Introduction

Steve Dale:
So good afternoon, welcome to the Winn Feline Foundation Symposium. How many of you have been to one of our symposiums before? Most of you. How many haven't? Well can everyone applaud for those folks that haven't, and we'll say welcome. How many of you happen to have a cat? Alright now two people in the room didn't raise their hands, as well as the bartender. So what I want to know is why those three people don't have a cat, and I do know that we have the Toronto Humane Society in the room, so we can hook you up by the way. Pretty much everything and most of you in this room I think probably know about the Winn Feline Foundation.

My name is Steve Dale, incidentally, and I have been on the Board of the Winn Feline Foundation now, I think for about 10 years. I have been moderating this, the Winn Feline Symposium for 5 or 6 or 7 years. It is always an honor for me to do, and it will be again today. I have the opportunity to introduce two of the best veterinarians in the field, in the world, literally on the planet, at what they do, and for us to give exposure to those veterinarians is an honor for us to at the Winn Feline Foundation, and hopefully you will find it useful, and appreciate the fact that these are superstars, and they are superstars in veterinary medicine. Are there veterinarians here? Great! So for you veterinarians, fill out the comment card that you got from Janet, and if you don't have one, Janet raise your hand back there the back of the room, and you can get one and fill that out, and then you can get your CE certificate back.

Talking about the Winn Feline Foundation, so I asked how many of you have a cat, and except three of you, you all raised your hand. So for those of you who have a cat, who knows someone who has cats, everything pretty much about that cat, that goes on with that cat, from the morning when the cat wakes up, well let me put that a better way, wakes you up is probably a better was to put it, until the end of the day when the cat finally allows you to go to bed. Everything in between all that, the Winn Feline Foundation has probably funded studies about, to what our cats eat, to the vaccines they get, to the health report that a veterinarian gives them every time they go to the veterinarian. We are not only talking about pedigree cats, and breed-specific illnesses, and we are, we are talking about all cats. The Winn Feline Foundation for nearly 50 years now, has had something to do with it. These symposiums have been going on for decades. Joan Miller is the lady who said, in her head, somewhere, somehow, I am going to start these symposiums. We are going to bring education to breeders. Joan isn't here this evening, but I still wanted to acknowledge her, a round of applause for her please.

We truly can't do it without my colleagues on the board I suppose, so for those colleagues on the board, I am very proud to be on this board all these years, I ask you to stand up. There we go, the Winn Feline Foundation Board of Directors, and our Executive Director is Dr. Vicki Thayer. Dr. Thayer helped organize this event. Dr. Thayer is past President of the American Association of Feline practitioners. She is a superstar in veterinarian medicine, we are grateful to have her, and boy does she work very, very, hard. It is not only her, it is our grant reviewers who are all experts in feline medicine that say, I wish we had more money. Every year it happens, we want to support more grants. We want to support more studies, there is good research out there, and every year we don't have as much money as we would like, and that is worked all of you can come in.

I am not asking you for donation, well yes I am asking you for a donation, but it is really you telling your friends, you telling people you know about the Winn Feline Foundation. If you are a breeder, someone purchases your cat, it would be great to tell them about the Winn Feline Foundation. You know what, we have two very specific ways people can donate. There are two funds, one is called the Ricky fund, which I have something to do with. That is for cats with hypertrophic cardiomyopathy, so that we can better understand that heart disease in cats. The other is for FIP, feline infectious peritonitis, that is called the Bria Fund. When a cat sadly passes away for you veterinarians, but also for anyone, when a cat sadly passes away from one of those illnesses, even if you made a $5 donation, $500 would be better but really $5 is fine, it is acknowledging to your client that this happened and I am making a donation in your cat's name to the Winn Feline Foundation, that is a great thing that helps all other cats helps of course the Feline Foundation, and that cat owner knows you care and this is all available on our website, which is winnfelinefoundation.org.

Before I introduce my first speaker, you know what, I am just talking, I am very bad for this. This is our mission, I talked about all this already, I am not even following what I am supposed to be talking about, but you can read and most of you know about it the Winn Feline Foundation, and yes, this is sort of hit list. If we had David Letterman here to count off the top 10 list, this would be top 12 maybe, of just some of what we have done. If you can read what they are, some of them are recent, some of them are not quite as recent, I will point out one or two. In the 1960s, cats were dying of dilated cardiomyopathy, a kind of heart disease and also getting blind, and it is like why was that? A veterinarian from the University of California, Davis, Dr. Paul Pion said, "I think I know why. I think there's not enough taurine in cat food." Some other organizations thought he was crazy, and we said, well you might be crazy but this actually makes some sense, despite your personality. If that is tweeted out you are in big trouble, he is a nice man. What I am trying to say is that, we said yes when others didn't, and it turned out he was right about that. Feline asthma was not very well understood until actually quite recently, and it was the Winn Feline Foundation that that funded those studies. If you are a Maine coon or ragdoll breeder, you know about the genetic markers for those cats for HCM, hypertrophic cardiomyopathy, and that was funded by the Winn Feline Foundation, and this is a very, believe me, small list.

Now, I ask you to turn down your cell phones if I didn't do that, I am supposed to say that. We have cards, comment cards, question cards the index cards on your table. You can fill those out, and this is the way to ask questions. I will read those cards after both speakers speak. Also we have these beautiful books that we made up for our 40th anniversary, we are close to 50 years now, so we have a lot of these left in our storage unit, between me and you, so please feel free to take any or if you have cat loving friends, relatives, family members, other breeders that would want these, veterinarians, technicians, please take these back to your homes, your practices, we appreciate that.

She is a superstar in veterinary medicine, we have funded Dr. Jodi Gookin many times. She is an associate professor at North Carolina State University. I will tell you, I speak at many veterinary conferences, I go to Veterinary conferences all over and there aren't many veterinarian conferences where Dr. Gookin's name is not there, and you are not getting one or two or three talks, but you are sometimes giving four or five or six talks, and she is as I said, a star. We know more about understanding the gut, when I say that of cats, because this lady has had the guts to do it, Dr. Gookin.

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Beginning of Dr. Jody Gookin Audio

Dr. Jody Gookin:
Alright, can you hear me? Well it is I that am proud and honored to be invited by the Winn Feline Foundation to speak to you today. What I am going to talk to you about is something that is near and dear to my heart, and it is something that would never have happened if it wasn't for the Winn Feline Foundation. It is a work in progress, but we stick with what we start, and we are going to help kittens with diarrhea and kittens that are dying with diarrhea. We have been going through this journey, looking at bacterial causes of diarrhea in young kittens, and we have, we think we have discovered an important bacteria that causes diarrhea and might kill young kittens due the diarrhea, and another that might even protect them against that diarrhea, and that death, but we are still in the process, but I would love to share with you this story.

First of all, why is what we are doing and what the Winn Feline Foundation is doing important. In the U.S. alone, there are 74 million owned, and 70 million feral cats. Each year, they give birth to 180 million kittens, and an inestimable numbers of these kittens end up at one of 4000 to 6000 U.S. animal shelters. In those shelters, it is estimated that 15% of those kittens will die or be euthanized due date illness before they reach 8 weeks of age. What is disheartening about this, is that an obvious cause is unknown in as many as 50% of these kittens, and hardly anybody seems to care, or has spent much time studying what is happening to these babies and why they are dying.

The few studies that we could actually find that look at mortality in kittens, we can find that the weaning period seems to be the most vulnerable. This study was published in 2002, out of the UK, and it was a necropsy study, so post-mortem examination of kittens that had died, and there were 274 of them, and actually these 4 kittens died in the perinatal period, 38 neonatal, 30 pre-weaned, but 202 post weaning. So it is that weaning period that seems to be very critical, and we think that is because of the stress of weaning, there may be a role in failure of immunity transfer from the queen to the kittens, crowding, sanitation, changes in diet. What do we know? We know that infectious disease appears to be the most common cause of death in these kittens. So these yellow areas are the percentage of kittens that died from an infectious disease and among those infectious diseases, 71% of them were viral, and we are looking at a lot of death from panleukopenia. So that is an area that needs some work, and 20% of those were actually bacterial causes of death. The other thing we know from these studies, is that these kittens often die with clinical signs or post-mortem evidence of intestinal disease.

So they die of vomiting, diarrhea, and we know that infections are common in these kittens, so it makes sense that focusing on infectious causes of diarrhea or GI disease in these kittens seems like a high priority area. The tragedy extends to the fact, and I am never lacking for amazement about this, that diarrhea is the second leading cause of childhood mortality in developing countries. So 8000 children per year die from diarrhea in developing countries.

There was a recent study done by the Bill and Melinda Gates Foundation and they demonstrated that among the infectious causes of diarrheal death in these children, most of them are caused by a mere four pathogens, and one of these is rotavirus, one of these is a protozoan parasite called cryptosporidium, and the other two are bacteria. Interestingly, these same infectious causes of diarrheal death in children in developing countries, cause diarrheal death in the children of our animals in the United States, our calves, our kittens, our puppies. So that sort of developing country is recapitulated in the environment that a lot of our young animals are growing up in.

So we have two bacterial causes, and in fact if you look at all of the infectious causes that they found in these kids, you will see that diarrhea caused by E. coli bacteria, was significantly associated with a hazard of death. So they found a bunch of different types of infectious diseases, but the two that had an increased hazard of death happened to be E. coli. We all have all have E. coli in our GI tract. Most of that E. coli is good E. coli, but there are bad E. Coli in there, and what makes a good E. coli versus a bad E. coli, is the presence of, in the chromosome, genes that make proteins that are toxins, or do bad things, and it is difficult to tell those good E. coli from those bad E. coli and we are going to go into that more.

What we do not know is very much about the role of E. coli as a cause of diarrhea in kittens or cats. What we do not know really much about anything about bacterial causes of diarrhea in cats or dogs. We as veterinarians only think of about four different bacterial causes of diarrhea when we see a dog or a cat, and those are salmonella, campylobacter, Clostridium perfringens and Clostridium difficile. The reason why those are the only ones we think about, well that problem is going to become clear, but we not only don't want to look for them, but we often cannot afford to look for them, because they are really cumbersome to diagnose. So if you want to diagnose an intestinal bacterial infection, what do you do? You start with feces, and you have to culture it on a Petri dish with media that fosters the growth of all the bacteria in there, and then you have to sort of pick a couple of those and find out what bacteria are those, and then you run each of them in a machine at $50 apiece, so they you can find out that is an E. coli, and that is an E. coli, and that is an E. coli. Okay we all have E. coli. So what is that mean?

Well to know what that means, I have to isolate those three different colonies, well there is a lot more on there than the three, right? Isolate those three colonies, take the bacteria, extract their DNA, put it in a polymerase chain reaction test, run the products of that test out on a gel, to see if those have the gene that codes for a virulent factor. That right there took a minimum of 3 to 4 days, and probably $150, and that is in house, right. So by that time, you cannot afford it, you are on a fishing expedition, and your patient is either dead or got better despite you.

So what makes it horrible, more horrible about finding bacterial causes of diarrhea, is what we now know about the gut microbiome, the bacteria that live within our bodies. In a human, 100 trillion bacteria in your GI tract, that represents over 100 species of bacteria that carry 3 million genes. So what do you think? Seriously? I am going to put little bit of poop on that dish, and I am just going to select three colonies, and I am going to find the bacterial pathogen. Well if you compare this microbiome to you and me, here is me, one human, one species, I have 23,000 genes, so guess what? I am only 10% human, only 10% of me up here is human, 90% of me is bacteria. So you have tons of these bacteria in the lumen of the gut. There are also other toxic things in the gut with the bacteria, and the only thing separating you from the 90% of other people that are there, these bacteria, is a single layer of intestinal epithelial cells. So there is a lot of potential, these guys are good guys, but there is a lot of potential for some of these guys to be bad guys, right. So how are we going to figure out who is bad? Which one of you guys is responsible for the diarrhea, raise your hand. It is not easy to accomplish. Therefore, very few bacterial causes have been identified, and there are so many potential causes that are sitting there, and the ones that we do know about actually vary from really common, to rare, sometimes are present in healthy cats and sick cats, and there are numerous tiers of tests and diagnostics levels you can do, so it is not very easy to get a hold of.

So why the heck would I start going to look at bacteria? I mean that is like a career ender right there, right? Go looking in that for something that might cause diarrhea. So the long-term goal of our work has always been to identify life-saving preventative or treatment strategies that help ameliorate the morbidity and mortality of gastrointestinal illness in cats. My training was as a GI person, not necessarily an infectious disease person. I am more interested in the cat than that bug that is in there. So work that we have done has centered around the relationship between pathogens and that intestinal epithelial lining. So most of you, if you have heard of me before, think of tritrichomonas, and that is a pathogen that's in the GI tract of the cat and it gets all up and close and personal on this intestinal epithelial cell that I have kind of drawn here, of one intestinal epithelial cell with its little nucleus.

We also do work on a pathogen called cryptosporidium, which is also a pathogen that sits on the top of the intestinal epithelial cells. So I guess I like things that like to sit on the intestinal epithelial cells. I like pathogens that like to go to the intestinal epithelial cell and get on it, which is inappropriate. These cells do not want to be touched, don't touch me, don't come near me, you're bad. So it was with this sort of background in mind, that I was asked one day to consult on a case. It was a pathologist from across the road, they got a kitten, and this kitten they wanted to consult on tritrichomonas. It was a 4-week-old Persian kitten that developed diarrhea 5 days ago, became inappetent and died. They did a post-mortem on this kitten, which is always a good idea by the way, to get a post-mortem on the kittens when you lose them, even though I am going to tell you usually don't find anything, still we need more help, we need more data.

They were interested in the tritrichomonas, and had lots of questions about tritrichomonas, but what interested me was what I have underlined, what interested me was that in the gut, in the small intestine of this cat, there were large numbers of bacteria that formed palisades along the intestinal epithelium, and they attached and effaced the epithelial cells. That is a buzz term, attaching and effacing for E. coli. For an E. coli that is abnormal in its virulence factors, and it gets all up in personal, and attaches on these intestinal epithelial cell. I am like cool, that is interesting. I like cats, I like infectious diseases and gut disease, and I had actually never heard of that before. I am like E. coli, like this kind of E. coli can happen in cats? I talked to pathologist, and they said, yeah, I see that sometimes. I am like wow, that is really cool.

I am going to put together a case series, give me all the cases you have ever seen. There were not that many, they came was seven, I will have one of my residents summarize the cases, we will take some pretty pictures, and we will get the word out there that E. coli can be the cause of diarrhea in cats. So we did that. First what we did was we read about what do we need to know about attaching in effacing E. coli? We learned that these E. coli attach to the epithelial cells, the green is the surface of a single epithelial cell that lines the gut, and the bacteria get all up and close to that plasma membrane. The plasma membrane actually comes up and worships the bacteria on this thing called a pedestal. It sounds like a good deal if you are one of the bacteria, you sit there and get worshipped. The way that they do that attachment, is that they have this receptor on them called intimin, and the reason why I am telling you this is because the gene that encodes that intimin, that allows that to sit on that cell is called EAE, and it is an important gene that we are going to be looking at as we go forward.

The other thing that they do is they efface the microvilli. Microvilli are like little hairs on the top of every intestinal epithelial cell, and if you look down on the top of the epithelial cells, this is a single epithelial cell, and this is another one, and another one, and you can see these fine hair-like projections, those are microvilli. On those is where the nutrient transporters are, that is where the gut absorbs nutrients and food, and when these bacteria here in increasingly higher numbers, here is a matt of them right here, they efface and flatten out those microvilli, which results in the intestine not absorbing normally.

So you get this malabsorption, and you get this diarrhea, and basically the kitten starts to starve and get dehydrated from diarrhea. There are basically two types of E. coli that do this kind of thing, one of them is called EHEC, and that makes toxins, and that is the E. coli that kids get when they go to petting zoos. It is carried by cows, it is O157:H7, so that one is nasty. So that one, EHEC, can sit on the intestinal epithelial cells like that, and then it makes these toxins. Then there is this other one called EPEC, enteropathogenic E. coli, and it also sits there, but it doesn't make toxins. These EPEC is going to be the one that we are going to be interested in, it is the one we are going to find, but this EPEC actually is one of the ones that is significantly associated with death of children in developing countries, that particular organism, this EPEC.

So we went ahead and we said, give us the cases you have of this E. coli that attaches and effaces, and we will have a look at them. So they gave us 7 kittens. So we got the kittens, and what the kittens were, were little blocks of paraffin with tissue in them. That is all we had, what was left over of this necropsy of these kittens. So we made slices, we looked at them under a microscope, and you can see, this is the intestinal epithelial layer right here, and this is the lumen where the bacteria would live, and all of this purple stuff right here are bacteria that are adhering to these intestinal epithelial cells.

When you Gram-stain that, you can see the bacteria right here, they are Gram-negative. They are negative because they don't look any different than anything else, right? Gram-negative means they do not take up any stain, and then when we look with a transmission electron microscope, we can see these E. coli sticking to these microvilli on the top of these epithelial cells. That was truly fascinating. What we did to figure out, who this is, is this EHEC or EPEC, is to carve out of that tissue with a scalpel, the organisms, extract their DNA, and do PCR on them and figure out what their virulence attributes were. They had EAE, which means they are an attaching and effacing E. coli. They didn't have the Shiga toxins, so they were not EHEC, they were EPEC.

So here we have a demonstration of an important E. coli, attaching to kittens that have died with diarrhea, the same type of E. coli that kills kids due to diarrhea in developing countries. What was the real game changer, direction changer for us, was that we saw this in 2 of the kittens, but in the other 5 kittens, when we got to the Gram-stain, we went what? Because this is what we found. So the pathologist told us that these were E. coli, based on this stain here and I believe him, the organisms are there, organisms are there. Then you do the Gram-stain, and go what? That is Gram positive, that is not an E. coli, that is something else. I have never even seen anything like that before, and when I look with the electron transmission microscope, it is doing the same thing that the E. coli does. So more kittens had this Gram-positive thing, than had this Gram-negative thing, whoa, what is going on here? Both of these are inappropriate.

So what we did was we took the scalpel and we carved out the guy, and we did PCR on him and we found out that he was an enterococcus, and a specific species of enterococcus called Enterococcus hirae. So at that point, we were like, well what do we know about enterococcus. I don't know anything about enterococcus. So let's look at the literature. This is our kitten, one of our kittens, one of the most dramatic examples. Like this is the intestinal epithelial layer, in a kitten's small intestine. Look at that, and that it is just remarkable. Those bacteria are not supposed to be there like that. It is inappropriate. I am thinking, hey, maybe we found a new pathogen of cats. What do I know? I found when we looked, that this actual activity of enterococci has been described in neonates of many domestic species, but not kittens. Chickens, foals, piglets, puppies, people, little descriptions here and there of this. In those descriptions, the association with GI disease is unclear. Everyone that looks at that is going to say, ewe, that is a pathogen. That is not supposed to be doing that. As we found, for the first time, no one has ever even made this connection before, it is nearly indistinguishable from EPEC infection, in the way it appears under a microscope. The thing in enterococci in general, is they are considered gastrointestinal commensals. We all have enterococci in our gut, just like we have E. coli. That is what makes it so difficult to identify these pathogens, because they are normally present, and they are frequently administered as probiotics. So in fact FortiFlora is Enterococcus faecium, that is a species of the enterococcus. I also am aware that the enterococci can be serious potential pathogens, so when enterococci get out of that gut and they get in the reproductive tract, or the urinary tract, or the biliary tract, or the lungs, they can cause disease, and really bad disease, and they can kill cats. They also could be antimicrobial resistant, they persist the environment in the form of biofilms on things that can be transmitted to other animals. What we don't know though, is could they be pathogens under some circumstances? So particularly in a neonate, under the conditions that we talked about, during weaning.

So this is when I went to the Winn Feline Foundation, and I said, I need your guys to help. I think we have something going on here. We have this EPEC, and we have this Enterococcus hirae, and they are both attaching seemingly inappropriately to the intestines of kittens. I think we are onto something here and I need help, and that is pretty much what the Winn Feline Foundation has always been for me, somebody that is willing to think outside the box, and invest in the future of something that is not certain, right? We are going to take some risks, we are going to follow the trail, and see what we get out of this. So our objective with this grant was to determine the association between these enteral adherent, these attaching bacterial infections, and terminal illness in foster kittens. Now we looked at terminal illness, because I can't look at this in a live kitten. In order for me to look at whether these are attaching to the gut, the kitten has got to be dead, right. So let's start by looking at kittens that died, and see if we can find it.

So Lenovo ThinkVantage Toolbox stopped working. [LAUGH] So our aims in this study were to define, what is the healthy enterococcal community in the gut of foster age kittens. I don't even know who is supposed to be there. Are you supposed to give them FortiFlora? What is up with this E. hirae, I have no idea what we are working with. We focused on kittens that were under 12 weeks of age, under 1 kg body weight. We looked in the small intestine, because that is the workhorse of the GI tract, its most important part, and we looked at the bacteria that were up close and personal with the epithelium. We wanted to know, how is that microbiota impacted by severe illness in this population, and is there any association between the presence of these attaching bacteria, and kitten mortality? Our approach was two groups of kittens. To get healthy dead kittens, we were able to work with our County Animal Shelter, who at the time had a very poor ability to foster their over slot with animals. It is hard to get kittens from them anymore, thank goodness, but at one time they had to kill a lot of kittens, because they had no place to put them. They allowed me to have those kittens and necropsy them and use them in this study, and we got 50 from them.

The other 50 came from the SPCA, lovingly cared for it, fostered kittens, who either died unexpectedly, or were dying and humanely euthanized. So that is our population. Each of these kittens, one at a time, were brought to NC State, and we did a full autopsy. We removed the entire GI tract; we took selected pieces of the GI tract and looked at them under the microscope. We Gram-stained them, we did special techniques called fluorescence in situ hybridization, that would make our bacteria glow if they were E. coli, or glow if they were enterococci, and we carved out the organisms and looked for the genes, like we did before, but we also took the small intestine and we cultured the surface bacteria for enterococci. We gathered the enterococci, we figured out who is there, what types of enterococci, are they virulent or not, and what is their antimicrobial susceptibility? Here is our kitten populations, 50 healthy kittens, 50 sick and dying kittens. They all meet the selection criteria, so their average is about 6 weeks of age, equal male and female, body weight averages 360 g in the sick kittens, significantly lower than in the healthy kittens, because they were really, really sick. Also, longer time for us between the time the kitten died, and the time we did necropsy in the ones that were sick, because they died in the middle of the night, whereas the other ones were going to be predictably be killed, and we could be there right away and do our necropsy.

Pre-mortem, the kittens got a fair amount of medications, especially the ones in foster care. So not only dewormers and vaccinations, but oral antibiotics, even probiotics, antiemetics, appetite stimulants and things, whereas the other group was pretty unadulterated, because there was not much invested in them, and they were not cared for for very long. The clinical histories are interesting, because we did not select based on any criteria, other than the kitten died. Here we find that in the sick kittens, 50% of them died with predominantly clinical signs of gastrointestinal disease, which is what we think, that most of these kittens do die with GI disease. The second most popular cause of death, upper respiratory, ocular, viral, another big, big, area we need to put time and energy into. Then a mishmash of wounded, disabled, failure to thrive, and so forth.

What we first did, was we did a routine autopsy, and we looked at all the sections, just with a light microscope, nothing fancy, of the GI tract, the stomach, the duodenum, the ileum, the colon, in the healthy and the sick kittens. I can summarize these pretty easily for you, it is what we already knew from the very few studies that have been done in this population, and that is that you can find minimal clues as to why a kitten died, by just doing a vanilla flavored light microscopic examination of the GI tract. When we hunkered down though, we found some very interesting differences between these two populations, and I want to summarize them for you.

First let's talk about what we found in the healthy kittens, and then we will talk about what we found in the sick and dying kittens. In the healthy kittens, and in all the kittens in general, we found six different types of enterococci in these kittens, by culturing their contents of their small intestine. Interestingly, we were not expecting this, in healthy kittens, the most popular, so to speak, enterococcus found in the intestinal tract, was Enterococcus hirae. That is the one that we found attaching to the villi. So Enterococcus hirae is the most popular enterococcus for a healthy kitten to have inside the small intestine.

Moreover, when we looked up close and personal with our special stains, and Gram-stains, and fluorescence in situ hybridization, in these healthy kittens, guess what? The healthy kittens were the ones that had the most, and richest, and largest surface area of these enterococci sticking to their gut. I got the grant from Winn telling them this is going to be a pathogen, now I am thinking this is the savior. What is going on? These kittens are full of this stuff, and it seems to be associated with health. If you make them glow, you get really pretty pictures too, all over that villus, attaching to the microvilli. What we did not find in any of the healthy kittens was EPEC. We did not find a single healthy kitten that had that E. coli sticking to the gut.

So what about the sick kittens? In the sick and dying kittens it was interesting, because when we cultured our enterococci that are found in that small intestine, all of the sudden we got less E. hirae, and more E. faecalis. So one term for this would be like a dysbiosis, a displacement, or an asymmetry, or an abnormality in these populations. If you look at it in terms of percent of isolates, in these sick kittens, our friend we think, E. hirae, is being displaced by other types of enterococci. When we looked up close and personal at the GI tract of these poor kittens that had died, an example right here of a typical perineal soiling, skinny, cachectic kitten that died, we see that we find Gram-negative glowing in the dark, enteropathogenic E. coli, in a significant number of these kittens. Interestingly, some of the sick kittens did have E. hirae stuck to their intestines, but none of the kittens with EPEC had any E. hirae around.

So how do we interpret this?

Well let's summarize, 60% of terminally ill kittens had either clinical or histopathological evidence of significant gastrointestinal disease at the time that they died, and the majority of it we could attribute to the small intestine. We also found that the healthy mucosa enterococcal flora in this population of kittens is E. hirae. We also characterized those E. hirae, and we found that they are pretty avirulent, they do not have virulent factors, they are susceptible to antibiotics, they don't form biofilm, they are relatively friendly on the scale of enterococcus.

In sick kittens though, we saw a dysbiosis of those enterococci, and a displacement of the Enterococcus hirae with more of this E. faecalis, and when we cultured that E. faecalis, that sucker was nasty. So the E. faecalis in these kittens came from multiple origins, they were very diverse, they were very anti-microbial resistant, they loved to form biofilm, and they made a lot of virulence factors. We are not right now looking at this E. faecalis as a pathogen, but I think we should, because we have got too many other things we are chasing, but that guy right there he might be bad, and what the heck is he doing in these kittens, and could he be hurting these kittens, this E. faecalis.

So what it looks like is that enteropathogenic E. coli, definitely in this study, is significantly associated with death in kittens when seen to attach to the gut, right. Whereas Enterococcus hirae seemed more extensive in the healthy kittens, and it was lacking in kittens that had EPEC. So the idea was, could the presence of this guy be friendly in a way that discourages EPEC from being able to attach. They seem to be mutually exclusive, so is this EPEC the bad guy, but if you have this guy, you are less likely to get EPEC because there is nowhere to land on the epithelium. So this is our E. hirae, a good guy. Moreover, could this E. hirae be a very nice probiotic in this group of kittens? What you will see is that E. faecium, which is what FortiFlora is, was not something that we found as normal inhabitant of the small intestine of kittens, in the manner in which we looked at it. What we did find is this. There is no commercially available probiotic that does something like that, and that might be that beneficial. It is obviously still very young in development, but we are very interested in looking at this E. hirae as potentially a really important thing that you could give young kittens that are at risk for developing diarrhea and EPEC infection.

So the next step really was, we have shown that in the dead kitten that has attaching EPEC in the gut, that that is a bad thing, right. That is associated with the death, but what we don't know is, how are we going to diagnose this? All we have right now is the attaching EPEC in a dead kitten. That is not going to help me diagnose that disease in a live kittens unless I kill it, and I look for it. Well that helped a lot, didn't it? I need a way to diagnose EPEC in a kitten before it is dead, so that I can try to do something about it.

So what we did then, was started looking at the feces, and using those really laborious old-school methods to culture E. coli, select colonies, figure out if they had virulence factors, and see if we could find EPEC in these live kittens that had diarrhea, and didn't have diarrhea, and kittens that died with diarrhea or didn't die with diarrhea. What we did, was we looked at the presence of not only our EPEC friend here, but all the other classic diarrhea causing E. coli. I mean you are there, you are isolating E. coli, you might as well look for the other kinds too.

So based on a PCR for all these different virulence genes, you can figure out if your E. coli is none of these things, which would be a good E. coli, or it is an enterotoxigenic E. coli, and enterohemorrhagic E. coli, a necrotoxigenic E. coli, or eeblablabla… So we did that, and so far we have got 14 kittens that were healthy at the time they were dead, and 26 that died with clinical signs of diarrhea, and so we are still gathering kittens, but we found pathogenic E. coli in 50% of these kittens, and they fell under the following categories. We found an ETEC and we found mostly EPEC. What we also found a lot of was necrotoxigenic E. coli, which is not the subject of our grant, but should be the subject of somebody's grant, because who knows what those guys are doing in kittens. More of the kittens had NTEC that even had EPEC. NTEC does not adhere though, so it was not the focus of our study.

So here are we are showing that kittens shed live diarrhea causing E. coli, EPEC being an important one. We also know that these EPEC are pretty diverse in these kittens. They have multiple serotypes, so these O and H designations, and when you look at their genetic composition, and you splice up their DNA, and you compare their pieces of DNA to each other they are very different to one another. That means that kittens are getting EPEC from multiple sources. They are not all sharing the same EPEC that they got at the SPCA, they are getting it from the environment, they are getting it from other kittens and so forth and we have even found that some of these sero groups, have been associated with diarrheal disease in kids in developing countries.

So the next thing we were looking at, and we are concurrently looking at, was, God can we make a diagnosis of EPEC any better than the old diagnostic of EPEC that no one runs anyway? Nobody, like I told you at the beginning, no one is going to culture feces, no one is going to want to pluck colonies, nobody is going to want to do PCR on the colonies. That approach is never going to work to help us save kittens lives in a foster care situation.

So what we simply asked was, I wonder if you could just take the feces, extract DNA out of it, and just look for EAE, because if there is EAE in the feces, then you know that E. coli is in the feces, and you know that there is also E. coli in the feces that makes EAE. EAE was that thing that made them stick to the cells. So can I jump, can I cheat and jump all the way to the virulence gene, and I was talking to Tori, and I am like, it is not going to work, but let's just do it. So we did it, jumped right to PCR. Oh my God, positive, positive, positive, positive, positive, positive, beautiful PCR, one product EAE, cleanest thing I have ever seen, right on the poop. When we did this, we found that this direct PCR detected EPEC in more kittens than the fecal culture did. So the fecal culture detected 8/40 kittens had EPEC, and when we used PCR 21/48, the number is a little different, because not the same number of kittens went into each assay, 44%, so twice as many kittens had EPEC than we found on culture. Moreover, if we compared the number of kittens that had this EAE amplified from their feces, based on whether they were healthy or had diarrhea, the presence of this EAE gene was significantly more common in the kittens that were dying with diarrhea, 58%, than the kittens that weren’t, only 18%.

So where have we gone? We have shown that both healthy and sick kittens, so some healthy kittens, shed live EPEC in their feces. Kittens do shed diarrhea causing E. coli in their feces, and EPEC is a really important one. EPEC is more common in kittens dying with diarrhea. Some of them may serve as a reservoir for infection in people, or maybe they came from people. EPEC is potentially very easy to diagnose by PCR, however some healthy kittens, and kittens with diarrhea due to other causes may test positive to EPEC, that is always a problem with these PCR tests, and with of diarrhea there is always going to be healthy animals that can test positive, and there is always going to be sick animals that test positive, but it is because they are healthy otherwise, and there is some other cause for the diarrhea. I know that is complicated, but it is not a litmus test for the diagnosis.

What we think the litmus test for this cat has EPEC, and the EPEC is causing diarrhea, might be that the EPEC in that kitten are actually attaching to the intestinal epithelium. So while healthy kittens can shed it in their feces, the sick kittens we hypothesize are the ones where not only their shedding it in the feces, but it is attaching to their intestinal epithelium, and causing the diarrhea and the disease. We also hypothesize that this dysbiosis of the intestinal enterococci, is what predisposes that gut to being vulnerable for those EPEC to adhere.

So where do we want to go with this? Obviously lots and lots of questions. Can EPEC that are attaching and causing diarrhea be distinguished from those who aren't? Can we take a cat and get EPEC from its feces, and ask something and test something about that EPEC, and determine that that EPEC is likely to be adhering inside that kitten and it is important, versus the one we might find in a healthy kitten? So maybe it is virulence gene expression, or serotype, or this DNA laddering, or presence of the ability to swim, or maybe it is some biomarker of the concurrent evidence of enterococcal dysbiosis, something that gives us another level of evidence for the importance of it in a particular kitten.

Is EPEC a direct cause of diarrhea, or is it a co-conspirator. Is there something else going on in these kittens that if we can fix that, they wouldn't get disease from EPEC. So is it a current parasite or a virus, or another bacteria that is affecting the pathogenicity, or the role of the microbiome, the diet, stress, failure of the maternal derived immunity. So is there something else we need to look at? These things we are also looking at right now in these same kittens. Finally, can a probiotic, especially this E. hirae, prevent or ameliorate diarrhea caused by EPEC in kittens? Can we get kittens that probiotic, fix that dysbiosis, and prevent them from becoming colonized and sick by EPEC.

I greatly appreciate all of your attention. I am proud to be here, and I would not be able to do any of this without the Winn Feline Foundation. I also would say I wouldn't be able to do anything about this without all of the people on this slide. It is been an enormous effort, between a lot of labs, a lot of people with a lot of expertise that goes way beyond my own, and also in other universities like Kansas State University. So I appreciate your time.

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Beginning of Dr. Craig Webb Audio

Steve Dale:
Before I introduce Dr. Webb, how many of you are cat breeders that belong to a breed club that have been previously, well not so previously. I will just leave it there. How many of you are cat breeders that belong to a breed club? A lot of you in the room. So I have got something, a scoop for you. I know we were talking about number two earlier and litter boxes. I don't mean that kind of scoop, I have a real scoop for you, and our president is going to talk more about this to all of you tomorrow. We want to make it easier for you, and we want to celebrate you as breeders. So the Winn Feline Foundation, at next year's symposium...oh my gosh [COUGH] sorry about that, I guess I ought to start smoking... So for breeders, what we are going to do is, next year at this symposium, you can just come in. We will welcome you if your breed club contributes throughout the year, to the Winn Feline Foundation. We will also recognize you in special ways, and all of that will be talked about tomorrow, but personally, and I know my colleagues on the board feel this way, we are fans of what you do, and we want to support in ways that at least we can support, what you do.

I am just such a fan personally of pedigreed cats. They are absolutely wonderful, and I try to in what I do for living, spread the word about pedigree cats. In fact I just wrote a story for my National newspaper column, about the most popular cat breeds, and received e-mails back about, what is an exotic, what is that? So I love talking about cat breeds to people. We love trying to help you out, and to support you in what you do, but we want to help educate you as well. We happen to have the best, and I am about to introduce one of the best.

Stem cells in human medicine, and I have talked on the web about this before, it's controversy in ways it is not in veterinary medicine, which we will talk about. Our scientific advisory board, when Dr. Webb's studies and other studies regarding stem cells came along, our veterinarians just looked at the science of it. There is no emotion behind it, there is no controversy behind it. Sometimes physicians, I hope there aren't any in the room, confuse me, because there really isn’t. It should be about what good medicine is about, and that should be about all. Review of the current therapies of dietary intervention, vitamin B12 supplementation, and probiotics, how fat derived stem cells work in treating chronic intestinal disease, and that word that we heard a lot about today, diarrhea, a pilot research study in the use of stem cells as a therapeutic approach to chronic inflammatory bowel disease, here is Dr. Craig Webb, from Colorado State University.

Dr. Craig Webb:
So let me start by thanking Vicki, and thanking Steve, and thanking the Winn Feline Foundation for inviting me here to gorgeous Toronto Canada. I was in Toronto once before, probably 20 or 25 years ago. I usually get invited to places like Nebraska. Nobody from Nebraska I hope, or Ohio, so to come to a place like this is actually really wonderful. You from Ohio? I love it, my wife, half of her family... She said she can't hear you. Oh you can't hear me. Oh, I thought you were about to start throwing things at me because I said something bad about Ohio. Does that help? You are from Ohio. Half of my wife's family is from Ohio, which is the only reason I am not really that anxious to go to Ohio. Nebraska, other reasons. So anyways, just thrilled, and the weather has cooperated, a beautiful place. So thanks so much just for having me here, regardless of what I need to do as far as thanking the Winn Feline Foundation for supporting research in cats. There just are very few outlets for people such as Dr. Gookin and myself, and any of us who want to try to research cats, and research things that will help cats, we have so few options, as far as places to go to try to get funded, that the Winn Feline Foundation is really a unique and very critical part of the future of the health and happiness of these little guys.

So, my talk is on the use of stem cell therapy in feline chronic enteropathy. I will start with a disclaimer slide. None of the kitty cats you will see in this talk were my own, or members of my family, nor would anybody pay me for what you are about to see, but again the one disclaimer, and I refuse to disclaim, you are supposed to disclaim your funding sources, I refuse to disclaim the Winn Feline Foundation. I embrace them, just as Dr. Gookin does. Thank goodness for us and for our cats that they are here to help us out. So they have funded everything I will talk about today.

The last time they actually had me talk, not that long ago, about this, I had with me my wife Dr. Tracy Webb, and that's important for this audience, and important to me for many reasons, but the most important being that she is the smart half of our combination. She is the brains of the organization and the brains of the operation. She knows what the heck she is talking about, and what she is doing. I am just trying to help cats with poop and diarrhea. That is about the level, I am honestly a clinician, but I work at an academic institution, so I have to convince them that I am doing some kind of research, and so thank God I have my wife to actually do that stuff for me. So that is important to you, and those little white cards that you are going to write down your questions on if you have them, please feel free to do that. You are welcome to do that, as long as I am welcome to take them home with me and have somebody smarter than myself help me with the answers. You can e-mail me, and I will provide you with that answer.

So I would like to start with what an ideal world would be for us, as both people, and then in particular as gastroenterologists and veterinarians worried about diarrhea in kitty cats. For me then, that starts or is exemplified by this particular case. This is how GI cases in cats should go. We have got presented to us, as 6-year-old female spayed domestic longhair, that may actually be a particular breed. I graduated from of a vet school that did not make us memorize breeds, and I am sure everybody in the audience is much sharper than I as far as picking out breeds in cats. So if this is something more specific than a domestic longhair, please write that down on a card and let me know, so I can be more accurate.

The problem that this owner brought the kitty in for was chronic intermittent vomiting. This was a great owner, as many cat owners are. Some of them are crazy, some of them are great, some are crazy and great. This owner gave a wonderful description. He said yeah, this has been going on for about a year, so right away you get some feel for the immediacy of the problem, not so bad, it has been going on for about a year. He said yeah, oh about once a week, maybe every other week, my cat, and it always happens about 2 in the morning, my cat starts making these noises. She wakes me up, kind of upsets me, and starts making these noises, and they actually do the noise for you, it sounds like this, augh, augh, augh, and I swear to God the cat is bringing its entire gastrointestinal tract up and depositing it on my carpet.

So after I get woken up by this, I fall back to sleep. Much to my surprise, the following morning my cat is still alive, and sure enough I have to carefully try to find where its gastrointestinal tract is, on what part of my carpet before I step on it. It doesn't seem to have the affected my cat otherwise all that much, the cat still wants breakfast. That is the history you get, and again this is an outstanding owner. Not only do they give you a great history, they bring with them a sample of what happened. This is what they brought off the carpet, so they have given you the history, they have given you the problem, maybe even given you the diagnosis. This is a cat vomiting up hairballs. Ideal. I haven't had to do a thing yet. I have just sat in the room. So I have the cat, I have the problem, I have the diagnosis, and because of that, because this is an ideal case, I have the treatment.

Sure, now I run into occasionally a few owners who are not all that compliant, and I am looking for an alternative treatment, well I even have some of those. Maybe it's diet, maybe it is a little oil, maybe you have got a grease up the intestinal tract a bit in this cat, and this is, this is a big deal, maybe not so much for the health of the cat, were not sure about that, but this is a common feline problem, right. Since the beginning of time, since the beginning of ownership of cats, we have been aware of hairball vomiting.

In fact, and I didn't notice till rather recently, there is a National Hairball Awareness Day. It happens, and my father was a physicist, it happens in the fall also on the National Physics Day, so throw the two of them together and that's exactly what my father would do at breakfast, if you were ever silly enough to ask him, well what happens when a cat vomits up hairball. He would show me, because he is a physicist. So that is how big a deal this is in our cat population, and yet we do not know anything about it, I mean next to nothing.

Quite a recent article by a wonderful vet from across the pond, and basically Dr. Cannon is telling us, is in these studies, or in any studies, the few that you could find, we still have, although there are a range of possible hairball diets or treatments, there is no objective information in the public domain regarding the efficacy, in fact studies to evaluate the success of other recommended, they just don't exist, highlighting that even in an ideal GI case, with a case or a problem that is so prevalent and so common in the feline population, we know almost nothing about it, and when I say no, I mean we have so little evidence to support what we are doing to try to help these cats. This is both highlighting where my own study is going, but again highlighting what I said at the very beginning, about the importance of people like the Winn Feline Foundation, because they are the people that are trying to solve this problem.

So how do we make decisions if we do not have the evidence? We do not have the studies, how do we decide how to treat these guys. Well unfortunately in veterinary medicine the most frequent strategy for making that kind of decision is anecdotal, anecdotal evidence. I tried it in the last cat and it worked so I am going to try it in the next cat or my buddy down the street talked to me about it and said, they tried this and it worked so I am going to try it. Of this I want to give you an example just to bring home again the strengths and or weaknesses of anecdotal evidence.

This is my favorite example. Vitamin E in inflammatory bowel disease, we have some vague appreciation for vitamin E as an antioxidant and so a few laboratory animals running around with a little less diarrhea because they were given some vitamin E. There is a dose we pulled out of our own diarrhea and came up with, it does not seem to have any side effects that are untoward so that is at least one good thing but there are actually no clinical studies in dogs and cats.

What I do have, though, is solid anecdotal evidence. This is a picture, there in the middle, that is Dr. David Twedt, one of the grandfathers of gastroenterology in veterinary medicine and just one of the guys. That is me on his left, and Dr. Ugo Lotti on his right. Dr. Twedt is so famous that frequently we have people coming from all over the world who want to spend time with him, just kind of trying to suck stuff out of him, just by being next to him you get a little smarter. Dr. Ugo Lotti came from Italy to spend a summer doing some research with Dr. Twedt, a great project. This happened to be the summer before Dr. Twedt starting taking vitamin E. Dr. Ugo Lotti spends the summer, he leaves. Dr. Twedt, realizing he is getting a little on in years, slowing up just a bit, says ‘Well, I had better do something to reverse those effects, I am going to start taking vitamin E,’ which he does.

The next summer we have another Italian come to spend the summer with us doing research with Dr. Twedt. Now, I want you to pay close attention. Although Dr. Twedt’s expression has not changed much at all, in many ways, the quality of the visiting Italian veterinarian has changed significantly. Anecdotal evidence that many of us, especially as we are getting a bit older, should consider taking vitamin E. Right, I mean what else would have made the difference? Perhaps not the most satisfying, I am certainly taking vitamin E now by the way, not the most satisfying way to get evidence. Let us take a look at another case and see if we can do just a bit better.

This is an 8-year-old female spayed domestic shorthair with chronic diarrhea. This is the cat we see all the time. Why is it? Because we will do all of those tests, Dr. Gookin mentioned a number of them, including, with the fecal exam, we will do our minimum database, our CBC, biochem profile, urinalysis, and you cannot get to the receptionist at CSU without having that done. Total T4, checking for some of those bad viral diseases, we do the Texas A&M GI panel which I will touch on briefly in a bit. We do all of this and that is a considerable investment, and boom, nothing, negative, nada, within normal limits, nothing, and yet I still have an adult cat who is having diarrhea. What do we do next? Well, let us spend some more money. It is a cost centered thing. Medicine will throw him to ultrasound because they are pretty confident ultrasound will tell medicine to go ahead and take them to endoscopy. We do just that, we get all the way into the cat with the scope, and grab some pieces of actual tissue, those things that Dr. Gookin would have loved to be able to do before the cats are dead, we can do them in adult cats, we can get scopes in them, grab some pieces while they are still alive. We do that, and in the majority of these kinds of cases, you already know the answer, you are going to be told if you are an owner or you are going to be doing the telling if you are a veterinarian, this cat has inflammatory bowel disease. I could do it at half-price, just give me the cat, I will give it back to you and give you the histopath even before you go through all of this stuff, it will be inflammatory bowel disease.

Critical part of this diagnosis is that we almost always forget the real name, which is idiopathic inflammatory bowel disease. Why is that important? Well, idiopathic in the Greek’s world comes from idios (one’s own) pathos (suffering), it is an adjective (whatever that is, my high school kid could tell me) arising spontaneously from obscure or unknown cause. Now stated that way, it sounds almost comforting to be able to walk back into the exam room and say, ‘well Ms. Smith, your cat has idiopathic inflammatory bowel disease,’ sounds Greek, right? Well, in the modern day, Dr. House reminds us that in fact it just means we do not know what the hell we are talking about, we do not really know what is going on, so we throw idiopathic on there. All of that effort and all that money, and we have gotten to the very same place we were when the client and the cat first walked in the door.

How might we treat, now that we have spent all of this money to put that diagnosis on the cat, how might we treat it? Well, it is not surprising that since we have very little idea of what is really going on, that we would have little idea or little evidence as far as what to effectively treat that cat with. We have a half-dozen things we will try, and half-dozen things we will put into that kitty cat, or try to put into that kitty cat, or hell, we do not do it at all, do we, we sign the script and then we say, ‘good luck’. So it is not surprising that either because the medicines are not the right medicine for the right disease or because the owner simply cannot afford the prescription diets and they cannot continue to try to slam two or three medications down their kitty’s mouth two or three times a day, that treatment fails. Again, even for one of the most, besides hairballs, for one of the most common GI diseases our kitty cats suffer from, idiopathic inflammatory bowel disease, we have little idea of what we really should be treating with and often not enough success in our treatment efforts. That motivates a number of us, whether we are clinicians or researchers, to try to take the next step, to move off anecdotal and try to find some actual evidence for what might work in these guys, and what could we reliably and confidently prescribe to owners of these kinds of cats.

How can we move from anecdotal to evidence? Well one of my favorite ways is to look at the human model of our patient’s disease. The whole world tries to look at our patients as models of their disease; I prefer to do it in reverse.

What human models are out there for the diseases that my patients are suffering from? Well, if you look at inflammatory bowel disease in humans, it is a much better and much more specifically and clearly defined syndrome than, ‘Well, it is an adult cat who has some diarrhea and I cannot figure it out.’ They have categorized these into several very specific forms, one of which is Crohn’s disease, and in many ways, or at least in some ways, Crohn’s disease looks reasonably similar to what our adult cats might be suffering from. Let us take a look, how does our human model deal with their version of inflammatory bowel disease, Crohn’s disease.

That is when I ran across this, and as soon as you run across one, many others, at least in the human field of research, study on stem cell therapy in inflammatory bowel disease. I highlighted the key line which I will read for you: “Mesenchymal stem cells have immune regulatory and regenerative properties and low immunogenicity. Based on these properties, these mesenchymal stem cells have been used in the systemic route in inflammatory bowel disease with promising results, even though it is still too soon to draw firm conclusions.” I thought, well, now that sounds pretty intriguing and there are a variety of important properties that we will touch on very briefly that might make it a pretty dang ideal treatment for these kitty cats.

Now, first to touch on very briefly, these stem cells are a particular kind of stem cell. They are not the embryonic stem cell that the various political administrations have made political livelihoods off of, they have nothing to do with embryos and all of that emotional and religious and those kinds of concerns. These are not them at all. So we get, whew, shove those right off the table. They also are not the stem cells that we might have learned about in high school biology that are actually in the gastrointestinal tract and in many other tissues. Resident stem cells are fascinating and absolute critical for the function of the GI tract. As Dr. Gookin pointed out, when those villi are effaced, the GI tract does not work at all, it is awful. It turns out the GI tract effaces itself even without the help of bugs, oh every two days you basically poop out almost all of your mucosa and you have to replace that or you are going to die, like those kitties, and who does that, it is the stem cells that are residents of those villi, the crypts, and regenerate themselves. So wonderful cells full of all sorts of critical properties, these are not those cells either. These are mesenchymal stem cells that in fact we can pull out of a piece of fat. Unlike the controversies that might happen when you get stem cells from embryos, who in this room would have any problem donating a little piece of their own fat, right? We would be happy to get rid of some of that. So boom, all of those issues, again, go by the wayside.

Mesenchymal itself means that it has something to do with connective tissue and that is where these cells enter the veterinary profession. You can see from this, mesenchymal stem cells here are shown (the dang toolbar still is not working; that is okay with me, as long as it goes away about now…good) so these mesenchymal stem cells are shown as being able to help or even regenerate connective tissues in bones and muscles, skin, tendons, ligaments, and so these stem cells first enter the veterinary field in a horse’s joint. That is still a huge area of research and effort is the use of mesenchymal stem cells in joint diseases in athletes, athletic horses, now making its way in to dogs, trickling a little just into kitties, but joint disease, whether it be tendon ruptures, tendon repairs, or osteoarthritis, more chronic stuff, that is really where they hit the veterinary profession first. But you will notice the gut is not on here. So, I mean, Dr. Gookin goes off on some potentially crazy tangents that Winn funded for, but this is nuts, right?

This makes no sense at all, until you look a little closer at more of their what you call biochemical properties. All of a sudden, all these black lines on the mesenchymal stem cells having some sort of impact or effect on almost every process in our immune system. If there is one thing we know about inflammatory bowel disease in adult kitties, we know that the immune system is messed up. It is probably reacting to the wrong thing, or it is overreacting, it is overstimulated, it has just become a very bad immune environment. Here we have a single little cell that, again, I will not bother you with these at all, the acronyms and names for cytokines and interleukins and effecting all these B cells and T cells and it goes on, the language of it is just overwhelming, but take my word for it, this is the immune system almost in total and the mesenchymal stem cell is affecting or can have an effect on each and every part of that system. So boy, if there ever was at least potentially a silver bullet that you might use if you have an immune system problem, the mesenchymal stem cell is one that it looks like it is raising its hand.

Then they found just that when they have used it looking at human disease, Crohn’s disease, and what mesenchymal stem cells (stromal is just another word for mesenchymal stem cells) can do and again, it is just a laundry list of impacting the immune system of that patient. Importantly, and I would have to come sit back where you are, they are, I think it is, yes, the very first, they are immunologically inert, so not only do you have a silver bullet but you have a silver bullet that you can shoot into an animal and it will not do any harm, it will not be recognized or thought of as foreign and ‘I have got to get rid of you’, so a critical component to this potential treatment. So, all of that inspired us or motivated us to think about using this in cats with chronic enteropathy. At CSU we are already doing some research with this with cats with kidney disease, and in Missouri, Dr. Marino is doing work on this in asthmatic cats, so there was certainly some background to the use of this in various feline diseases. We wanted to look at it in cats with GI disease, and we called it a proof of concept study, meaning, it means a couple things, but meaning for us that before we got crazy, we wanted to start and see is there any reason that we might go forward with this. Does giving this to some cats give us any hope that it might make a difference and motivate us to again put in, again, research dollars are so hard to come by, we wanted to get some idea in a small number of cats that at the very least this was safe, and then potentially effective. So that is kind of what we call a proof of concept study.

Now, again, I started out by reminding you, I am a clinician. I am interested in cat poop, and the owners of cats with diarrhea, and I want to make both of those people feel better. I have some science behind me just because I married well. So, unlike the Dr. Gookin studies which are done correctly, do not tell everybody who is from Winn, put their fingers in their ears now, I rarely do this nearly as correctly as I should. So what I did, I said, well, instead of making all sorts of entry criteria, and maybe getting one cat every six months, I want to open my clinic door to the cats I actually see. I say, if your cat has chronic diarrhea, come be in my study. So I added a little bit about, well, we have got to make sure that it does not have a significant other disease that is still causing problems, so if the cat is gasping because it has cardiac disease, maybe not. Or, if it has got asthma that is bad, or if it’s total T4 and thyroid hormone level is 12 or something, maybe not. But how many of you have ever seen or know of an older adult cat who only has one problem, please raise your hand…yeah, one, and she even took her hand back. These are not the kinds of cats we see in the clinic, so it made no sense to me. So we opened the door. The one thing we did that helped in my mind tremendously is we also had a placebo group, and these owners were blinded to what their cats were getting. Too pretty for us, pretty critical because again, any owner that you say, ‘Yeah, I am going to give your cat something that is going to make him feel better; I need you to come back in two weeks and tell me it made him feel better’, that does not work. Hence the blinded owner and the placebo group being critical parts of this.

Here is our study design. We have the cat come in and again, we did as much as was feasible or financially feasible to make sure the kitty did not have other bad diseases, it was not in bad chronic renal failure, so we did the bloodwork, we did the Texas A&M GI panel I mentioned earlier, mostly for cobalamin or vitamin B12 which is, at least in some studies, a great indicator of the severity of your GI disease, and we had owners because again, I am a clinician, and what the owner thinks is a pretty critical part of what is going on with their cat, so we had questionnaires that then would help us put various scores on their poop, everybody has seen the chart, number your poop. That day, if they met the grade, they got the first injection of adipose-derived mesenchymal stem cells, so stem cells that have been pulled out of a piece of another cat’s fat. We culture and do various things behind the scenes, and we have a syringe ready to give the cat a certain number of these stem cells. Then two weeks later the cat gets injection number two, because we wanted to see if these cats could do well with multiple treatments.

Now remember, the placebo cats would get injections with just basically sterile saline, and the owners are not told which their cats receive. Then two weeks after that, we have them return, we repeat some of the bloodwork, we have them fill out that questionnaire again. These times are coming from again, just wisp and whims from human studies, so little is known, that most of this we are making up, how many should we give, how often, those are all open questions, but we took our best guess. Then, because again we had no idea really what time frame we should be looking at, we kept the owners, and this is critical, we kept the owners blinded, although we were not treating them anymore, we kept them blinded for at least a month, sometimes 2 or 3 months, for follow-up later, and that is indicated here for those of you who are younger than I am, this is what a phone used to look like, and this is just meant to represent usually it was actually email contact, but some kind of contact with owners still blinded to what their cat had received. So that very simply is the study design. Let us meet some of the stars.

Very first cat to enter our study was Kiff, owned by Knut, and I am not making that up. You have no idea how many times I walked into that lobby going, damn, is the cat Kiff, or is the cat Knut, or is the owner Knut or Kiff. Fortunately super guy, he did not care what I called him or his cat, and I am sure I frequently got it wrong. Seven-year-old male castrated orange tabby, chronic vomiting for years, it had gotten progressively worse, that is what attracted this guy to our study. He had tried so much stuff. He had given up, the cat was on nothing and clearly this kind of last straw for this poor guy. He was on a natural balance diet but no meds for some period of time. He had been losing weight, his appetite was off, this is sick cat not feeling good. Stool was soggy and here is that little scale, so the owner considered the stool soggy. Turned out this guy did have a concurrent problem. He had a heart murmur that got worked up. He had obstructive cardiomyopathy, but everything seemed to be well-controlled on Atenolol, so doing fine, although we noted the murmur. He had previously had ultrasound and histopath, and sure enough, got his kitty diagnosed with the classic diagnosis, moderate lymphocytic plasmacytic enteritis, otherwise known as inflammatory bowel disease. It was a great first study cat for us, just what we wanted.

It turns out, thank goodness, the research gods were smiling on us, this cat actually got stem cells. So, we start with a CBC/chem; that was all unremarkable, kind of the baseline stuff. He was not hyperthyroid. His cobalamin or vitamin B12, this is a very low number, 166. This guy had serious gastrointestinal disease. He was vomiting up to three times a day, his stool was soggy, had diarrhea at least once a day, pretty miserable. Got his first injection, at two weeks got his second injection, and then two weeks after that, and having had nothing but stem cell therapy, those two injections, his cobalamine had gone from 166 which is kind of in the toilet to 528 which is great, normal and plenty. From vomiting much more frequently, he is now only vomiting about once a day, and when asked on the questionnaire ‘is your cat having diarrhea’, the answer was ‘no’, and the number put to that answer was 3.5, so 3 being normal, and the cat’s appetite has increased. So Knut was going to take us to Stockholm. This is Noble prize, we are feeling so good about ourselves at this point, we are figuring, n of 1, that is all we need, let us end the study now.

So, just when we were about to follow up with Knut about a month later, we learned that the little guy had had an acute onset of dyspnea unresponsive to medical therapy and was euthanized. I mean, this close to our final follow-up, and we thought, we just went from Stockholm to I am looking for another job, because of course although we had all the reason to believe this was a safe therapy and would never do anything else to this cat, this raised the specter of oh, what if something has gone terribly wrong. Again, I told you this guy was a fantastic owner. He was. He allowed us to necropsy, which Dr. Gookin highlighted the importance of that. Our pathologist, the one working this cat, they knew of the previous histopath, they knew nothing of this cat’s entry into any study. They found, thank goodness, I mean it is still awful for little Knut or Kiff, he died of basically heart failure, and clearly classic heart failure. But then what was particularly fascinating for us, the histological evidence, remember previously it had been moderate lymphocytic plasmacytic enteritis, IBD, was not identified in sections of the small intestine, perhaps due to treatment or resolution. Who has ever heard of resolution of inflammatory bowel disease? So again, we are booking our flight.

A couple other examples. This is Sesame and Pilar, because of course who owns only one cat, it is almost unheard of. Wonderful owner brought both of them in. They were I think just siblings in family only as opposed to birth. Both were, they were phenomenally similar to each other, both were 13 year olds, chronic diarrhea with intermittent vomiting. This owner was treating them with prednisolone every other day, FortiFlora that probiotic Dr. Gookin mentioned, but the stools were still moist and soggy and having diarrhea four to five times a week. These guys had no histopathic diagnosis, but I tell you right now it was IBD, it just did not cost as much. Here is classic treatment for these problems in these two cats, and it clearly is not working, so she was motivated to bring them to our study.

To make a long story short, neither of them were hyperthyroid, their cobalamin was ok, but they were, again, having lousy diarrhea, they were vomiting some. They get our first injection, they get our second injection two weeks later, two weeks after that, eh, it is not looking great, did not seem to have much impact. We said okay, well, she remained blind, and the owner remained blinded. We will get a hold of you in about a month, two months from now, who knows, we will just see, but again the enthusiasm is a little bit dampened at this point. Well it turns out, just about a month later, Pilar came back in for what the owner presumed to be a urinary tract infection, inappropriate urination outside of the box, so we wanted to do a cystocentesis and grab some urine for a sample for culture. Our nurse made the first mistake at this point, she had me restrain the animal. Never have the doctor restrain the animal, they suck at it. I was trying to restrain and this cat was wiggling and squirming, and I said, “Ah, I am not doing a very good job, am I, I am sorry…’ and lo and behold the cat pooped, so who would not be straining if they have to poop. Then we laughed just fine, got the urine no problem. I pick up Pilar, okay can you handle the samples and I am going to take Pilar back to the lobby to mom. This happens with age, I had this delayed response because just as I am going to hand Pilar back to mom in the lobby, it dawns on me, ‘Jeepers, that was the most normal cat stool I have ever seen.’ I ran back into the exam room to make sure I could get a hold of it, and sure, it was beautiful, fully formed. So I asked the owner, so, how has it been going, right? Diarrhea now a full month or might have even been a bit more after we kind of had lost, after we had taken our last samples, diarrhea now is only once or twice a week, otherwise all stools were normal, had only vomited once in the past 2-4 weeks. So way out here at a time point that we had almost written off, boom, this cat was a new cat.

Another example, Nora, how many of you have cats show up your clinic, or I guess I won’t even ask, how many of you do this with your cat? Right, this is how Nora shows up, shows up with chronic diarrhea of six months’ duration. A little bit of again the kidneys are not working as well as they should but that had been stable for quite some time. No specific meds but using again a probiotic and a little slippery elm. Moist, soggy diarrhea once or twice a day. This is one of those owners who is heavily into the diet stuff, various different diets, no histopath done on Nora. Boom, Nora’s cobalamin was just fine, but the stools are soft and yucky, and we are having diarrhea way too frequently. We get our second injection, and then two weeks after that, stools eh, not a whole lot going on there either, but now with our previous experience we are thinking, well let us now give up hope yet, and sure enough, by the time we got out to between one to three months, no diarrhea, normal stools. This is one of those cat owners that you do not have to email, right, you can just sit at your desk and the emails will come, and they came quite frequently. No change after first treatment, but since the second treatment, completely normal stool with no cling-ons, has not happened since January. Nora is doing great, one episode when there was stress in the house, but otherwise firm and formed.

Now, at this point, you could certainly argue, well, all these cats would have gotten better anyways, had nothing to do with the stem cells, they just finally got better, their disease resolved, but…oh and I am sorry, the owner was so happy that she brought me a stool sample just to prove that the emails were not lying. That brings up the importance of the blind placebo control. There were 7 kitty cats that ended up getting stem cells, 4 that ended up getting the placebo which was just saline, went through everything, was the same process, but they got saline. Again, unsolicited, this was 1 of the 4, I just got to say I hope she is in your control group, still battling with all of the problems, trying to work with food, little to no change and that was the recurrent theme. In fact 1 of the 4 cats got quite a bit worse. Adding strength to our claim that since the only thing that was different between the two groups and the only thing that was changed, so even if they came in on treatments already or diets already, they were told do not change anything during this three-month period that we are trying to see what the stem cells or the placebo are doing, so nothing else was changed, adds to the strength of our trying to claim that, well, at least these stem cells are something worthy of study. From this group of about 11 cats, we got the proof or proof enough to continue. We found no adverse reactions at all, either during the giving or with time, because they were getting these stem cells, and so we are ready then to now take the next steps study-wise to advance this possible treatment.

You might think that, well, obviously now there are all sorts of clinical things we can do in our next studies, so we can demand that the cats have the histopath, we can bring uniformity to their treatments, etc, etc, but in fact, what we are doing is, I would not call it a big step backwards, but we are now catching our breath. We have the proof to say, okay, this has promise, it is worthy of further study, but think about the huge number of questions that we could answer even before we get to the cats again, and that is what we are doing now at this period of time. These cats all received stem cells, and another important part of the study, from the same donor cat, a specific pathogen-free donor cat who happened to be getting spayed, and that is also important it was a female, and they took a piece of her fat and through again the magic of my wife, that produces enough stem cells to treat about 7, 8, 9 different cats. So all of those numbers worked out, but it begs the question, well who would be the best donor? Is it a boy, is it a girl, is it a healthy cat, should it be from another cat who is having diarrhea because those cells are jazzed up ready to go…all of those questions, even just to kind of come up with who should be the best donor.

Then there are questions, once you take it out of the donor, this little piece of fat, are there things you should do or could do to that fat to make it even better or more specialized or more prepared for the job you are going to ask it to do when you put it into a cat with diarrhea. Those are just some of the things for instance, it turns out that fat you get from a female cat, stem cells you get from a female cat, are distinctly different in a number of potentially important ways then the stem cells you get from a male cat, and so right away, just from the benchtop research we are now doing, you lean toward, well, I think we had better get it from Fred instead of Ethel. Then pre-treating these with various cytokines or putting them into a little well with a little bit of funny stuff sure enough influences what they are more likely to do or not do to the immune system. Again, there are all sorts of possibilities and questions to be answered now before we even get back in to more cats. And, they are highlighting that actually. It turns out there are some viruses, or this particular virus which otherwise has no consequence on a cat, they do not care, has no clinical consequence if they have got this foamy virus rolling around your kitty cat, but oddly enough, it does seem to affect the properties of the stem cells you would get out of that cat. Right there you are saying, well just like in a blood donor program, there probably needs to be some kind of prescreening process for kitties that are going to be donors of stem cells.

The same individual, Dr. Borjesson from UC Davis, her group has done this with stem cells, how phenomenal. This is a kitty cat with this chronic gingival stomatitis; you just look at that and you cringe. Injection of adipose-derived feline mesenchymal stem cells, and this is what that cat becomes. It is amazing. So if there were any veterinarian in this country who should be yelling for the use of stem cells as a treatment for kitty cats, it should be Dr. Borjesson, right? I mean, she has seen this, she has done this, she has seen stem cells do this, she ought to be standing on the top of the Westin yelling, ‘stem cells, stem cells, stem cells,’ and yet what is she telling us. She is telling us whoa, be careful now, slow up. Veterinarians offer stem cell therapies to satisfied demanding customers because your clients have seen these pictures. So clinicians are sucked into giving treatment even in the absence of research to support such treatment, taking us right back to the very beginning of all of this, where so many things are unknown, so many things about this treatment need further research before we start throwing it out there and that is where people like the Winn Feline Foundation come in to help us out and fill in many, many important gaps because as it is now, you can go on the internet and find stem cells for your cat, no problem, piece of cake. Any client who wants to can get their cat injected with some stem cells. There are 2 or 3, but 2 major stem cells companies, right? Boom, boom. So it is a no-brainer to get; if you got the money, you can get stem cells into your cat. But, is that the right thing to do, is it an effective thing to do, and perhaps most importantly of course, is it a safe thing to do.

We had no side effects in our kitty cats, but what I will tell you is that again, it was my wife who prepared those, and it was my wife who handed me the syringe that I was then going to spend the next 20 minutes injecting into the cat, because you do not want them to clump, and I was standing there at the cat cage doing this with the syringe for 20 minutes. But if my wife, you would have to know my wife which I do quite well, if my wife hands me a syringe of something to inject into a cat, I know I would have no problem injecting that into myself, because I know my wife. What you have to ask yourself, and what we as a profession have to ask ourselves, and what we have to ask our clients, you send a piece of fat off to company X, they send you back a syringe, would you inject that into yourself because you are about to inject it into your cat. So, again, huge number of questions that need to be researched to make this truly, hopefully, potentially a silver bullet and it is the Winn Feline Foundation that undoubtedly will continue to take our field forward in just that direction, so thank god for people like them and thank you for joining them at a symposium like this, and again, going on to support them in the future. So, thank you, thank you Winn, and I will turn it back to Steve. Thanks very much. [applause]

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Beginning of Question & Answer Audio

Steve Dale:
Our board members get to get some exercise in lieu of joining a health club and run around from table to table to grab questions. Dr. Thayer just read my mind, but then she knows that is very light reading. There is a photo contest that we have ongoing right now, so for those of you who have a cat, how many, raise your hands again, I just wanted you to move around so you are awake, okay keep those hands up, how many of those who have a cat, raise your hands if you have a cat, right, dress up your cat for the Fourth of July? I do not see quite as many hands going up, but if you can think of a way in which your cat is patriotic, or you know one of those cat owners that Dr. Webb was talking about, that do things that might be a little different, we do have a photo contest going on. It is WinnFelineFoundation.org. If nothing else, it is entertaining to see some of the submissions of these cats, but you can enter the photo contest as well. We also have a newsletter that goes out with information that is the kind of information that you just saw, information from scientists, so I am going to call Dr. Webb and Dr. Gookin back up here, and I will ask you to share the microphone perhaps, and I will ask these questions; thank you.

The first question goes to you, Dr. Webb. I do not quite understand this, but I will read it as it is. This person wants to know if, in the study that you did, “Were you blinded as well as the cats?”

Dr. Craig Webb:
That is a great question, and yes I was.

Steve Dale:
So I think you referred to that actually, so you did not know.

Dr. Craig Webb:
When I was handed the syringe, I did not know what was in it.

Steve Dale:
“Did the stem cell cats, did they stay well, those that got well?” So that was a while ago, how are they doing now?

Dr. Craig Webb:
That is a great question. Again, being cat owners, I do not have to elicit opinions, they come on in, and so I am aware of at least three of the, so 7 got treated, 3 of the 7 have continued to do very well, but a number of the 7 have developed other problems and other conditions. But the GI signs, although we have not formally followed them up long term, the GI signs from what we have informally gathered have continued to do quite well.

Steve Dale:
When you showed that slide from the human medical community, of stem cells and all of that, there was, the way I read it, in fact there was a red star next to it, I do not recall the exact wording, but essentially it said there may be side effects in people we just do not know enough. Right, can you talk about that?

Dr. Craig Webb:
Right, one of the biggest concerns, because they are stem cells which means they have all the potential to do all sorts of stuff, one of the overriding big concerns is cancer, or neoplastic processes. If you are putting an ignorant cell that just wants to divide into a body, that is basically the definition of a cancerous cell. So, that is a concern where longer term follow-up will be critical, and it is an absolutely critical, unanswered question.

Steve Dale:
I am going to try to pronounce this, and also read the handwriting, this is for Dr. Gookin, “Treating a kitten with diarrhea and suspected sepsis and epithelial translocation, what antibiotic would you recommend?”

Dr. Jody Gookin:
Wow, I should not say it, my veterinary colleagues would kill me, but if I had a septic kitten, I would treat them with the best stuff I had, because you just do not have any time to waste, and you cannot culture and figure out what you should be using, so if a kitten came into my clinic and it was septic I would give probably something like, well, Clavamox, but in IV formulation, and a fluoroquinolone. What do you do, Dr. Webb?

Dr. Craig Webb:
Yes, Unasyn and injectable Baytril. [Audience: We cannot get Unasyn in Canada, I believe, I have not been able to find it…]

Steve Dale:
“Would you ever recommend antibiotics as a preventive or prophylaxis?” Controversial question I suppose, but a good one.

Dr. Jody Gookin:
For bacterial diarrhea, no I would not recommend preventing with antibiotics, because there are so many players involved, there is so much bacteria in the GI tract, the antibiotics are very promiscuous, they are not going to just take out the one thing that you want to kill. They are going to take out the good guys too. So for example if the enterococcus we are looking at is a good guy, and you give it an antibiotic to kill the E. coli, and that antibiotic also kills the enterococcus, you could make it worse. So, no, I would not do that. You will also actually select for that cat to have intestinal bacteria that are resistant to that antibiotic that you gave it.

Steve Dale:
By the way, I caught your Fred and Ethel, yes, yes, it is the reruns, I am not that old to personally remember, but I caught that. We had a fish named Fred, and he did not make it very long. He is buried in our back yard, though, it is true.

“Do you ever think there is a relationship between the progression of HCM and stem cell therapy?”

Dr. Craig Webb:
No, I think the two were unrelated, to be honest.

Steve Dale:
Some cats though before they succumb with HCM, so they are doing okay, they are doing okay, they are doing okay, and for a few weeks before they die, they do have GI issues, and/or other issues as well.

Dr. Craig Webb:
Absolutely, yeah, cardiac diseases are on the rule-out list for vomiting and diarrhea, for sure, so that is not surprising.

Steve Dale:
That is a good question. “How do I find out about studies that my cat could be in? She is a domestic shorthair, she has food allergies, seasonal allergies, diabetes, asthma, ear infections, bowel problems (laughter). Her poop goes from black to…(well, I won’t even, I will stop there)...vomiting 3, 4 times a week”

Dr. Craig Webb:
So, yeah, it may require a little bit of searching, but places I would start would be whatever veterinary teaching hospital is closest to you, and I would just ask them, or dive onto their websites. Winn Foundation publishes the studies that they are funding, not just the ones they have funded, but the ones that are receive current funding that will list the researchers as well, so that is a potential route. It is somewhat difficult. I get emails all the time from people living all over the country who want to be a part of that study, that unfortunately often cannot happen just because being in the house is an important part of making the study work, so it is certainly worth you searching for it, but do not be too disappointed if your cat just cannot be helped because of the logistics of it.

Steve Dale:
“Can you provide more detail about what the Texas A&M GI panel does and provides?”

Dr. Craig Webb:
The Texas A&M GI panel has a TLI, trypsin-like immunoreactivity, that is looking for EPI, which is exocrine pancreatic insufficiency. It has PLI, which is pancreatic lipase immunoreactivity, which helps you look for pancreatitis. Then it has cobalamin and folate, two important basically vitamins that give you a measure of what might be going on, either the GI tract is not working well or as Dr. Gookin mentioned, a measure of dysbiosis potentially in the GI tract.

Steve Dale:
So I asked about antibiotics as a preventive earlier. Dr. Gookin, I need your help in saying this again, it is E. hirae, so do you ever envision the day, this is my question kind of, but I will get to this one which is similar, sort of, follows it…envision a day where as a prophylactic, I asked about antibiotics earlier, that E. hirae might be used?

Dr. Jody Gookin:
I think it will be…can I envision that day? Sure, but I also am a scientist, and I need that evidence. With the right evidence, if things progress the way they could, then absolutely. I think that probiotics are a really, really hot topic. People are using them. What we really lack is hard-core evidence for their efficacy, and when should we use them, how do they work. So I think that that is growing exponentially right now, and I do see a future where probiotics will have specific indications and maybe even specific contraindications as our understanding of what they are doing grows.

Steve Dale:
So speaking of which, is E. hirae (notice I pronounced it every time differently, I figured I will hit it right eventually), is it vulnerable to antibiotics? Healthy cats did not have antibiotics, sick cats did have antibiotics, therefore perhaps sick cats did have E. hirae prior to medicine?

Dr. Jody Gookin:
Yeah, that is a great question. So the E. hirae is pretty susceptible to antibiotics, and correctly, some of the kittens that were in the sick group had received antibiotics. We did look statistically to see if there was a relationship between those kittens having had received antibiotics and them being more likely to have gotten that EPEC and we could not find any relationship. The thing I do not know, though, is specifically do we have enough kittens to actually answer that question, because once you look at subsets of the data, like just the kittens that got the antibiotics, or did not, and then just the kittens that had the EPEC, and did not, you are talking about small numbers of cats. So with large numbers of cats, could it be that antibiotics predispose to colonization by pathogenic bacteria? I think that that could be possible, yeah.

Steve Dale:
So that cat that I mentioned that is the domestic shorthair that has food allergies, seasonal allergies, diabetes, asthma, ear infections, bowel problems, as well as vomiting, could one day stem cells be used for that cat? And could that cat that you are treating for problem A among the fifteen problems there, could it treat 14 of the 15 just because it is stem cells?”

Dr. Craig Webb:
Right, it is certainly possible, because many of those that you rattled off clearly have an immunological or immune system basis, kind of as the final common pathway, and so, yeah, that may be very well a treatment you would consider.

Steve Dale:
Similarly, most felines have multiple issues it says, along with IBD, concurrent problems such as stomatitis, coronavirus maybe, herpes, those are the ones listed here, same thing, could stem cells then miraculously, or are we putting too much faith in the stem cells, putting the cart before the horse or the mouse before the cat?

Dr. Craig Webb:
Right, and that is what we are specifically trying not to do, for sure, and so, no, will I start kind of willy-nilly offering these injections to any concurrent disease-riddled cat that comes in the clinic, absolutely not. But is it certainly a possibility and therefore worthy of further study? Yes, absolutely yes.

Steve Dale:
Did any cats get cobalamin?

Dr. Craig Webb:
No, so any changes in cobalamin were not because they started receiving supplementation. We did not allow owners or have owners change the treatment, any treatments the cats were already on.

Steve Dale:
So you said that, I think you said one of the cats in the control group died…

Dr. Craig Webb:
Got worse.

Steve Dale:
Got worse. So were those cats given stem cells after the study?

Dr. Craig Webb:
Right, one of them, when everything was unblinded, one of them came back and asked for stem cells. That cat did receive stem cell therapy, and did not get any better, which is, again, in a client’s world is unfortunate, you feel bad, as a clinician you feel bad. As a researcher, you are thrilled, really, because to have 100% response to any treatment is just unrealistic and something is not right. So in fact 2 of the 7 that received stem cells, they did not get any worse, they got moderately better, 5 got significantly better, and then the 1 who was unblinded did not get much better and that is exactly what we would expect and in fact hope for in a complicated biological system like that.

Steve Dale:
“Dr. Gookin, does the timeframe after death greatly affect bacteria distribution, number, etc.?”

Dr. Jody Gookin:
I think it would, absolutely. The question is, how long does it take for that to happen. So I think that has to be a consideration and one of the reasons why we specifically kept track of whether there were differences in the amount of time between death and us looking at these bacteria. It is also one reason why now the studies we are transitioning into are in live kittens. So having made the observation in dead kittens, and needing to see that GI tract to make that distinction, whether those bacteria are there or not, now that we can find them by other means like PCR and culture, we can study live cats and remove that death variable. Are they dying for some other reason and then the EPEC adheres, so that is a very legitimate question. We did look at that in the study. The study that I showed today is actually published, and we looked at whether there was any relationship between the time between death and the necropsy, and the bacterial populations present, and we could not find any.

Steve Dale:
That was actually the last question. So I have one more thing to do, and I want to ask you to help me do it, and that is thank our great speakers [applause]. I hope you consider learning more about the Winn Feline Foundation, like our Facebook page, as they say, that is what the kids say, right, but do like our Facebook page, and I also I do hope you check out the website. Thank you very much for attending another Winn Feline Foundation Symposium.