Good evening, everybody, and welcome to tonight's webinar. My name is Bruce Stevenson and I have the honour and privilege of chairing this evening's webinar. Before I run through a little bit of housekeeping, I just want to say a big thank you to our sponsors, Animal Care, and in a few minutes, we will hear from Paul Colton from Animal Care.

But before I hand over to Paul, just a little bit of housekeeping. For those of you that haven't been with us before, please remember that we are recording this webinar, so it will be up on the webinarvet website in the next 24 or 36 hours and available for you to go and rewind and fast forward and relook at slides because we are not able to go back and re-look at slides afterwards. We will take questions, so if you have any of those, just move your mouse over the screen.

A control bar pops up, normally a little black bar at the bottom. There's a Q&A box there. Just click on that, type your questions in there, and we will get to as many of those as we can tonight.

Before I introduce our speaker, Doctor Sue Patterson, I'd just like to hand over to Paul Colton from Animal Care. Paul, welcome to the webinar, Vet, and thank you for your company's sponsorship this evening. That's great, thanks very much Bruce.

Thank you for everybody listening. Tonight, I'm just gonna give you all a very brief introduction to two products that we launched onto the UK market, last year, related to the microbiome in skin and ears. First off, I'd like to talk about ProAtop, which is a skin spray directed to for dogs.

And this skin spray and across both products, the skin spray, contains two live probiotic bacteria, in the form of lactoacilli. One being Lactobacillus rhamnosis and the other being Lactolantobacillus plantarum. The spray itself is a 50 mL pump spray, and it's directed to apply, the spray onto the skin once daily for 4 weeks.

The products really exist, as a microbiome support. Now, whether that's in an atopic dog, and there's a irritated skin. And things like folds, skin folds, irritated paws, any area where there's redness or irritation, then we know there's going to be, a high chance there's going to be a dysbiosis with bacteria such as Staphylococci.

So, the lactobacilli being live, effectively, are applied to the skin, and produce metabolites, compete with the pathogens, and also produce lactic acid to acidify the skin. So ProAop is there really to maintain a healthy balance. And it can be used until the expiry date anywhere on the dog's, the trunk of the dog or the paws.

The other product we've got is for ears, Pro Aris, and that is directed at ears. And again for use in dogs, same two strains, as Pro-Atop, slightly smaller bottle at 20 mL, and that is, to be administered twice, once, every two days. And again.

Very similar to Proattop and that is there to compete with pathogens, implicated in otitis, externa, or very irritated ears prior to infection setting in, or post antimicrobial treatment, to maintain a period of health or optimise wellness. And again, proborus can be used until the expiry date. If you do have any more questions about the products or want any technical information, please do email us at [email protected] and we can answer any questions you have.

Thank you very much and I will hand over to our speaker. Paul, thank you very much. I'm gonna jump back in again before we go to Sue.

So, just, give you a heads up, Sue is feeling a little under the weather. She's got the dreaded Logie, so, she, we'd like to really thank Sue for making the effort of being here tonight because, I have seen you mute yourself and, try and displace your lungs, but, thank you, Sue. So for those of you that don't know Doctor Sue Patterson, she holds both the UK and European diplomas in veterinary dermatology.

She is a Fellow of the RCVS awarded for its contributions to veterinary dermatology. Sue is a past president of the ESVD and current president of the WAVD and has lectured in more than 60 countries worldwide and has more than 90 articles published in peer-reviewed journals. Sue, over to you.

Thank you, thank you so much, that's, that's really kind and thank you very much Paul for that presentation. Just to set the scene, I'm now going to see if I can just share my presentation for you, hopefully this will come through OK. How are we doing, is that all OK, can we see all that?

Yep, we can see it clearly, thanks Sue. Oopsir Daisy, let's go back then. OK, so one thing I should just say before I start, you just heard Paul there talking about the two products that Animal Care launched last year, and he mentioned the word, and I've written it down so I don't get it wrong, Lacticai bacillus rhamnosis and lactic plant.

Bacillus plantorum, and those are the new terms for Lactobacillus as far as I understand. So where you see me put Lactobacillus in this presentation, those are the bacteria that we're referring to, which are the bacteria that we've actually got in that particular product. So, as always, these things are very fluid, we often see name changes occurring.

So as I say, thank you so much for inviting me to talk about this. This is a subject that I find really interesting because I think the more that we know about the microbiome, the more we can start to understand as clinicians and as dermatologists, why we actually see the presentations that we see when we're examining our allergic dogs, particularly in this case. And it can also help us when we're looking at long-term management because we know it's one thing to get an allergic dog under control, it's much more challenging to keep them under control, and I think a knowledge of the microbiome can certainly help with that.

So these are our learning objectives tonight, so just a, a, a slide really on what we mean by the microbiome. I'm sure we have an educated audience today and you know what that is, but just to remind you. Then what I'm gonna do is talk a little bit about how the micro microbiome differs between normal and allergic dogs.

We're gonna talk about why we see shifts in the microbiome. I'm gonna look at therapeutic interventions and what effect they have on the microbiome, and then a little bit of sort of blue sky if you will, thinking about future. Strategies, how we can influence the microbiome because we know that it's really important in both some of the diseases that we see, but also increasingly it's being recognised in human medicine as underlying many of the very common skin conditions that they recognise, things like acne for example, and allergic dermatitis.

So first of all, what do we mean by the skin microbiome? Well, the microbiome really is everything that sits on the surface of the skin, it's the bacteria, it's the fungi, it's the viruses, it's the parasites. Demodex, for example, is part of the microbiome, and it's the metabolites that they produce that are found within the most superficial layers of the skin, so the epidermis.

But we also see that microbiome extending into the glands in the skin, so we see that microbiome in both the sweat glands, the sebaceous glands, and of course the hair follicle. And the microbes that we find within each of those areas will of course be affected by a range of different factors, so heat, pH, salinity, moisture, and nutrient levels will affect the growth of those particular organisms. And therefore we will see different organisms at different sites on a dog or a cat or a rabbit's body because those, those environmental factors will change depending on the site that we're dealing with.

Something just to, just to flag to you, this is something that you may, you may see, if you're looking at any papers, whoops, if you're looking at any papers on the microbiome, you'll see things like beta diversity mentioned, and you'll see things like alpha diversity mentioned. Alpha diversity is referring to. To a, to a specific, specific sample from a specific site, and they will look at the richness, so the amount of organisms there are there, and the evenness.

So do we have similar numbers of each of the microbes or are we seeing increased numbers of some at the expense of others? And then beta diversity is the other thing that you will see described, and that is very much a comparison between two different sites. So how does, for example, the microbiome on the dorsum compare to the microbiome in the groyne?

Very different environmental factors there, so we'd expect to see marked differences in the microbes that we identify in each of those sites. And we will see these sorts of terminology referred to as we go through our presentation today. So let's start off first of all by thinking about what we see with the microbiome in a normal dog.

An excuse to show you one of my dogs, one of my chocolate Labradors, this is Truffle, who I hope has got a very normal microbiome. And this study here is one of the very first studies that was actually published, gosh, over 12 years ago now from Aileen Rodriguez Hoffman from the States, looking at the skin microbiome in both healthy and allergic dogs. And we're gonna talk about allergic dogs in just a minute, so I want to concentrate at this stage about the microbiome on a healthy dog.

And this is one of the first studies that use what we call next generation sequencing to actually look at what was happening on the dog's skin. And what we know when we talk about next generation sequencing is it's a really sensitive way to identify bacterial and indeed fungal colonies on the surface of the skin. When we're looking at some of the older papers that we see, the way.

That we identified what we thought were then the commensals on the skin was by doing culture, and cultures are very insensitive, it's a bit of a blunderbuss way of actually identifying organisms, because it relies on the organisms being picked up on the sample and then them growing on whatever, whatever medium you actually use. And we know some organisms like Staphylococcus will grow well in a laboratory. And other organisms, for example, like Kaani bacteria may not grow anywhere near as well.

So next generation sequencing actually avoids the problem that we see with culture techniques and is a very much more sensitive way of doing it. And what Aileen found using next generation sequencing is that the skin of the dog was inhabited by a far wider range of microbial communities than we ever thought possible using these much blunter techniques such as culture. And what she showed was that there was a high degree of variability between samples collected from different patients, and also as we would expect, we saw different populations of bacteria at different sites on the animal's body.

And this is taken directly from that paper, it's a really lovely open access paper, I would urge you if you're at all interested to have a look at it. I put the breeds that she, she sampled at the top there, it's a bit of a sort of mix and match, some labs, some Boston terriers, pugs, pit bulls, healers, terriers, etc. So a real mix of different breeds that she's looking at.

And what you can see from the picture there is you can. See looking at those little pie charts from each of those different sites, you can see they're very, very different. So the bacteria that we're finding in that dorsal perianal area is, for example, very different to the bacteria we might find take from a sample taken in the conjunctiva.

What she found was a wide range of bacteria there, but looking at the different phyla that she could see, she found the phyla that were present most commonly were the proteobacteria, the actinobacteria, the, the Fermicues, and then the bacteroides and the Fusobacteria. So those were the main phyla that she found on a normal dog's skin. Of course the protobacteria is the phyla in which we find the Pseudomona family, so Pseudomonas, the Fermacues is the phyla in which we find the Staphylococci family.

So those are well represented on the skin of a dog. And so we'll come back a little bit later and look at and look, look and compare this to what we see on the skin of a, a dog with an allergy. So just a brief summary of that paper, what Aileen found was the highest diversity was seen on haired skin, so particularly areas like the groyne, dorsum, ear pinny, dorsal aspect of the nose, and that was much more diverse compared to mucosal surfaces, so things like the conjunctiva lips.

The nostrils, and the area that was the most diverse was the dorsal aspect of the nose, and I suspect you can see from the picture on the right hand side that a dog's nose is an area of its body which goes into all sorts of sometimes most unpleasant places. So that's perhaps why we might see the highest diversity on that particular area. What I also think is important though is there were some further studies that I'd like to share with you, looking at different breeds, looking at microbiome within breeds, and looking at differences between different breed groups.

And this is a nice paper, a little bit more recently published in 2017, and this looked at the microbiome within a particular breed. And this was 35 Labrador slash retriever crosses, and interestingly, there was lots of factors that were kept exactly the same. So they were similar age, they had a similar genetic background, and they're all kept in a very similar environment, fed pretty similar diets, and what they found that despite the fact that they're.

Environment, their ages and the genetic material were all pretty much identical. They found that each dog has its own unique skin microbiota. It varied, of course, as we'd expect to see between the different sites, but every single dog was an individual, every single dog was, was completely different.

This is one of the dogs taken from that paper, and what you can see there on the left hand side, you can see the different areas that they sampled, so ranging from the inner pinna at the front there, the chin, the nasal skin to the perianal area at the back. And what again you can see there, looking this time not at pie charts, but looking at bar charts, you can see the major phyla that they identified. And again, you can see it's very similar to the data that was shown in Aileen's paper, showing again you've got large numbers of protobacteria, you've got large numbers of of firmacues and bacteroides being very common.

So again, similar signs, which is actually really quite reassuring that we've got a different study reproducing very similar results to those earlier studies. The other thing I think which is even more interesting though, is looking at whether there's any trends that you can see within breeds and any differences between breeds, and this is a 2nd study by the same, same group. This is looking now at 3 different breeds of dog.

That this is comparing French Bulldogs, German Shepherd dogs, and West Highland White Terriers, so 9 of each of those breeds, and again, these are healthy dogs that we're looking at. What they found again was they found there were certain phyla that were very much shared between all those different breeds, so again, the same groups as you can see before the Fermacues, the Protobacterium are there, the Actonobacterium are there, so those core microbiota are very, very similar between those different. Dogs, but what they found again was there was very little in the way of trends within the breed, and very little when you compared one breed with another, and that's quite surprising because you might find, expect to find, for example, the microbiota in a Westie is very different to the microbiobota in a German Shepherd dog.

They're all different from each other, there's no trends that are seen within those particular breeds. And this is again a chart just illustrating that for you and what you can see here, so what I've got there in the first set, those are the French Bulldogs, that's a French bulldog, the 1st French bulldog in the study, you've got the 2nd French Bulldog in the study, and the 3rd French bulldog in the study, and what you can see within that one breed. There's a massive difference in the bacteria found on the surface of the skin.

You can see, for example, in that first French bulldog, there's an awful lot more of the protobacteria and the firmacues than there is in the second and the 3rd 1, where we're seeing actually quite a lot of fusobacteria. And for me, I think this is really interesting because what you then start to think about, as a clinician I start to think about, well I think about well what happens if these dogs develop allergies when we're looking at the microbiome. If these dogs develop allergies, which of these three dogs is going to be more susceptible potentially to developing a staphylococcal infection?

And when you look at bull, French bulldog #1 with those large numbers of firmma cuties, if those are representative of Staphylococcus, you can see potentially why that dog, if it developed an allergy, may be the one that develops a staphylococcal infection, whereas perhaps bulldog 2 and bulldog 3, because they perhaps don't have the same numbers, might not develop signs of a staphylococcal infection. And we'll come back to that in a little while. This for comparison is between the three breeds, so the last one I showed you was 3 different French Bulldogs.

This now is comparing a French bulldog, a German Shepherd dog, and a West Highland White Terrier, and again you can see there's very marked differences between the patterns between these 3 breeds. So it, it just shows that the microbiome is individual. We all as, as humans have our own individual microbiome and.

Every single dog has its own individual microbiome, and when it's a healthy dog, the only point of similarity between one dog and another is that it's very diverse. The types of bacteria, there is a core microbiome there, but other than that, there is little else, that's similar between different dogs of different in one breed or between different breeds. This brings us right up to date, this is probably one of the most recent papers I could find looking at microbiome, and this talks in more detail about this core microbiome.

This looked at large numbers of dogs and looked at which phyla were the predominant phyla when we were looking across a whole range of different animals with a healthy skin microbiome, and again. Overlap with what we've already talked about, the protobacteria, the bacteroides, and the Actinob bacteriota were the three phyla that were found to predominate when we were looking at the skin of healthy dogs. They went further than that in this study, and as well as looking at the three predominant phyla, they also went down beyond the class and the order and the family in the genus right the way down to species, and these were the 15 species that appeared on more than 80% of normal dogs.

So these are the bacteria that we'd expect to see occupying the microbiome with a dog with a normal, healthy. Bacterial population that's there, so the core microbiome is pretty set for more than 80% of animals, it's very diverse, and that diversity is something that helps protect that animal against infection with a diverse microbiome and become far less susceptible to develop infection because you have this balance between the different bacteria that sit on the animal's skin. So having talked about normal dogs, let's go on now and talk about allergic dogs.

Of course our classical allergic Westie is the one that we see really commonly. And let's go back to Eileen's paper that we talked about before, and let's look at the second half of that paper which looked at the microbiome in allergic dogs. And what they found, and this is the first time to my knowledge this was reported back in 2014.

They found that whereas normal dogs have a really diverse microbiome, that diversity is lost when an animal becomes allergic. We see increased numbers of particular bacteria at the expense of others. And again, a nice illustration taken from that particular paper, I think shows that really nicely.

From what you can see on this particular chart here, you can see on the left hand side for those four sites, so we've got axilla, we've got groyne, we've got interdigital area, and we've got nostrils. On the left hand side we have the bacterial flora for the allergic animal, and that's compared to a healthy individual. And what you can see with each of those healthy animals, you can see there are much more lines across it.

There's a lot more colour in those, in those histograms there because what you're actually looking at is a very diverse bacterial population. Once we start to look at the allergic animals, you can see there's far fewer colours. It's far less diverse, and then of course the other thing that stands out, particularly in this, in this example here when we're looking at the groyne and we're looking at the nostrils, you can see we've got much larger numbers there of Staphylococci on the skin of allergic animals than we have on healthy.

Yeah, and these are not animals with infection. These are animals that are allergic and the and the and the samples are taken from the allergic skin but there is no evidence of infection on these particular animals, so we're not taking samples from an area that's got pustules and papules and epidermal cholerettes. We're taking it from an area of skin that potentially has just got a little bit of erythema that's there, and what we're seeing is this very marked difference in the populations in those sites in these allergic animals.

And again you can see once an animal starts to get inflammation, why potentially this abnormality, this loss of diversity can then progress through to bacterial overgrowth and bacterial infection. Couple more studies really just for comparison, again, these show very, very similar signs. The first one again, showing that same lack of diversity in allergic animals, and the second one again, 2025 this time, again, shows again a lack of diversity in these allergic animals, and again, looking at the difference in the.

Bacterial flora, they found a very different bacterial flora in healthy skin compared to those from allergic animals, and where we had inflamed skin, not only was that loss of diversity evident, but they saw a lot more Staphylococcus as the abundant genius, abundant genus in those particular cases. So again, this reinforces the work of Aileen. Looking at loss of diversity in allergic animals and relative increases in the numbers of staph on the skin in those particular animals.

So as we said, these are dogs that don't have infection, these are allergic animals, but what we're seeing is we're seeing this change in their microbiome, this loss of diversity and this tendency to see increased numbers of Staphylococcus. Let's think about why that actually occurs and what the mechanism is behind it, and we certainly know when we look in the human field, we know that we're seeing these abnormalities, this proliferation, particularly in humans of Staphylococcus, associated with a wide range of diseases. We know that it's really.

Important in human atopic dermatitis, but we also know abnormalities, dysregulation, dysbiosis of the skin microbiome is also important in people, not just in atopic dermatitis, but also in acne, in psoriasis, it's also thought to play a part in impaired wound healing as well. But what's really interesting is human medicine is starting to move away from, you know, getting rid of infection by using antibiotics and antiseptics. What they're trying to do now is move much more to restore a normal microbial balance rather than just wiping out bacteria.

So what about the information that we have when it comes to dogs and cats? Well, this I think is a really interesting paper, and this is a paper looking at allergen induced canine atopic dermatitis, and again this is using this very sensitive next generation sequencing. And this was animals challenged with Haus mite.

These are house dust mite allergic dogs, they were challenged with Haus mite, and what the investigators did was they looked at the changes in the levels of staphylococci on the skin after that challenge, and I'll show you the chart in just a second, which I think is really interesting to see. But what they found after a single allergen challenge, they actually saw a really dramatic increase in the staph levels on the skin. And those numbers persisted for up to 3 weeks after the challenge before starting to return to normal levels, so let's just show you that in a diagrammatic form.

So here we have the first two charts and what you can see here is you can see there's representation of the different phyla there, the Staphylococci is there in orange, and what you can see pre-challenge, there's relatively small numbers of those orange Staphylococci on that, on that bar that we can see. On day 1, so challenge occurs on day 0, on day 1, we can start to see a slight increase in Staphylococcus, and you, you have to remember this is a single allergenic challenge. What we see at day 7 though, is quite a dramatic change.

We're starting to see much more in the way of Staphylococcus, and that change drops a little at day 14, but it's still significantly higher than the levels are pre-challenge. Still quite high at day 21, dropping down at day 28, but still higher than it was at the beginning. So that's with, with a dog, with a lot of diversity, an allergic animal, challenged with what they're allergic to, you see that dramatic shoot up of those numbers of Staphylococcus after just a single challenge.

And for me that really starts to explain why we start to see infection in many of these dogs. So it might be that it's something like this, we get a, a this terrier, this English bull terrier here, English bull terrier has an allergy, we look at the microbiome on different areas of its body, we look at the microbiome in its groyne, and in its groyne it's got this increased numbers of Staphylococcus in its groyne before it develops an infection. It has an allergic reaction and we see increasing numbers of staph as we showed you in that previous slide, and that's what pushes this animal over the edge and starts them to develop bacterial infection.

And from, from my point of view, I think it's this increased numbers of staph on the skin, this inflammatory response, and this change in the microbiome at different areas that helps start to explain why we see the clinical signs that we see in particular dogs, and what I've done is I've pulled some of the data from those previous cases that we looked at. So this was one of those French bulldogs that we looked at before, and this was the French bulldog that had really high levels of firmacues, which is the phyla that contains Staphylococcus, in its interdigital space. So the sample taken at site 7 was its interdigital space.

So when this dog develops an allergic reaction, the area we'd expect to see with its bacterial overgrowth is going to be the interdigital space, and that may be why this dog develops signs in that area and doesn't develop it, develop it in other areas as a result of that. And let's look at one of those German Shepherd dogs. The German Shepherd dog doesn't have that overgrowth in area 7.

It has that bacterial overgrowth in area 6, which is the groyne. So there's very poor diversity on the abdominal skin, large numbers of the firmacues, when this dog gets an allergic reaction. It develops bacterial infection on its ventrum rather than for example, in its interdigital space, because we don't have that overgrowth, that increased numbers of staph potentially at that particular site, whereas we do have them in its groyne.

So I think it very neatly starts to explain why we see some dogs with ventral pyoderma, why we see some dogs with interdittal pyoderma, why we see some dogs with superficial folliculitis. On their dorsum and on their flanks. I would suggest it's due to these differing levels of bacteria in the microbiome at different sites, that means when that inflammatory response occurs, it drives this whole process and pushes the that lack of diversity over the edge, going through increased numbers to bacterial overgrowth to full blown bacterial infection where we start to see signs of primary lesions.

What about the effect of therapeutics though on the skin microbiome, what effect will they have on the microbiome and how can we best use them to actually ensure that we are doing the very best that we can for our patients, but also being mindful of the fact that we need to use these drugs responsibly. We know overuse of antibiotics. Is something that can lead to antimicrobial resistance.

We know they have an environmental impact. We know antiseptics are probably our, our modality of choice, looking at topical therapy for bacterial infection, but again, many of these have an environmental impact. So what can we use best to actually have a beneficial effect on the microbiome in our clients?

And this is one general paper I pulled out, this isn't actually from dogs or from people, this is actually from mice, but I think it, I, I put it up really cos it just illustrates a couple of points. These are actually animals which have got normal skins, but what it shows first of all is where you use a broad spectrum, antibiotic attack on the skin, so that top. Antibiotic combination there, that's a combination of three different antibiotics, it's a combination of neomycin, of, of polymixin B and a bacitracin, something I hope people wouldn't routinely use to treat infection, but that broad spectrum attack causes really dramatic reductions in bacterial diversity.

If you use something like mupirocin, which is a much more narrow spectrum antibiotic, specifically with just activity against Gram-posit organisms, it still causes disruption of the microbiome, but it's far less. If you use topical antiseptics, and in this particular study they used 80% ethanol, so alcohol, and 10% peidone iodine, they tend to produce less loss of microbial of of the diversity of the microbiome. So I think a nice example that shows us that antibiotics will have profound effects on the microbiome, so we really do need to use them with care.

And it also illustrates why if we are going to use an antibiotic, we should try and use a narrow spectrum drug because they're going to have less effect on the microbiome than broad spectrum drugs do. And of course the take home message from this paper is that of course by treating the microbiome like this with an. Antibiotics, we may potentially if the animal's got infection, resolve the infection, but we also remove all the commensal organisms as well, and they compete with the skin pathogens, so actually this blanket approach potentially isn't the best way to deal with infection in the domestic species that we deal with.

So let's have a look at some papers from some dogs now, and this is a cohort of 15 dogs with allergy, and with skin infection. And of course we know from the very start that these are dogs that don't have a normal microbiome. So when we look at the pre-treatment levels of these dogs, the fact they've got reduced bacterial diversity is perhaps something we'd expect to see, and the fact of course they've got increased numbers of Staphylococcus, particularly Staphsuud intermedius, is exactly what we'd expect to see.

In this particular study, they did culture and sensitivity, they selected the antibiotics on the basis of that, so they used a wide range of different antibiotics here. In the study they used clindamycin, they used cephalexin, they used Clavamox. Worryingly, they used enrofloxacin as well as trimethoprine sulphur, and they gave antibiotics for 4 to 6 weeks, and we could argue.

Very strongly, that's totally inappropriate way to use antibiotics in these cases. But what they did find, the positive thing from this study was that the oral antibiotics did restore that bacterial diversity as well, of course, in reducing the population of staph and obviously improving the clinical signs of the animals that had the allergy. So antibiotics helped restore the microbiome, but we would hope not to give 4 to 6 weeks, particularly of critically important antibiotics like enrofloxacin in our allergic animals, so there are benefits, but I would really worry about the long-term effects of using those types of drugs on these allergic animals for maintenance therapy and ongoing treatment.

This is another study again taken from, from dogs. This is a comparative study here looking at healthy control dogs with 3 dogs who have atopic dermatitis, and again, they looked at the microbiome before and after therapy. This time though, they didn't use antibiotics.

They used topical therapy, and they used a shampoo, I'm sure you'll recognise this particular shampoo with 2% chlorhexidine and 2% miconazole. They applied it twice weekly for 3 weeks, and what they found was in these animals, this also increased the diversity of the microbiome. So really nice thing to show that our topical treatment was dealing with infection and was actually improving the diversity.

And the other positive thing from this was of course that after they stopped the treatment, after the animals infections had resolved, they continued to monitor the animals and what they found was that, That diversity was not maintained, they found that it took quite a while to return back to the, the, the, the pre-treatment samples, but they found over the next 3 to 4 weeks, they found that microbiome slipped back to the air, to the levels that it was before. And I think this again is a nice illustration. You know, we are so lucky with the range of drugs that we now have to control our allergic animals, using our various JAK inhibitors, using our monoclonals or using even something like steroids, we can control itch, we can control inflammation, but if we have an animal with bacterial infection on the skin, Treating it is not enough because what we will find is that once we stop that treatment, that animal potentially will relapse as that microbiome reverts back to its pre-treatment levels.

So for me this study is really powerful evidence to suggest the need for ongoing maintenance therapy for animals which have got allergic skin disease to try and keep that normal microbiome there. So if we think about that and put all of that together, when we have allergic animals, I think it's very much a balance of protecting our antimicrobials versus restoring the microbiome. We need to treat infection responsibly, thinking about our appropriate antimicrobial stewardship, and the other thing we also need to be aware of is the effect that those drugs are going to have on the environment.

We know we see antibiotics in waterways in the UK we know all over the world that there are pharmaceuticals in the waterways, so prescribing these drugs potentially is getting into the environment, so we need to use antibiotics as and when we really need them. But we do need to restore the microbiome, and we know that using antibiotics and using antiseptics will restore the microbiome, so is there a balance we can get to restore the microbiome in these allergic animals without compromising, antimicrobial stewardship without compromising the environmental impacts that we have. So when it comes to antibiotics, you know, we know about the overuse of, of antibiotics.

We know certainly the most recent WSAVA guidelines suggest the indications for using systemic antibiotics in allergy is really quite small. We really should only be using these in cases of deep pyoderma on the basis of culture and sensitivity. And we know that they will reduce the skin resident from that mouse study I showed you, and we know that they can exacerbates dysbiosis on an ongoing basis.

So certainly, I can see little justification for antibiotics in canine atopic dermatitis to help control infection and to help restore the microbiome. Anttisptics though I think there is a part for these to play, and certainly we've shown that they improve microbiome diversity, without, but what we do know from that study I showed you with the Myconazole chlorhexidine shampoo, that we need to keep using antiseptics in order to maintain that diverse microbiome in these animals. The problem I have with that is that pretty much all of the antiseptics that we use are ecotoxins, so chlorhexidine is a fantastic drug, it's great for resolving infection, but it is an ecotoxin.

It will pollute watercourses, it will reduce biodiversity, so for me, antiseptics should be placed to treat infection, but not to maintain that microbiome afterwards if we can find other ways in which we can do that. So what we should try and do is try and maintain a healthy diverse microbiome, but we should do that by optimising the skin microbiome rather than these massive nuclear options of wiping out all the microorganisms in a non-targeted way. And so what have we got, what's available to us, what strategies are in the offing, what innovations are there to help us to try and do that in the most successful way we can.

So what strategies to influence microbiome? And there are 4 things I wanna talk a little bit about. One of those is diet.

One of those is skin micro microbiota transplant, which is being done in people. I wanna talk a little bit about prebiotics and I wanna talk a little bit more about probiotics. Let's start off first of all with diet.

There's not much in the literature as far as diet and its effect on the microbiome, but this is one really interesting study that I found back from 2022. We have to take it, if you'll excuse the pun, with a little pinch of salt because these are very small numbers. We're looking at 8 dogs and we're looking at 8 healthy dogs.

But it's interesting data and I certainly think it's something that may be worth exploring perhaps in more detail. These were dogs that were fed a fresh cooked diet, so not a raw diet, it was a fresh cooked diet, they were fed for 30 days. They had a 4 day washout period and you can argue whether that's enough.

And then they were fed for a dry food on 30 days, 30 days after that. Next generation sequencing was done. After each of those different periods, and what they looked at, they looked at the alpha diversity, so the, the diversity within each of the samples for dogs who were fed a fresh diet, dogs that were fed a dried diet, and what they found was that animals that had a fresh diet had.

Increased proportions of commensal Staphylococcus, so staph epidermidis, potentially Staph aureus and Staph pseudonomeus. So they found increased proportions of Staphylococcus, and they showed decreased proportions of some of the other phylo that were there. What they found with the dogs that were fed a dry food was they show a relative decrease in the skin microbiome.

So interestingly, the fresh diet seemed to promote an increase in the microbial diversity compared to the dried food. Now I say, it's difficult to know why that occurred because if you look at the paper in a bit of detail and you look at the components of that fresh diet, it's very variable. The fresh cooked diet contained a wide range of things, it had chicken, chicken livers, egg, carrot, rice, sweet potato, pumpkin, fish oil, so it was higher in moisture, it was higher in protein, it was higher in fat, it was higher in omega 3 acids, and also things like pump.

And it probably had some prebiotics as well in the form of sort of indigestible carbohydrates. So it's difficult to know why the dogs had this increased diversity of their microbiome, but the fact is they had, so potentially something to look at and something to explore in the future and of course if we could maintain a normal microbiome just by feeding dogs particular diets, I think that'd be a great way for maintenance therapy in some of these animals with really difficult skin conditions. This is interesting though, this is some of the work from people looking at skin microbiota transplant, and this works on the same principle as we see with people doing faecal transplants, so aiming to transfer in inverted commas, the healthy donor's skin microbiota to a, to someone with skin disease to try and restore the balance on their skin.

And certainly some of the early studies, one of which I've highlighted here, shows that it is a potential way of actually managing some of these cases, particularly people with atopic dermatitis, where they have got bacterial overgrowth with staph aureus, and certainly using this technique they've been able to make people more comfortable, reduce their pruritus, reduce the severity of their skin lesions. The problem is, of course, with this sort of technology, is that there are lots and lots of challenges associated with it, because of course we know the microbiome is site specific, so you'd have to take a sample from a site on a donor that corresponds to the same site on the recipient. It can be difficult to get the right numbers of, of, of bacteria on it and of course there is always the safety concerns when we're transferring micro microbes from one person to another that we may be transferring organisms that we don't want to.

So until we have some form of regulation of this where we have appropriate laboratory conditions, standardisation of processes, this is something that will remain very definitely experimental, but it's something we may be able to use at some stage in the future when we're looking at our dogs and cats and potentially even our horses. Prebiotics, these are indigestible carbohydrates, things like fructans and galactans, are what we would recognise, and these are thought to selectively stimulate the growth and activity of beneficial gut bacteria, and the aim being that that promotes microbial diversity. They can be used topically where they're acting via the gut skin access, sorry, orally where they're acting via the skin gut access, or they can be applied topically, and they're thought to be able to promote these beneficial skin microbes, so things like staph epidermidis is thought to be a beneficial microbe, and that is, it's certainly been shown to suppress some of the pathogenic organisms, they're also thought to have other benefits as well.

When you go through the literature though, there's a lot of anecdotal information about the benefits of prebiotics, but there's very little in the way of a proper evidence base to support their use. And when I looked through, there was only one paper I could find looking at their use in canine atopic dermatitis, and this actually is, is used where we got a prebiotic used in combination with a probiotic, what we call a, a symbiotic, and this is dogs. That will give them Lactobacillus, paracacea with with kestose, so the lactobacillus is obviously our probiotic, and the kestose is our prebiotic, and what they showed was that where they had dogs who'd gone, who'd been well controlled with prednisolone, using a combination of those two particular products, they were actually able to make those dogs more comfortable in reducing their, Purtis and reducing their canine atopic dermatitis lesion score and were able to reduce their total prednisolone, so not able to take them off prednisolone, but used as an adjunct therapy in order to support that in combination with the anti-inflammatory drugs that were there, so potentially, but who knows whether it's the lactobacillus, that's produced the benefit or the kestos as the prebiotic.

Probiotics though are things there is more information in the literature about, and these are very different to our prebiotics, these are live microorganisms, and we know that these can produce a wide range of different effects on the skin. We know they're able to modulate the skin microbiome, and they can be used again either topically or again working through that gut skin access, they can be given orally. And the ones that you will see most frequently, the stain, the strains most commonly seen, in publications, whether they're human populations, publications or whether they're veterinary publications are Loctobacillus, Bydobacterum, and then Staph epidermidis, there's staph we know that that competes with Staph aureus on the surface of human skin.

These are a couple of papers in the literature looking at probiotics in human medicine and some really very encouraging results here, looking at the use of Lactobacillus, in allergic women, sorry, in pregnant women, given Lactobacillus, they showed they could reduce the incidence of atopic dermatitis in children, very dramatically, a 50% reduction compared to those in the placebo group. A second paper there is looking at children with moderate to severe atopic dermatitis, and again they show the use of a probiotic, the Lactobacillus probiotic there was enormously beneficial in treating those animals and produced a significant reduction in those animals' clinical signs. So there's certainly evidence in the human literature to support the, Use of probiotics, certainly in allergic individuals.

What about dogs? Well, in dogs, most of the work's been done looking at Lactobacillus, and there are several studies looking at both killed Lactobacillus, and now we're starting to see some studies based on the back of the new products developed by Animal Care, looking at live bacteria in those preparations. So when we're looking at those products that we have there on the market, which Lactobacillus should we choose?

Should we choose killed these tinderli bacteria, or should we use live bacteria? Well, I put this chart up for you to come back and have a look at at your leisure, perhaps if you have a look at this after the presentation, but broadly speaking, when we're looking at the different lactobacilli that we have here, or the new names, the Lacto Cai bacillus or lactilantiba bacillus, so whatever, whichever of the ones we actually look at, they all have got significant benefits. They all seem to be able to reduce pro-inflammatory cytokines.

They all appear to be able to suppress this T helper 2 response, which is this abnormal aberrant response we see in atopic individuals, so driving the process away from those pro-inflammatory mediators. We know certainly they seem to be able to compete with Staphylococcus on the skin to rebalance the microbiome, and we know that they seem to be capable of producing a range of anti-staphylococcal components to reduce the Staphylococcal colonisation of the skin. And then in addition to that, we also know that they can improve barrier function, which we know is a problem in atopic dogs, and we also know that atopic animals have an increased transepidermal water loss, their skin is very dry, and again, these products have been shown to increase hydration.

So lots of really good positives looking at these different products and the potential that they can have in helping us to manage some of our allergic animals. So let's have a look what's in the literature, what's there. So these are two studies looking at the use of the so-called tinderla, these heat treated lactobacilli, and both of these studies actually show they produced significant benefits.

They had, they produced a reduction in pruritus, they produced reduction in the allergic response, they produce a rapid decrease in clinical signs, and that clinical signs improvement can last up to 14 days. The one thing though the studies don't show, certainly the first study, which is a clinical study there, is that the Lactobacillus actually produced change in the microbiota. So it would seem when we look at tinderli lactobacilli, although they do seem to have good anti-inflammatory effects, and potentially these could be added into our regime to improve the management of a particular animal, particularly if they're used topically, they certainly on those studies that are there in the literature, they don't seem to have any direct effect on the dysbiosis that we see, this lack of diversity in the skin microbiome.

So they seem to be useful to control clinical signs, but for ongoing maintenance therapy to try and correct this lack of diversity that we see, they don't seem to be able to do that based on the information that we have in the literature at the moment. There's not so much about in the literature about the use of live lacto bacilli, but this is one paper that I can share with you, it's just about hot off the press, it has literally just been accepted for publication in veterinary dermatology, so those of you who've got access to that, please do have a look when it's, when it's, when it's available, freely available. This actually isn't looking at canine skin disease, it's actually looking at canine otitis externa, but I think it's really useful because we know allergic dogs don't just have dysbiosis on their skin, we know they have dysbiosis in their ears as well, we know all the same conditions occur in an allergic dog's ear that occur on the skin of an allergic dog, so I think we can probably extrapolate quite widely from this study to a study potentially on the skin.

But there were 15 healthy dogs randomised into 5 different groups according to the time of sampling, and they were given 6 drops of an ear product containing those two organisms that we mentioned before, so Lacticaa bacillus rhamnosis and Lacti. Planti bacillus plantarum, so L. Plantarum and L rhamnosis, those went into the ear, and then swabs were taken at 24, 48, 72, 96 hours and 7 days.

So each group had a sample taken at one of those time periods. No, none of the dogs had samples taken at each of those times, each group had them at those particular times. What they also did in addition to that, they looked at clinical isolates of pseudomonas, of staph.

And Malacasia, and they actually put up some Kirby Bauer tests, looking at testing the probiotic eardrops against a range of different antimicrobials, and this is based on the fact that we know these lactobacilli produce lactic acid, they also produce bacteriocydins, and it, It was, it was really a nod to see if the ability to produce the ability of live bacteria to produce those antibacterial components actually could be reflected in looking at its effect on the growth of bacteria on one of these Kirby Bauer tests. So let me share with you some of the results on these. And the first part of the test was really looking when they took these ear swabs, as to, first of all, putting those lactobacilli on the skin, did they stay on the skin, did they multiply on the skin, and how persistent were they on the skin.

And so this is a graph taken from that particular study, and what they showed at time naught, when they looked at Lactobacillus numbers on the skin, they found they were relatively low. But really interesting, 24 hours after this single application of lactobacilli, they actually found the numbers increased significantly, and what they found was that those numbers remained high for up to 72 hours before they then started to trail off. The last of the samples taken in the study was at 168 hours, and even then you can see the levels are still higher than they were right at the beginning.

So I think this is really interesting, this shows that putting live bacteria onto the skin, that those numbers stay there, they persist on the skin, and of course we know therefore we have the potential for Lactobacillus to have an effect on staph on the skin where the Staphylococcus overgrowth. Growth there, potentially Lactobacillus can act competitively with the Staphylococcus and potentially if they're producing lactic acid, if they're potentially producing other antibacterial compounds, they have the potential actually to help reduce directly those staphylococcal numbers. Let's have a look at what they found with this sort of Kirby Bauer type of test, and what you can see here, I've just shown you three of those plates.

What you can see there is you have got the Staphylococcus seeded across the plate, confluent growth of Staphylococcus, Pseudomonas, and Malacasia respectively, and then we have. Little discs that contain either the probiotic, the antibiotic of choice, or there's a negative control there, which is a, a, a culture of Micrococcus luteus, and what they found in the study was they found that the Lactobacillus had better inhibition of Pseudomonas than gentamicin and marbifloxacin, better inhibition of Pseudomonas of pseudointermedius than gentamicin, neomycin and marbifloxacin, and similar inhibition to Myconazole and tabinafine. So what this suggests is not only is Lactobacillus colonising the skin and staying on the skin, it also seems to have the potential here to produce compounds that have got antibacterial and actually antimycotic effects on the skin.

So some really interesting studies that really start to look at alternatives that we may be thinking about considering in the management of these cases. So live lacto lactobacilli for me I've got an edge over our tinderli bacteria, we know they've got this potential anti-inflammatory effects, know potentially they can improve clinical signs, but we've got the potential of an awful lot more when we're looking at live lactobacillus with these potential benefits of inhibiting the growth of pathogens on the surface of the skin. So finally, how could we use our knowledge of the microbiome to help in the therapy of canine atopic dermatitis?

Let's just have a look, finally at 22 cases for you. 2 year old Jack Russell terrier. Highly pruritic Jack Russell, it's got really significant areas of self-inflicted trauma, so this is the dog that whips around and nibbles at its, at its flanks, it's chewing at its feet, it's rubbing its face, but this does not have evidence of secondary infection, it's got no primary lesions.

So what this suggests to me is that although it's got problems with the diversity of its microbiome, it's probably not too bad, it's probably got just mild, abnormalities in its microbiome. We're going to put something in place to control its itch, that might be a jack, it might be a monoclonal, it might be steroids, cyclosporine, and allergen specific immunotherapy, but in addition to that, you know, we know that these allergic animals can have really acute flare-ups, they can, you know, they can move in and out of pollen period, the central heating can go on and can go off and housedust mite levels can go up and down, so we know they've got the potential to have flares, so for me, This would be where a topical probiotic with live bacteria would be really useful, so some, putting something like this on the skin, even though this dog doesn't have infection, I think has got real potential to help try and improve the diversity of the skin and we know that certainly those lives. Bacteria stay there for 72 hours, so putting that on twice a week I think is a really useful adjunct, not to treat this animal's pruritus, but to actually add in there as an adjunct therapy for maintenance treatment to try and prevent this animal developing evidence of bacterial infection in the future.

Let's go to that English bull terrier that I showed you before, and these for me are the single most difficult animals to manage. These are the animals with allergy that also have secondary infection as well. We can control their itch with a wide range of different products, we talked about our jacks, etc.

But we need to treat the infection with these animals as well, and from Choice I'd use a topical antiseptic following those WSAVA guidelines, I'd use some chlorhexidine. That might be straight chlorhexidine, concentration of 2 to 4% or chlorhexidine, potentially with miconazole, or I might even use a hypochlorous acid spray to treat this infection. But what we know with this animal is that once we stop our topical treatment, once we've resolved that infection, we know from those studies we've shared with you, this animal's microbiome is gonna bounce back, we're gonna lose that diversity, and unless we've got 100% control of this dog's allergy, we're gonna see infection coming back again.

So this again for me is a really good indication of adding in a probiotic to help maintain that diversity that's actually produced by our topical treatment. So our topical treatment treats our infection, it increases the diversity on the skin, and then our probiotic maintains that diversity to try and prevent this animal relapsing, prevent this animal developing infection in the future. So take homes We know that dogs with atopic dermatitis have have dysbiosis, they have this lack of diversity of their microbiome, right the way back to that very early study from Aileen Rodriguez Hoffman, it's been shown over and over again.

We know that both antiseptics and antibiotics can improve that lack of diversity and restore a more normal microbiome. But we know both of those come at a cost, antibiotics, I would strongly suggest we shouldn't be using in routine skin disease in dogs because of the implications of antimicrobial resistance and the environmental impact. Antisptics should be our preferred means of choice, but again, where we have alternatives for ongoing treatment, that is going to be much more sustainable, it's going to be a much greener approach, and I know we've not got much evidence at the moment, but certainly I think, looking at some of the clinical studies we're seeing coming out, looking at probiotics, Particularly with those that are using live bacteria, where we have the potential for them to colonise the skin and compete with the pathogens that are there, I think they form a very attractive way for ongoing maintenance control of our allergic animals to reduce the risk of bacterial infection.

So I think that just gives us just a few minutes, this is another picture of truffle. For some questions if anybody has any. Sue, what do I say besides, wow.

That was brilliant. it, it, it's such a, an unbelievable approach to, CAD that, yeah, I'm just sorry that I, I kind of didn't hear about it before. It certainly is something that, I think is going to make a huge, huge difference to our, dermatitis patients, and, I really thank you for sharing all your knowledge and experience with us.

Thank you. Also, congratulations on holding out to the end. That was, that was pretty impressive.

Yeah, absolutely. Well I mean hopefully, I I think it's a very, I think it's a very exciting field. I think if you pardon the expression, we're just scratching the surface, we need to know, we need to know so much more, but I think it starts to explain so much of what we see in the way of clinical signs, you know, why that dog's get interdigital disease, why that dog's get ear disease, why that dog's get a ventral pyoderma.

I think it all starts to make so much more sense and it helps us to manage these cases and also. You know, there's, there's been so many quotes about the biggest threat to mankind is resistant antibiotics, you know, regardless of anybody who lives in America or Russia or Ukraine or Israel or any other part of the world for that matter, antimicrobial resistance is the thing that is going. To wipe wipe us out, so I think the more responsibly we can use antibiotics and antiseptics, and the more alternatives we can explore and use to the benefit of our patients, the better it's got to be.

Definitely, definitely, there's no doubt about that. Unfortunately, we have run out of time. So, what we're going to do, folks, is we are going to forward your questions through to, Sue and the team at Animal Care and, ask them if they wouldn't mind, answering those for us, at a later date.

But I do remind you that this webinar has been recorded and will be up on the webinar vet website in the next 24 or 36 hours. So, If you are sitting with your mind a little bit blown like I am, you can go back and and watch it again and listen to Sue, and her brilliant, knowledge sharing with us. A big thank you to our sponsors, Animal Care and, Paul Carton for joining us and giving us that presentation in the beginning.

Thank you to all of you for attending tonight's webinar. I do hope that you enjoyed it as much as I did. And, to my controller, Dawn in the background, as always, thank you very much.

And once again, last but not least, Susan. Sue, I won't call you Susan. Sue, thank you for your time tonight and thank you for, sharing your knowledge with us.

From myself, it's saying good night.