Good evening everybody and welcome to tonight's sponsored webinar. My name is Bruce Stevenson and I will be chairing the webinar tonight. It's, with great pleasure and great thanks to Purina that we, welcome them and thank them for their sponsorship.

We have Jennifer from Purina with us tonight. She's gonna give us a little talk, but before she does that, Just a little bit of housekeeping. If you do have a question for our presenters, simply hover your mouse over the screen.

You'll see there's a little control bar that pops up. There's a Q and A box. Click on it, type in your questions, they'll come through to me and we will hold them over to the end so that we don't interrupt our presenters.

And depending on how time goes, we will answer and get to as many of those as what we can. Like I said, tonight's webinar is proudly sponsored by Purina. And it's my privilege to welcome Jennifer from Purina in France.

Jennifer, welcome and over to you. Thank you, Bruce. Thank you for the introduction and good evening, everyone.

So, first, I want to thank also to the people who have joined the webinar because I know that it can be difficult to find a time maybe at the end of the day for some of them. So, but I think that you will really enjoy this talk because we have a very well recognised expert in the field, as is Doctor Francesca Casaniga, and she's also a fantastic speaker, so I want also to thank Doctor. Casaniga to be available for giving this webinar on this really fascinating topic for us.

So we are really looking forward to, to hearing her. Thanks also to the webinar bed for the organisation of the session and it's always a pleasure for us, for Brina to sponsor webinars that could promote science and innovation because this is really aligned with our company mindset. We are really focused on helping pets live their best life possible.

So. And also as a science-based company, I'm sorry, as a science-based company, we are really especially proud of our veterinary products that you can see now in your screen. We have our preplan Purina proplant veterinary diets which offer a complete nutritional solution also for canine and feline patients.

We have a conscious, comprehensive and effective range of dry and also wet products that could be adapted to promote our pets' well-being. So really if you are interested or you want more information about our products, please, you feel free to. Contact Purina representative in your country or you can leave a message after the webinar and we will be really happy to contact with you.

And without further delay, I will let you enjoy the webinar on the Cara Axis. Thanks again to everyone and Doctor Casaniga, we look forward to hearing you and Bruce, if you want to introduce Doctor Casaniga, will be great. So thanks a lot and enjoy.

Jennifer, thank you so much and once again thank you to Purina for their sponsorship of tonight's webinar. Francesca, if you can share your screen in the meantime for us, that would be great. So Francesca grew up in Vermont and she got her bachelor's degree in biology from Dartmouth College in 2007.

She went to the University of California, San Francisco for her PhD where she studied the effects of stress on telomeres and telomerase in ageing in the lab of the Nobel laureate Elizabeth Blackburn. She's currently a post-doctoral fellow in the lab of Dennis Casper at Harvard Medical School, where she studies how gut bacteria affect the immune system. She also works at the Weiss Institute for Biologically inspired Engineering at Harvard, where she developed a micro microfluidic mouse gut on a chip to model intestinal infections.

In 2018, she was selected by the defence Advanced Research Project Agency. As a riser and especially promising early career scientist and presented at DARPA's 60th anniversary conference. In her free time, she loves hiking, camping, and cross-country skiing with her husband and her 3 year old son.

Francesca, welcome to the webinar vet and it's over to you. All right. Thank you and thank you so much for inviting me to speak here.

I had a lot of fun a couple years ago at the Purina Companion Animal Summit in Vancouver. And so it's fun to be back thinking about how my research and gut microbiota research in general can be applied to companion animals. Now, one thing I noticed at the, at the summit a few years ago is I seem to be the only speaker who does not have their own cat or dog and My presentation was missing any sort of cat and dog pictures.

I told this to my family, and they were incredibly embarrassed for me. And so then they sent me all their pictures of their cats and dogs, and then it became a whole competition of who could send me more pictures. So now this presentation is filled with adorable pets.

And so to get started, I'm going to be talking about some models that we use now to study the gut brain axis and then understanding the difference between bacterial species and bacterial strains. And then I'll give a few examples of published papers showing that diet gut brain interactions in mice, but I think these could all be applied to companion animals. And then finally we'll talk about how we actually make a good probiotic that works in animals, and then I'll end with some future studies I think would be interesting to test to see how, the efficacy of probiotics in certain companion animals.

So we'll just get started. And so there are 100 trillion bacteria that live in the human gut, and they influence a wide variety of diseases, but since this seminar is focused on, the gut brain axis will focus on neurologic diseases here. And so, it's been shown that gut bacteria play a role in multiple sclerosis, in autism, in Parkinson's, and sensing pain, in Alzheimer's, depression and mood disorders, viral encephalitis, stroke and epilepsy.

Now, to researchers, it just seems completely surprising that a bacteria living in your gut has some effect on, on your mood. But to many patients, this isn't surprising at all. For example, in Parkinson's, many Parkinson's patients actually start feeling gut symptoms before they feel any other motor symptoms.

And so the big question we're now trying to ask is how does the signal from the gut get to the brain? And there are two main thoughts behind this, 2 main theories right now. Both seem to be true.

So one is that gut bacteria actually create metabolites, and these metabolites enter the bloodstream and stimulate specific cell types. And so there are many examples of this in the for the immune system. So if you eat a lot of fibre, gut bacteria can metabolise it into short chain fatty acids which affect the immune system.

If you eat a lot of high fat foods, you can. Gut bacteria actually digest bile into secondary bile acids, and then they can also digest tryptophan into many different types of metabolites. And so we know this is definitely true for the immune system, and we're now starting to see that some of these gut bacterial metabolites can act as neurotransmitters and affect neurons in the brain.

Another thing we now know is that gut bacteria can directly affect immune cells, and the immune cells can influence neurologic diseases such as multiple sclerosis and autism. And so how do we study these interactions? So one way we can study them is to study them in humans.

And so the nice thing about studying humans is this is noninvasive. So the way you do this is you collect faecal samples and you do 16S sequencing on the bacteria in the samples. And so 16TS sequencing is a gene that only bacteria have, and you can get the relative abundance of bacterial genera and species in your sample.

So the cons are this is all just correlative. So while you understand what bacteria are associated with the disease, you don't know if it caused the disease or is an effect of the disease. Which is why many researchers use mice.

So here's a picture of me working with an isolator, and so we can actually, breed mice in these sterile isolators, so they're free of all, all bacteria, all viruses, all fungi. And then we can deliver specific bacteria to these mice and induce disease, and that way we can actually figure out a cause and effect if this bacteria enhances disease or can protect from disease. Another nice thing about using mice is that we can sacrifice the mice and analyse the cells.

So not only do we know if a bacteria is working, we know how it's working in the mice. But the cons are, some bacteria host relationships are animal specific. So just because it works in mice doesn't mean it will work in humans or cats and dogs, and you still need to eventually figure out a way to translate this research to other species.

And so one way we try to do this is with the microfluidic chips. And so here I'm showing you a picture here of a microfluidic chip. It's very small, can fit in between your fingers, and it has two channels.

And so, and they're separated by a membrane, and you can, and we can seed gut epithelial cells onto this membrane. And we get the gut epithelial cells actually by harvesting stem. Cells, and you can get enough from just a colonoscopy to do this.

And then here's an example of, of one of my chips, a cross section of it. And so you can see that you, it forms a polarised, mouse colon epithelium. And then the, and I've added an invasive bacteria in this case, an invasive E.

Coli, so you can see that the yellow dots are the are the E. Coli invading into the chip. And so, the nice thing about this is you can identify specific interactions between specific cell types.

You can add one bacteria, one mammalian cell, or whatever different combinations you want to understand what the interaction is. And you can derive stem cells from any animal that you can do a colonoscopy on. And so you can test this in multiple species to see how a certain bacteria affects multiple different animals.

And so the way we think about using chips now is it can be used as a safety test before going into the whole animal or a whole human. So you identify a bacteria, you think it works really well in the mouse, and then, you go and you try it first in a human chip or you try it in a dog chip and make sure it doesn't damage the cells in this animal before moving on to the entire animal itself. And the cons are it is an in vitro model, so eventually you need to validate it in an entire organism instead of just, instead of just the gut cells.

So another way we can study this is to actually study the bacteria themselves. So here is a picture of bacteria that one of my students fluorescently labelled. And I'll talk about this bacteria later.

It's a, it's a good one. And so it's nice because bacteria are really easy to grow in test tubes, and you can determine their nutritional requirements. So what is, what food would be needed to supplement if you were going to give this bacteria as a probiotic.

You can also analyse what the bacteria produce, their metabolites, to identify different functional molecules. And again, the cons for growing only bacteria by themselves is you need to make sure they would grow that same way inside an organism. And so, now I mentioned this at the last Purina conference, but I thought this is important because we still have to talk about this to other scientists, strain is incredibly important when we're talking about bacteria.

And so lots of people just say the species, or sometimes even just the genus of the bacteria. And this is why I think Many probiotics might not work on the market because you might, someone might list a species and say, oh, this bacteria works really well, but if you use a different strain, then it might not work. So for example, our lab studies bacteroides fragillus.

The strain is 9343. And this is a good bacteria. It was originally isolated from a healthy human, and it protects from mouse models of ulcerative colitis, of multiple sclerosis and autism.

So this is a bacteria you would, you would want in your gut, and it would be a good candidate as a probiotic. There is another strain of bacterias fragillus that is actually toxic. And so this strain causes colitis in colon cancer in mice.

It causes diarrheal disease in humans and is associated with colon cancer in humans. And so you can't say all bacteroides fragillus is a good bacteria because this strain is good, while other strains can be very bad. And so if we use this analogy to when we think about animals, you can see on this screen, you have many different dogs, and you can see right away they they look differently, they act differently, they have different nutritional requirements, different Exercise requirements are susceptible to different diseases, but they are all the same species Canis familiars.

And so, many probiotics might list one species, for example, be fragilist, but you don't actually know if you are getting this B fragilist versus this B fragilist. And so some probiotics even go even more vaguer or more vague, and or if you get 16S data back, they might only give you the genus, which is Canis, which is, yeah, even less helpful because while I wouldn't mind playing with this canis, I would not want to play with this one. And so now, when we start thinking about the gut bacteria in our body, there are 10 trillion gut bacteria in, in the gut, which comes out to about 200 different species or 1000 different strains.

So we're not even talking about different types of cats or dogs. We're talking about 200 completely different species and understanding the interactions between all of these species. And so, what we want to do is identify the bad species and eliminate them and identify the good species and make sure they can grow well.

And so, again, when we're going back to the 16S data that we get a lot of times, we wonder, well, why is it helpful if we're only getting the species and we don't even know the exact strain. And the thing is, or or the or the genus in some cases, but it actually is helpful because you have, you have your starting sample, and you might only be able to be told that it's a whale, but a whale has a very different growth requirements than a horse. And so then you can start narrowing down.

From your sample, even if you only know the genus to start with, what type of, what I and isolate your own bacteria, and then you have your own isolate that probably acts differently than an isolate from a different clinical sample. And so, now that I've gone over that, the strain is very important and all published papers report what the strain is. And so then that that you can, and then you have to deposit it for you can buy it.

But then if you isolate your own strain, it's probably very different, and then it's And then you can use it for whatever, whatever you want. And so, I'm just gonna go through some examples of diet gut brain interactions that affect mice, but I think could be applied to companion animals. And so again, when we work with experiments, we work with germ-free, we can work with germ-free mice bred in these isolators, so they have no bacteria, no viruses, or no fungi.

And then, a lot of people refer to conventional mice as SPF mice or specific pathogen-free. So these are conventional lab mice. They have a, the regular lab microbiota, and all the animal facilities routinely test these mice for the set of known pathogens.

There could be others, but at least all they're free of known pathogens. And so when we do these experiments, we take mice that are germ free, so free of all bacteria, and we colonise them with specific sets of bacteria. Then we can induce disease, and then compare the bacteria and the healthy mice versus the sick mice to start narrowing down what bacteria might protect from disease or what bacteria might cause disease.

And then we give mice these. So this is still at the association level, but then we give mice that are, what we think are the pro-health bacteria and induced disease again. And from there, you can end up with bacteria that actually protect from disease.

So, this is, I think, a pretty cool study that really links the diet to, to gut microbiota and the brain. And so, in humans, women who are overweight or obese during pregnancy actually do have a higher risk of giving birth to a child with autism. And so these investigators work to see if they could mimic that and model that in mice.

And so they took mice and they fed them either a regular diet, RD or a high fat diet. And so this high fat diet was also extremely high in calories. So the mice become fat on this diet, until they're 8 weeks old, and then, so once the mice were fat, they were, or still on the regular diet, they were bred.

And then they tested the offspring of these mice for social deficits. And so here, is what they use to test for social deficits in mice. And so, it's a three chamber cage, so you put the mouse in it, let it get used to the environment, and then you add either a new mouse or an empty cage.

And the typical lab mouse is considered social and it will spend much more time exploring the new mouse in the empty cage. And then you can also test how much the mouse is interested in social novelty. And so the typical lab mouse is more interested in in new things.

And so now you have that old mouse that it's already explored, and you add a new mouse and it will spend more time with the new mouse. And so then the researchers tested to see whether or not this is actually if there is a decrease in the social behaviour, if they're fed the high fat diet, if the mothers were fed the high fat diet, not the pups. And so sure enough, what you can see here is mice on a regular diet, spend more time.

This first graph is showing they spend more time with mouse one versus the empty cage, and then they also spend more time with the new mouse versus the old mouse. But mice that whose mothers were on the high fat diet and were overweight, actually spent an equal amount of time with the empty cage versus the mouse or the new mouse versus the old mouse, showing that they are less social. And so then they compared the gut microbiota in these two groups.

So even though the pups were all on the same diet, their mothers were on different diets, and that affected the pups microbiota. And so this is a principal coordinate analysis graph, which is just showing the difference in the microbiota. So the closer they are, the more related that two different types of microbiota are, the farther away they are, the more different.

And you can see they're barely overlapping at all. And what they found is that the mice whose mothers were on the maternal high fat diet were missing Lactobacillus rori. Significantly missing this in their microbiota.

And so then the researchers wanted to test if this alone could, could be responsible for these social deficits. And so now looking at these, the blue shades are all mice on a regular diet. So if you give, so mice on a regular diet, spend more time with the, with the mouse versus the empty cage or the new mouse.

And if you give them either heat-killed, Lactobacillus reduri or live Lactobacillus reduri. They, they stay the same. It doesn't really affect their behaviour, doesn't make them particularly more social, but they're still, the same social levels.

But if you give them just, controlled media alone, they're not very social. If you give give them the heat-killed bacteria, they're not very social. But if you give them the Lactobacillus roduri, that's able to rescue the effects of the high fat diet, and they become more social spending more time with mouse versus empty cage or the new mouse.

So this was pretty cool because it was linking the diet to the changes in the microbiota, but saying you can also reverse these changes by giving one specific strain of bacteria. And there's another way that you can study autism and social behaviours in mice, and that is, again, mimicking humans, which is that pregnant mothers who get very sick, and this is very sick to where you're hospitalised with an infection, do have an increased risk of giving birth to a child with autism. And so they're able to recapitulate this in mice by Injecting the pregnant mouse with a viral mimic.

So this is called poly I, but it mimics very strong viral infection. And then these mice, give birth to autistic pups and the way they define autistic pups are they have decreased communication, decreased open field exploration, and decreased sociability, as I showed you in the grass before. And they also have increased repetitive behaviour, a leak in your gut, and, altered gut microbiota, or which is called gut dysbiosis.

And so again, the researchers looked to see if there's a way to rescue this behaviour. And so the first group was looking at some marble burying or repetitive behaviour. And what they found in this graph is, so saline is if you inject the the pregnant mom with saline, so that shouldn't make the, shouldn't give rise to any autistic pups.

And so the normal amount of marbles varied if you give a mouse, an area with a bunch of marbles is about 30%. But if you mimic a strong viral infection in the mother, then that the marbles buried go up to about 45%. But if you give the strong infection and then give the pups, be fragils, the bacteria is fragilous orally, then that actually is able to rescue this behaviour.

So showing it again, another type of gut microbe that can rescue this behaviour, rescue social deficit behaviour. And so then another group realised that during this process, when these mice, the, the mothers give birth to these autistic mice, there's actually a lot of inflammation going on. And so, and specifically IL-17, which is a pro-inflammatory cytokine, released by immune cells.

And so, in this case, the researchers were able to block IL-17, to block this pro infla inflammatory response. And then the and then they were able to rescue a lot of social deficits. In this case, I'm showing the marble, the marble varying behaviour.

And so just applying what we've learned about sociability or the autism models to what could be possible for companion animals is a maternal high fat diet does decrease sociability of the offspring. Lactobacillus reeride supplementation can rescue these social deficits. And maternal infection can during pregnancy also increases the risk of autism-like behaviours.

And what I didn't mention before is that a high fibre diet can actually reduce the risk of infection, in mice, at least this has been shown. And so, if you give oral bacteress fragillus, you can rescue many of the autism-like symptoms. And so all of these data taken together suggests that, breeding animals on a high fibre diet to protect from infection, and active Lactobacillus roderi and B fragilist strains could promote these specific strains that the researchers showed to promote socially adept offspring.

And so now I'm gonna go on to a model of mice that's for Parkinson's symptoms. And so this is more modelling motor deficits. And so this is a So this model is an actual genetic deletion a genetic mutation.

So there's a mutation in the alpha snucleon gene. And so mice with this mutation are slower to cross the beam. They're slower to descend the pole, slower to remove a sticker from their nose, and more constipated.

So this mimics a lot of symptoms that Parkinson that human Parkinson symptoms have. And so These researchers kind of found the opposite, where generally bacteria actually seemed to worsen a lot of the Parkinson's symptoms. And so again, SPF mice means mice colonised with a regular microbiota and GF means germ-free.

And so, Wild type mice, whether or not they are SPF or germ-free, spends an equal amount of time to, to cross a beam. But the the genetically mutant mice actually take a lot longer to cross the beam. What's interesting, if there is no bacteria present, then the genetically mutant mice are fine.

And so then time to descend a pole, it takes them longer to descend if they're genetically, if they have the genetic mutation. Germ-free mice, it actually is increased a little bit, but nowhere near the levels of mice with the conventional microbiota. And then this graph is looking at the faecal pellets for mouse, so the more constipated you are, the, the less faecal pellets you produce over time.

And so the ones that produce the least, the least are the conventional mice with the genetic mutation. So all of these results suggest that bacteria are actually making the motor symptoms worse. And so then the researchers went on to figure out what is it about bacteria that are making the motor symptoms worse.

And what it is is that bacteria metabolise fibre into short chain fatty acids, and one of the short chain fatty acids is butyrate. And so when they added butyrate to to germ-free mice, they were able to exacerbate the symptoms to the level of colonised mice, either by looking at the time to cross the beam, time to descend a beam, or time to remove a sticker. And so this is interesting because we normally think of fibre as good, but in this case, a high fibre diet could make Parkinson's symptoms actually worse because it would increase the amount of butyrate produced by bacteria.

And so then just taking this together, the high fibre diet promotes bacteria that produce short chain fatty acids. It, it's a high fibre, this high fibre diet and short chain fatty acids protect from intestinal infections, and they actually also protect from models of colitis and asthma. So generally, we like this, but in this case for Parkinson's, the short chain fatty acids exacerbate motor deficits in the Parkinson's model.

And so I think the what we know so far we could take from this to apply to companion animals is that high fibre diets can benefit younger animals, but maybe once animals start displaying motor deficits, or are at higher risk of developing motor, motor issues that they should avoid high fibre diets. And so another great pretty recent example of how diet affects microbiota, which then affects the brain is the ketogenic diet and epilepsy. And so again, in humans, the ketogenic diet seems to protect from epilepsy.

But the ketogenic diet is extremely hard to follow. It's extremely low carb and high fat and to pro to protein ratio. And so for the mouse diet, it was 6 parts fat to protein.

And so this diet was a little bit different than the diet I presented earlier, which was the just the general high fat diet, which is also extremely high in calories. This diet is high fat, but not necessarily higher in calories in the controlled diet. And so what you can see here is mice fed this ketogenic diet.

It actually doesn't affect their weight at all over time. And this is whether or not they are colonised or, or if they are germ-free or if they are given antibiotics. So this diet does not affect their weight.

So KD is the ketogenic diet, and CD is a controlled diet. And so, again, so this, and then the researchers used a model to induce seizures in mice with electrodes. So this is called the 6 hertz seizure model.

And, this is measuring the milliamps it requires to create a seizure in the mice, and mice on the ketogenic diet were actually protected, meaning that it took more, more current in order to induce the seizure, the seizures in the mice. And this is just days, how long they were on this diet. And so, again, the first thing the researchers did was look at how does this diet affect the microbiota.

And what they found is, while generally, the microbiota on the ketogenic diet was less diverse than the controlled diet, there were a few species that were much higher in the ketogenic diet than in controlled diets. And these were acromancia, mucinophila, and Pyobacteroides. So in this case, only the, the genus was given.

That they were able to get the information from. And so then they wanted to see if these bacteria alone could protect from seizures, seizures. And so in this case, again, here is the SPF mice fed the controlled diet, and that's the level that they need to induce a seizure.

But if they are given both the acromansia and the yobacteroides, they are actually protected from seizures. You need a higher amount to induce seizures. If you're, if you give only one of the bacteria, it actually wasn't enough to protect.

And if you give heat-killed bacteria, it didn't work. So in this case, the bacteria needed to be live. And what they found is that bacteria actually produce metabolites that then seemed to affect the brain.

And so this actually changed the gaba glutamine ratio in the hippocampus of the brain. So, some sort of metabolite going on the guts made it to the brain. And so this is pretty So this is also pretty exciting because it shows that the ketogenic diet does reduce seizures in humans and mouse models.

But since it's very hard to follow in humans, I actually don't know how it is in other companion animals. The researchers wanted to see if you could fix it in another way. And so the ketogenic diet definitely alters the gut microbiota without altering weight, and these two bacteria are very important, and sufficient to confer protection.

So you can be on a regular diet and only have these bacteria, and then you're actually protected from seizures. And the way they do this is they alter metabolites in the colon and they seem to actually reach the brain and to protect from seizures. So, again, diets that were supplemented with a mucinophila and pyobacteroides can protect from seizures in animals, it seems.

And so, again, these researchers only report a mucinophila andyobacteroides. It's not that they're being vague, that's just all the, the information they're getting, but it is that exact strain that they isolated from their sample that is protecting. And so now that we've had a few examples of what bacteria are good could really help animals, some actually, or some could make it worse, but some could really help.

How do you make a good probiotic? How do you make one that actually functions in an animal? And so, in some cases, the bacterial surface molecules can modulate the immune system, and to confer protection from disease.

So again, in our lab, the bacterios fragillus protects from colitis, and it's actually a surface molecule that protects the bacteria can be dead as long as the bacteria is still intact, which is dead, they can actually signal to the immune cells to protect. And so the first thing that many researchers do, and as you saw in a couple of the examples I gave, is test if dead bacteria can confer protection. If your dead bacteria can, that's great.

You can just add dead bacteria to to food and be done. But it seems to be more the case that, bacteria can't rescue with being dead. And so then, if that's the case, then you have to deliver live bacteria or figure out what is the bacterial metabolite.

And so the next step is to keep the bacteria alive, but in some sort of vegetative state, so that they grow in the gut, but not in the pill or in the food, because if they were to grow in the bottle, then they would just continue to grow until they ran out of nutrients and then die off. So you want them to be vegetative until you deliver them to the gut and then alive. And so, the biggest problem with that is many gut bacteria are anaerobic, which means they die in the presence of oxygen.

And so when a lot of the studies we do, we isolate a great bacteria, it's, but it's anaerobic. But we, it works for all of our studies because we grow them in a big anaerobic chamber in a test tube that's anaerobic, and we deliver it directly to the mouse. This isn't, this wouldn't work to translate into food for humans or for pets because this is, this is small scale, this would not work at the large scale.

And so the first thing you have to do is identify are the bacteria that you've identified as as potential probiotics anaerobic. If they are, then what a lot of companies have started to do now is, is to design capsules that block oxygen, and so they keep the bacteria alive and so that they can be in this vegetative state, but not exposed to oxygen, so they won't die. And if not, then you can just go on immediately to testing the vegetative state.

And the only thing you have to test is that most bacteria ideally grow at 37 degrees C, but some can grow at room temperature, but most don't grow at 4 degrees C. And so it's figuring out what temperature you would need to store your food or pills to keep the, bacteria alive but not growing. So And then the next question you have is once you've figured out this, you have to test if your bacteria are actually stay alive in this pill form that you've discovered.

And if they are, then the next thing to do is to determine if there are any specific nutrients that would promote its growth. They're alive in this form, but you want to make sure that they stay alive in the gut. And so, again, just because I know a lot of bacteroides fragylus, what's really interesting about bacterios fragils and a few other bacteroides species is that they can metabolise xylose, a sugar that most other bacteria cannot.

So if you were to be giving this bacteria with xylose, it would create an environment that it could actually keep growing. But if your bacteria, if you cannot figure out a way to keep them alive in pill form, then what you would have to do is actually identify the product that the bacteria makes. That is being active in helping the immune system or, or the brain in some way.

And I'm saying this like, oh, just do this, but this usually takes several years because you have to compare this bacteria to related bacteria that don't work to try to understand what molecule it's making and use a mass spec to identify what the molecules are, and then slowly narrow down what it is that's active in your bacteria. But hopefully you don't have to go through this and you can find something from one of the earlier ways. And so then the next question the next thing I want to discuss is some future studies that you could do to test whether or not your candidate probiotics can actually work in companion animals.

And so the easiest way is to do 16S sequencing. So you identify two groups, healthy versus unhealthy, active versus inactive, social, non-social, whatever you want to compare, and collect the faecal samples for 16S sequencing. And the most important thing when you collect faecal samples is to immediately freeze the faecal samples to preserve the ratio of bacteria.

Again, because many gut bacteria are anaerobic, the second they come out of the gut. On the outside of the stool sample, the bacteria will start to die. And so the longer you leave it at room temperature, aerobic bacteria will start to grow, and, anaerobic will die off, and that will change your ratio.

So for all of this, we immediately freeze our faecal samples. And so this is nice because it's a non-invasive study, and you get cor correlative information of if there a difference between gut bacteria, and your two groups. But one of the cons are, again, it's correlative, so you don't know if that bacteria is actually the cause of the effect.

And many cat and dog microbes are unknown, so you get your sequencing results back and it comes back as a bunch of unknown bacteria. And so a way to kind of fight this is to collect these samples, but then put them into germ-free mice. So put healthy companion animal or diseased companion animal faecal samples into mice, induce whichever disease you're trying to study.

And then compare the healthy versus sick. And you can do this by trying to sequence, see if you get any 16S data back. But if you don't, the other way you can narrow down what bacteria is active is by using selective antibiotic treatments.

So different antibiotics kill only specific types of microbes. And so then you can figure out which, what antibiotic your, your active bacteria are susceptible to, and then from there, start to isolate your single strain. And so while you might have at first an unidentified bacteria, which might not be exciting, it's actually super exciting because you can from your stock sample, you can actually isolate that specific strain.

Bacteria, and then you get to name it and characterise it, and hopefully it will be able to promote health in a companion animal. And so once you have your active bacteria, I think the way that everyone wants to go is to first try this in these microfluidic chips before going on to whole organisms. And so again, we can isolate intestinal cryps from a biopsy from human, from a mouse, from any sort of animal.

And you can grow up the cells to get to, because they're stem cells, you can grow them up as much as you need and seed them onto these microfluidic devices. And so here's now looking top down, one of my chips, so this is a mouse colon. And so the blue is the one which measures the tight junction, so to keep the gut.

So not permeable, so that it's not leaky. And then the yellow is looking at an endocrine cells and the purple is looking at goblet cells which produce mucus, which are an essential part of the colon. And so you can see we have a nice healthy.

You can start adding bacteria to see if that bacteria either promotes the health of this gut ship or could damage it. And I'll just show you an example of one of my experiments that I've done. So this is looking at the entire chip, here, and so this is looking at a healthy sterile chip and the blue is looking at the epi the gut epithelial cell nuclei.

And so you can see that a healthy chip is covered with, with epithelial cells. And so then I infected these chips with salmonella typheerium. So, in mice, this actually makes them extremely sick to where they die, from extreme diarrhoea and dehydration.

In humans, this also affects humans and makes, and does make you have bloody diarrhoea, but it's usually not fatal. And what you can see here is that in the chip, it, It actually destroys the epithelium. And so these black parts here are all just empty.

It's just the membrane with no cells. So it actually causes the epithelium to detach and destroy. So you wouldn't want to use salmonella, obviously as a probiotic.

But then, this, example here, this chip is a probiotic I was testing. So it was one I had isolated from a faecal sample. But I wanted to make sure that it alone would be, would be good and would not be harmful to human cells.

And what you can see here is when I colonised with this potential probiotic, the cells remained healthy and intact on the chip. And so I think this is a really nice way to start the transition from mouse work to the whole organism is to test first if it's toxic or helpful in these chips before moving on to the next organism, humans or mice or humans or cats or dogs. And so just to summarise what I've discussed today is, the different models we use to study the gut brain axis, and then understanding the difference between just being told a species or the actual strain, strain is very important for activity.

And then we've talked about a few different ways diet and the gut brain, interactions affect mice and how this might affect other companion animals. And so we've talked about social behaviours, motor functions, and seizures, and then how to make a good probiotic, so testing its activity in different conditions. And then finally, looking at future studies of how we can verify these incompaning animals.

And so, I do have a pet. He's not a traditional companion animal, but I felt guilty about not showing a picture of him at least. So, this is Max.

But I will, and thank you all for listening and I'm just gonna go back to this slide, to see if anyone has any questions. Francesca, I think Max is very cute. So lovely, lovely.

That was, that was absolutely fascinating. Thank you so much for sharing that with us. It really has been very, very interesting.

We do have a couple of questions coming through. We've got one that says, does this sort of fibre, in other words, soluble or insoluble fibre, seem to make a difference? I am sure it does, and, definitely from a chemical perspective, it does.

In most of the mouse studies, the fibre is from apple pectin. But I'm sure that different fibres will be metabolised differently and have different effects. Interesting question coming through here about changing bacterial compositions and that.

And it's to do with the difference between gut bacteria and skin bacteria. Is there a variation that you can affect from that? Yeah, so, Gut bacteria and skin bacteria are very different.

The skin is definitely less diverse than the gut, and then obviously, it's composed of aerobic bacteria or facultative anaerobes, meaning that they could grow in oxygen and not in oxygen. In mice, and so, I guess if your question is whether or not you alter the gut, does it alter the skin? And that's, that's actually harder to test in mice because, as I'm sure you all know, mice are coprophagic and they're living in their cage with their faeces.

And so when we give antibiotics to mice, it's kind of hard to say whether or not the skin is affected because it's just, they're, they're also sitting in their faeces. And not, not for very long. We changed the cages once a week, but for a little bit there.

So it's a little bit harder to dissect that out in an experimental setting. Interesting question coming from Zao, in Portugal. Hey, Ziao Zhao's one of our regulars on these and a shout out to him.

commercial products that are available with these bacteria and I know we're sponsored by Purina tonight, but is there a wide variation between the products and what would you recommend? So that's, so this is what I don't know, because, you know, we publish with a strain and then usually the strain name is not mentioned on the probiotic itself. And so I don't actually know which, what, what works.

And so what I tell people now is if you find something that really seems to make a difference, it might very well be working. It's just, that strain might be good, but I just don't know what it is. OK.

Jennifer, if you're still with us, if you want to unmute yourself and jump in here and give us, some insights if you can, that would be great. . While we're waiting for Jennifer, if she's still with us, to come back.

There's an interesting question that says, does this mean that depending on what is wrong with you, you should look at your diet and would it mean that you may require different probiotics for different conditions, depending on what the individual disease is, that's going on in your body and how would you make that decision? Yeah, so that's a great question, and we definitely think that seems to be the case because, the gut bacteria definitely stimulate the immune system, and you can imagine in some cases you want a more stimulated immune system, in some cases you want a less stimulated immune system. And so there are bacteria that are really important for colitis or autoimmune diseases that you wouldn't really want, in those, those downplay of the immune system.

And then those actually wouldn't be very good and in an infection model or in a cancer model where you actually want your immune system to be stimulated. And so we actually see that all the time in a different project I have in the lab where I'm looking at . A cancer strain.

I've isolated a strain of bacteria that, seems to promote, cancer killing cells. And my lab mate isolated that same bacteria, and he was using it as a control because it did nothing in his colitis model. So it definitely, you do need to start taking into account what disease you might be susceptible to or have, and then what, and then exactly what diet promotes that specific bacteria that would help you.

It sounds like that is a a huge topic that probably needs further studies. I should imagine there's a, there's a commercial proposition out there for different companies to be doing market research on on probiotics for different conditions. Right, yeah, I think so.

I think there should be. Maria wants to know, is enterococcus fecium a good bacteria to promote brain, gut brain access health? Specifically knowing that it's one of the most common, in nowadays common probiotics that we get at the moment.

Yeah, so, Interococcus specium is very interesting. So it's in, so most humans either are colonised with the inococcus faum or Eococcus vicalis, and they're actually, Some people consider them a pathobiant, meaning that they live healthily in our gut for a very long time, but in very sick people that are hospitalised, it actually can overgrow and they usually, and then people can end up dying from this infection. So a lot of people think.

It's actually kind of a sign you are dying and not a cause. And so, because that bacteria, a lot, a lot of times is resistant to antibiotics. But it does seem to do good.

And so it seems to help in some infections. And then it also recently, there was a paper showing that, Cancer patients who had inococcus, facium were better responded to immunotherapy than those that did not have that in their stool samples. And so I, I think that's a perfect example of one that could probably do a lot of good in some cases and maybe, but maybe not in every single case.

Interesting. Very, very interesting. Steph wants to know, do you have a website that you could recommend where people could go to, to look up these different things like, you know, what to look for for Parkinson's or for IBD or these sorts of things?

No, I've actually been thinking, I've, I've been thinking about trying to make a website for this very reason because it's not, you have to, you have to look at the papers and then go from there, but there isn't a collated area where you could say like, what should I do for this bacteria. Or for this, for this disease. There isn't that out there right now.

Again, a commercial opportunity. I should imagine it's gonna be a very difficult one because, you know, in the beginning you were saying how, how species-specific it is. So, you know, somebody who wants to go on a website to look for something in, in people, I mean, I don't know.

Do, do people have cultural differences to these as well as we have species differences in the veterinary field? Yes, so what, so for a lot of, a lot of times what we do is we take human faecal samples and put that into a mouse to see if it has an effect. So at least if you do isolate something, it was originally a human bacteria and it might go back to a human bacteria.

And I would say a lot of the human differences so far between like, if you live in a different place, seem to be tied to the diet. And so they, where, so who you live with does affect. So if you live with people, your bacteria is, closer, to your gut bacteria is closer than someone you don't live with, but also what you eat dictates a lot.

So I think that probably would be the biggest difference. Wow, OK. Interesting statement or question that's come through.

Would nutritionists be able to have this knowledge and help people? Yeah, so I think, so, again, anyone who has access to, to reading all these scientific papers could have this nutritional information, but a lot of it is just being discovered now. And so when you think about nutrition, again, like the example of xylose or some other sugars that we don't metabolise at all.

Nutrition hasn't really focused on because we can't metabolise them, but it turns out gut bacteria can, and then they affect our health. And so I think this is a new field for nutritionists that they're starting to look at and starting to understand. We actually have to think about how to feed the microbes, even though we can't metabolise it, they can, and then that affects us.

Along those notes, I mean, could, could any of these prebiotics and probiotics actually be detrimental to someone's health? So, I think possi, so I think the way they might be detrimental is if you already have a leaky gut or have been exposed to many rounds of, of antibiotics to where your bacteria, your gut is damaged from the antibiotics and, And your gut bacteria is altered, because if the bacteria get into your bloodstream, that could, that could cause sepsis. If your immune system is healthy, it'll clear it.

But if you are some reason very immunosuppressed, then adding more bacteria might make things worse. OK. Just a, an interesting question and not really to do with probiotics specifically, but there was a question I've just lost it somewhere along the line, about how do you breed the mice to have no organisms, no bacteria.

Surely they're getting some passed down from their parents. No, so we, so. The first germ-free mice were reared by hand.

So the way you get them germ free is you do a C-section. And I have performed this. This is the craziest thing I've done is to do a C-section on a little mouse pup, revive it sterilely, transfer it into an isolator.

And then the very first ones were then reared by hand, which for mouth. Pups is every hour giving them sterile milk. And, but then once they are sterile and living in the isolator, then you can just continue to breed them in the isolator.

And so we have these ports where everything is autoclaved, everything is sterile entering it, you spray it, and then you wait 3 hours for the sterilising solution to work, and then you pull in the sterile food from the other side. So it's a, it's a crazy process, but it does work and we check to make sure that they are free of all microbes every week. There we go.

So that answers that question. Just a message through, from Jennifer to say she's having some technical issues. She can't come back on with us.

But she says that the Purina Pro Plan 40 Flora, contains live enterococcus fecium SF 68, and it is encapsulated as, as Francesca was telling us earlier. So, that's a, a, a good one to use. Yeah, that sounds good, yeah.

Yeah. Folks, that's all we have time for tonight. Francesca, this has been fascinating and I really thank you for your, your time that you have spent tonight talking to us and, and discussing this.

So thank you for coming onto the webinar vet. Thank you. To Jennifer and Purina, thank you so much for sponsoring.

It's, through their proud sponsorship that we actually get to have these free webinars for you. So, let's support the companies that support us. And, Purina, thank you so much for your sponsorship.

To my controller in the background, Dawn, as always, thank you for making everything work so effortless effortlessly. Now I've got my teeth back in and from my side, it's good night until the next time. Thank you.

OK, so this is the answer for the question that was raised during the webinar last Tuesday of Francesca Casaniga. We had some technical issues, so I was not able to answer at that moment. So the question was, why the companies, or it seems like the companies are not communicating the strain of the probiotic, in our products, and in fact, we do.

So we as a reliable company, we communicate the strain of 40 fluoride in our pack or or level. So our product which is Purina pro plan fortiflora is a probiotic, which can be used for dogs and cats and contains life lactic acid bacteria, which is enterococcus phys SF 68, which is strain is the next, which is the NCIMB 10415. So this is our strain and this is communicated in OAC.

And for sure we ensure the survival of the bacteria through a proprietary encapsulation process, as also Doctor Casaniga mentioned, and conducting exhaustive quality controls also of the product. And indeed, most research in animals have been done with these bacteria with Interococcus viation. So its effect or efficacy on intestinal health and balance has been widely proven, which is really good for us.

And now I have to say that we are also looking at the role of probiotics on behaviour with promising results for the moment, I can say, but it's really still in progress, so I cannot say more for the moment, but we are in the process also of studying these in dogs specifically. So, the thing is that for sure our probiotic accomplished all the steps that also Doctor Casaniga has remarked for creating a, a reliable probiotic. So I think maybe it's a probiotic that you can use maybe in, in dogs or, or cats.

So thanks, thanks for the attention and thanks for being in, in the webinar. See you in the next webinar.