Melanie Cole, MS (Host): Metritis is an inflammatory uterine disease found in about 20% of dairy cows after delivering calves. Studies at the University of Florida College of Veterinary Medicine have found a distinct link between uterine disease and the uterine microbiome.

Melanie Cole, MS: Welcome to UF Vet Med Voice with the University of Florida College of Veterinary Medicine. I'm Melanie Cole. And joining me today is Dr. Klibs Galvao. He's a Professor in Food Animal Reproduction and Medicine in the Department of Large Animal Clinical Sciences at the University of Florida College of Veterinary Medicine.

Dr. Galvao, it is a pleasure to have you with us today. I'd like you to start by just telling us a little bit about your background and area of interest. Tell us about this interest in Uterine Health.

Dr Klibs Galvao: Sure. Great to be with you, Melanie. So, my training has been mostly in animal reproduction, particularly in dairy cows. And I got my interest in uterine health during my residency and later during my PhD training. And that's when I found out that uterine disease was very prevalent in dairy cows and that had a huge negative impact in their fertility. And that is important because if cows do not get pregnant on a timely manner, they end up being discarded or sold from the herd, and that is a big loss to dairy producers.

So in the case of metritis, as you said, this is an acute inflammatory disease that happens shortly after parturition in dairy cows. And that is a very acute, severe infection that can get cows very sick, so some cows may even die because of that condition. Now, most cows recover from that and they go on to have lactation. But the problem, as I said in the beginning, is that their fertility is greatly compromised. So, we really struggle with those cows first to know what are the main risk factors and, as you alluded to, how does the microbiome of the uterus, how the changes in the microbiome predisposes the cows to develop that condition. And once they develop that condition, what can we do to help them?

So, the first thing we want to do is to try to prevent the disease from happening in the first place. But if the cow ends up developing that disease, how can we best treat that condition so the cow doesn't suffer from the negative consequences. So, one of the main consequences that we have in mind is the welfare of the animal. So as you can imagine with any infection, there is pain associated with that, and that is a very painful condition for the cow. So, we have to keep that in mind and try to alleviate any symptoms associated with pain, so anti-inflammatories are used in that situation. Because it is an infectious disease, we also use antibiotics and that is the most effective treatment for the condition.

Now, there is also a concern about antimicrobial resistance, so we try to be very judicious with the use of antimicrobials. Keep in mind that we want to avoid the problem of antimicrobial resistance. So, that's why we studied microbiome to see first if we can prevent the disease from happening and, later, are there anything we can do in regards to changes in the microbiome that can potentially avoid the use of antimicrobials?

Melanie Cole, MS: That's fascinating, Dr. Galvao. So, explain a little bit more about the intersection between the uterine microbiome and the immune system in dairy cows with this goal, as you've been explaining, of understanding the etiology of uterine diseases. Tell us a little bit about your work.

Dr Klibs Galvao: The intersection of immune function or the immunology and the microbiome. Any disease happens because of an imbalance, an imbalance between the host, the immune system of the host that cannot prevent an infection from taking place or a pathogen that is very virulent or that we are naive to that pathogen.

An example would be the COVID-19 virus, that is a new virus. We didn't have any immunity to that virus. So, it's not that our immune system was naive or deficient, but the virus was novel and very virulent. Now with the course of the disease, some people, we found out that the immune system responded differently. So, it is a very important component. Naturally, each of us have a different immune system, so we try to understand how the immune system of the cows that develop disease and how is the immune system of cows that do not develop disease and how that impacts the uterine microbiome.

So, now, we understand much better that intersection. We learned that cows that have problems pre-partum, that start before parturition, they have issues handling the demands for energy. Because dairy cows, once they give birth, there is very high demands for milk production. These cows produce a lot of milk. As you can imagine, a dairy cow will produce eight to 10 gallons of milk a day. So to produce that much milk, it requires a lot of energy. Now, those energy demands start even before parturition. So, the adaptation of those cows before parturition and what we call the transition period is really a key factor. So if the cows do not adapt well getting close to parturition, that will have a negative impact on their immune function. So once they give birth, they cannot really control the pathogens that will go on to cause disease. So, what we observe is then a dysbiosis in the uterine microbiome. So at around two to three days after giving birth, we have a shift in the uterine microbiome where some pathogens, mostly Gram-negative pathogens, take over the command cells in the uterus. And we see bacteria such as Fusobacterium, Bacteroidetes really grow exponentially and take over the command cells, and that's what end up causing disease in those cows. So, it's really a matter of balance between the immune system, and that starts before calving and then carries over after calving. And those cows that have this impacted immune function, impacted immune system cannot control bacterial growth of these certain pathogens, particularly Gram-negative pathogens and we see this dysbiosis, which end up leading to disease development.

Melanie Cole, MS: Dr. Galvao, tell us about your two intertwined projects funded by the USDA that's investigating causes of metritis and how you are using unique integrative approaches to investigate better ways of understanding the bovine uterine microbiome and, hopefully, prevent uterine disease.

Dr Klibs Galvao: The first thing we had to do was to understand the shift in the uterine microbiome after cows give birth. So, that was not known exactly how the uterine microbiome progressed after calving, from the time of calving until development of disease. So, we seek out developing disease around day seven, eight, nine, 10 after giving birth. But we didn't know exactly what happened between the time of calving, parturition until disease development. We had a good idea of what the microbes were like during disease, but not what happened before.

So, our first step was to start sampling cows, collecting a uterine sample, the uterine swab, right after a cow has given birth. So within 20 minutes of a cow giving birth, we collected the first sample. Then, we came back two days later and then got another sample when the cows developed the disease. And that gave us a very good idea of the shifts in the uterine microbiome that were associated with disease. And that's what informed us that cows had developed disease had a dysbiosis in the uterine microbiome that led to disease development. So, that was the first step.

Then, we followed up on that study. We knew that's what happened, so then we wanted to understand why it happened. So, we knew that the immune system had to be a component. Like in any disease, the immune system, the balance between the pathogen, trying to cause disease and the immune system trying to prevent disease happening had to play a role. Also, we knew the metabolism. As I said, cows have a very high demand for energy for milk production. So, we knew that the energy demands and the metabolism could impact both the immune system and the uterine microbiome. And that's the second part of our studies and that we are just completing now where we investigated the immune system. We saw all the changes that happened from pre-partum all the way until disease development. And what we observed in that is actually because cows start to lose weight, it's very interesting that cows that then go on to develop disease, they start losing weight before they give birth, and that's not what is supposed to happen. Cows are supposed to maintain their body weight before calving or even gain a little bit of weight.

Now, we saw that these cows that went on to develop disease, they were having a hard time adapting, so they started losing body weight. And that's because they were not eating very well. So, we don't know exactly why cows don't eat very well. We just observed that they were not eating very well, that's why they were losing weight. What happens is that when cows lose weight, that creates an inflammatory process. So now, it's known that adipose tissue, just presence of adipose tissue, it's inflammatory. Also, losing body fats is also inflammatory. It activates the immune system. So, the immune system of those cows was being activated even before they gave birth. So, you would think that's maybe a good thing, the immune system is activated once they give birth, they can control bacterial growth and prevent disease, but the opposite happens. Because the immune system is activated when it's not supposed to, the immune system becomes a refractory to infection after calving. So, the cows cannot control bacterial infection because this immune system was activated when it was not supposed to. Then later on, when it's supposed to get activated, it is already refractory and does not respond very well to infection. So, we see this dysbiosis in the uterine microbiome, overgrowth of these gram-negative pathogens and establishment of disease.

Melanie Cole, MS: This is such important work that you're doing, Dr. Galvao, and it's so interesting. As we get ready to wrap up, how do you envision this research translating into care for human to veterinary medicine and vice versa? I'd like you to tell us how you feel this research will ultimately lead to the development of better prevention strategies for uterine disease in cattle, which is expected to improve the health of dairy cattle, benefit the dairy industry, improve the sustainability of the dairy enterprise as a whole and, really, the public at large.

Dr Klibs Galvao: The first thing is that this disease, it's very costly. So, it has a huge detrimental effect on herd profitability. It's very prevalent, so the negative impact is really enormous. It's just to keep in mind with a prevalence of around 20% in a total herd size in the US of about 9 million cows and a cost of about $500 per case, the overall cost is about $1 billion per year to the dairy enterprise. So, our goal is really to prevent this disease. So, we are trying the best we can to understand the disease and try to find ways to best prevent the disease.

So, we foresee in the future potentially using probiotics or even prebiotics or even something that can be fed to cows as part of their normal ration that can help modulate the uterine microbiota in ways that are beneficial to the cow. Now, we know the bacteria that can be helpful to cows and bacteria that can be harmful to cows, the goal would be to find ways to modulate the uterine microbiome towards the bacteria that are beneficial to the cow. It's not that we do not want any bacteria in the uterus that's part of their normal flora. Just like cows have a normal vaginal flora, they have a normal uterine flora right after calving, and that changes over time. But we want those changes to go towards a healthy uterus rather than towards disease.

So, that's what we are working towards and we think we're going to have to start working with those cows way before calving, trying to get them in a more stable energy balance, so as to control their metabolism, prevent that loss of weight and maybe even prevent them even before from gaining so much weight because those cows are the ones that are more exposed to lose weight, and identify those cows that are at risk and may have interventions at calving. So if we know which cows are at risk, maybe we do interventions at calving.

Now, probably even with our best efforts, some cows still develop disease. So, our goal as well is to, once cows develop disease, what can we do? Are there probiotics or prebiotics that can be used instead of regular antimicrobials that can be used to treat those cows. That way, they can have a similar outcome with cows treated with antimicrobials, but without the worry about antimicrobial resistance.

Hopefully, in the long term, our main goals are to decrease the prevalence of uterine disease, and also to decrease the negative effects of having the uterine disease on fertility, on milk production, on survival in the herd and, hopefully, improve profitability in the dairy enterprise.

Melanie Cole, MS (Host): Thank you so much, Dr. Galvao, for joining us today and telling us about your research. It's so interesting. And for more information about the UF College of Veterinary Medicine, please visit vetmed.ufl.edu/ufachievers. And to listen to more podcasts from our experts, you can always visit vetmed.ufl.edu.

Melanie Cole, MS: That concludes today's episode of UF Vet Med Voice, brought to you by the University of Florida College of Veterinary Medicine, advancing animal, human and environmental health. I'm Melanie Cole. Thanks so much for joining us today,