and when we use mucosa, so intranasal vaccination routes. It is worth noting, however, that if there are very high levels of NDA that will still, suppress the responses. And then finally, why do some vaccines have a single dose claim?

And I suppose the simple answer for this is because somebody did a protection study during licensure and they demonstrated satisfactory protection. And, you know, we killed vaccines, the best protection is nearly always seen after 3 doses, and most vaccines are only 2 dose courses, and there's a lot of market pressure to reduce the number of doses, and that's sort of why we end up at sort of 2 and ideally 11 dose. But if you look at the feature of vaccines that have a single dose claim, they're either live vaccines, so things like Davila, for example, or the IVR PI3 and NRSV live vaccines, or they're potentially primed through field exposure.

So, the, scal vaccines and also Manheimir vaccines where it's probably, a relatively high level is commensal within the population. And really, we only want a relatively short duration of protection for the sort of scour, pathogens, for example, following vaccination. So in the last few minutes, I just want to go over immuno stimulants because they are likely to be more coming onto the market and understanding their mechanism of actions.

It's quite important to appreciating how they can be used appropriately. So they were classically developed as adjuvants for vaccines, and they're really targeting innate immunity. Just think back to those graphs at the start of the talk in terms of the time course of an immune response.

And of course, things like Levamazole have been used by vets for their sort of non-specific immune stimulation effects for decades. So now in, in, a number of countries, including the EU there's bovine, a granulocyte common stimulating factor, BGCSF, which has been licenced to reduce the risk of clinical mastitis during the peripartuan period. And then in the states, Zelmate, which is a CPG liposome, has now been licenced to help reduce BRD lung lesions and the mortality associated with manhya.

So the background of GCSF is that it's a cytokine and it stimulates bone marrow neutrophil production. It's been used in humans, for years for chemotherapy, and it, it does enhance some neutrophil functions in periparur calves, cows, sorry, but not in calves or in humans. And it was originally investigated, if you look in the literature as a pneumonia preventative.

So here we've got, the percent of lung affected following Mannheimer challenge, which there's a big reduction of these animals are being treated with GCSF before challenge in low tier, low antibody titer animals. For some reason, it never got brought to market for this use and it's been brought forward for mastitis, and it's been peggyated, so it's had a molecule attached to it to increase its half-life in the blood, although we don't actually know the pharmacokinetics of, bovine GCSF, in vivo. So it's been developed to counteract this peripractuan, reduced neutral function.

Remember, peripractuan cars are not neutropenic, but when you give it, you get this huge neutrophilia, during that peripractuan period. Unsurprisingly, it didn't provide any protection following intramammary E. Coli, challenge, 5 days postpartum.

I think that's probably fair enough if you put a very large number of the E. Coli into the edge of a cow, be huge amounts of endotoxin there, and nothing you do is really going to protect those, those animals. But, given 7 days prior to and then around, the first, day, of, of milking, it does actually reduce mastitis in, in some of the field trials.

But administration simply, around 1 day milk had, no effect on mastitis. So this product had quite a ropey, ride through licence. So when it went through the Committee of Veterinary Medicine Products in the EU, it was approved by 24 out of the 31 committee members.

But 7 members did actually sign a divergent opinion, noticing that on one farm, there was actually a 7% rate of hypersensitivity, even though the overall rate was about 0.7%. They felt the benefit was relatively marginal, and that the product was, had a narrow therapeutic index and I think they're probably being a bit hard on this product, given the number of animals actually in the test groups, but they felt that the increased number or apparent increased number in, amass er erosions was a sufficient concern for them to sign a divergent opinion.

So it's not to, sort of knock or be particularly pro, any one of these products, but just to point out that a lot of these, responses, a lot of these, products are quite non-specific and there may be potential and certain concerns for side effects. Just finally, the CPG liposome, so the Zalmate product that has been developed in the States. This is essentially a pump, you know, it's, it's bacterial, DNA, and it's recognised by the tolar receptor TLR-9 and in green here, we've got what happens in cattle, and it results in production of a wide range of pro-inflammatory responses.

So, we're stimulating B cells to proliferate and to produce cytokines. We're stimulating NK cells, and we're also stimulating, pro-inflammatory. Sites kind production.

When it's given to cattle, there's not a major effect on sort of temperature. It's not a major effect on, on sort of peripheral neutrophil count. There's sort of a bit of a spike here after the first administration, but really not much after the second.

But there is an increase in, in haptoglobin production, by the liver, so you are seeing a, inflammatory response following the administration. And in cattle, they've, this is not peer reviewed, but in their technical manuals, they've, given their product to, beef animals before challenging them with Mannheimer, and then looked at the lung scores and, and seen a significant reduction in, in lung scores following challenge due to presumably activation of, of innate immunity in the respiratory tract. In feedlot trials, again, being in North America, they're sort of slightly, different to what we might be used to.

They've compared Zeln administration to group treatment with Mycoil on arrival to the feedlots, and haven't really found any difference in terms of time to, BID treatment or mortality, when, when looked at statistically. So really being considered as A product that can be given as an alternative to antibiotics on the way into, into feedlots, at the high risk of respiratory disease. Don't worry too much about this slide, but it's just to point out, if you look in the literature, there's now lots more on the horizon.

So this paper has screened lots of different TLR agonists, against their ability to increase the amount of tracheos, so, antimicrobial peptides, so tap production. And you can see here that TLR 21 and TLR26 agonists produce or stimulate and increase production of, of, this antimicrobial peptide in the respiratory tract. So there's an area of ongoing interest and, and one where I would suspect we'll see new products coming to market.

So it was a very quick whistle stop tour through immuno stimulants there, but, in terms of, general principles and, and how to sort of interpret, the data on these products, just remind yourselves that they're non-specific in their action. They're quite varied in the way they're working. The effects are likely to be quite transient, and for that reason, that time of administration is absolutely critical, so you have to understand disease risk in the herd, and when you're trying to target it.

And just be aware of, of potential side effects that we might see with some of these products. So that's it. I've run over by 4 minutes, but I'll hand back over to the moderator if there are any questions.

Thank you, Alex, and I think we'll let you off those 4 minutes. Don't worry, you're very disciplined there. Never, I was coming from a non-veterinary background, never fails me, to, never, I'm never failed by the, wide range of topics that we have available in this one today was, one of those that we haven't covered that much, so it's great to, to put it out there and to, it'd be great to hear your, the people who are listening, your feedback, any questions you may have, please don't be shy.

I suppose, just looking at it, Alex, obviously, you know, farmers traditionally very traditional in their approach, and very cost sensitive. So in terms of, you know, when you're talking to the farmers about the different methods, and also, as you say, as new ones come on online, how are you finding their response? Are they very much, well, this is the way I've always done it, so I'm not interested in what the science is behind it, or are farmers becoming more receptive to alternative ways and introducing new methods?

So I think, you know, depending on how sensitive they are to the pressure to reduce antibiotics in their systems, they are generally becoming more receptive. So I work with a lot of supermarket aligned producers now, and they're under a lot of pressure and therefore, you know, really understanding, how to optimise the use of vaccines in their system is something that, you know, they're, they're paying a lot more attention to and, and showing more interest in. And, and not just vaccines, you know, some of the earlier parts of the webinar where we're talking about optimising immune functions through sort of nutritional management or periprourant management, becoming sort of increasingly aware of.

And also the genetic side of things, you know, we're not the only people out there talking about immunity in livestock, you know, genetics companies are out there as well. And it's something that certain dairy clients we have are are sort of paying increased attention to. So it's not always the vets that are out there talking about it, and it's not always the products we're selling that are potentially, Raising the profile of of considering the immune function in these animals.

No problem, that's fantastic. So I think they've got quite a lot of shy people on their line tonight. There's no questions coming in, at all.

So, as I say, it's, I, you know, I think you've obviously covered all the areas. The webinar is going to be up, on our website within the next 40 hours. So for any of those, who maybe missed a couple of bits, whatever, then you can log back in in 48 hours' time and rewatch it, and it will be covered there for you again.

. As I said at the beginning, we do have our feedback survey. Dawn has kindly put the survey in our chat box, with a link to it, but also it should appear, when you move away from the Zoom platform, it should be in your browser as well. So please do take time to complete the form and return that to us.

So what it leads me to do is to thank Dawn for being online tonight and supporting me and, helping with all the, any issues we've had. And then obviously, a massive thank you to yourself, Alex, for taking your time to, present this great topic tonight. And, we look forward to welcoming you all on a webinar very soon.

Enjoy the rest of this pleasant evening. Good night.