Podcast

Key takeaways

• A problem mare is one bred three or more times to fertile semen without conceiving. Roughly 5 to 10 percent of a broodmare population ends a season open (not pregnant).
• According to Bojesen and Petersen, around 80 percent of problem mares carry a dormant Streptococcus zooepidemicus infection, a hidden ("latent") endometritis that produces few or no clinical signs.
• Dormant streptococci shut down their metabolism, so they do not grow on standard culture. The bacteria also hide inside the mare's own uterine cells and as deep as about 10 mm into the uterine lining, which is why standard swabs return false negatives.
• Standard swab culture detects only about one third of positive mares. Flush (lavage) or biopsy raise this to roughly 60 to 70 percent. Dormant streptococci are still missed unless an activation medium like bActivate is used.
• Three risk factors each independently flag a high (about 80 percent) probability of dormant infection: age 12 or older, three or more foals, or a previous episode of endometritis or abortion. Any one is enough.
• Hagyard Equine Medical Institute study (Dr. Kristina Lu, ~64 problem mares, from 2010): about 70 percent became pregnant, and at least 32 delivered live foals (some were sold pregnant and lost to follow-up).
• Kildangan Stud / Godolphin study (Dr. Meta Osborne, 19 problem mares): 16 were activation positive and treated with penicillin; 14 of the 19 carried a live foal the following year, at an average of 1.1 cycles per mare.
• Re-activation study (19 activation-positive mares): after activation and full local plus systemic treatment, 18 of 19 were sterile (no growth) when activated again the next cycle. The single positive mare had received only local, not systemic, treatment.
• The protocol fits a normal reproductive practice: sample the uterus, infuse 10 ml of bActivate, wait about 48 hours, re-sample and culture. If streptococci are activated, treat locally and systemically with penicillin (three days), since streptococci are almost always penicillin sensitive.
• Biofilm is a superficial bacterial colony on the uterine lining; breaking it up with acetylcysteine only treats the surface and does not reach the dormant infection deeper in the tissue, so it is "not even halfway" to fixing the problem.
• Because infection builds up with each foaling, the researchers also use activation proactively (for example resting a mare in autumn) once a mare has had two or more foals, to stop the dormant infection accumulating.

Introduction

Martin Hinz: If you work with broodmares as a vet or owner, you have surely met the problem mare: trying to get that mare pregnant, only to end up with no result and big disappointments. What if the answer is something you have overlooked? Welcome to From Headache to Hope. My name is Martin. Joining me are two of the leading minds in equine fertility, Professor Anders Miki Bojesen and Dr. Morten Ronn Petersen, equine specialist. Morten, introduce yourself.

Dr. Petersen: Thanks. I am a vet and have worked with fertility for many years. My final project in vet school was on equine reproduction, I worked in clinical practice on a very large stud farm, then did a residency in Davis, California, and a PhD at the University of Copenhagen, where I joined up with Miki and did a lot of research on endometritis in mares.

Prof. Bojesen: I am Anders Miki Bojesen, professor of microbiology at the University of Copenhagen. I am also a vet, but I work with microbiology and infections in various animals, especially horses, and Morten and I have done a number of projects together.

What is a problem mare?

Martin Hinz: What is a problem mare?

Dr. Petersen: The clear definition is a mare bred three times without getting in foal. Many of the mares we have looked at were bred more than three times with fertile semen and still did not conceive. Our work has primarily been on infectious endometritis, especially the chronic, hidden cases. These mares keep being empty, or barren, season after season, some of them year after year.

Martin Hinz: And owners are spending a lot of money on them.

Dr. Petersen: A lot. These mares will have seen a reproductive vet several times and been through many treatments, antibiotics and otherwise, over and over again. A mare that is barren over a long time is very costly, especially when the bloodline is valuable.

Martin Hinz: Out of 100 broodmares starting a season, how many end up as problem mares?

Dr. Petersen: Depending on the age profile, somewhere between 5 and 10 percent will be open following a season.

The hidden problem: dormant Streptococcus

Martin Hinz: What are these owners overlooking?

Prof. Bojesen: People who work with broodmares know streptococci are a major cause of infectious endometritis. What many do not know is that mares can be infected without showing it. We call this a dormant or hidden infection. Surprisingly many mares have an infection that is not easy to detect. Sometimes they show an infection, are treated, it disappears, and yet the mare still cannot get in foal, because the infection is still there, unseen.

Martin Hinz: Can you explain what dormant streptococci are and why they are so hard to handle?

Prof. Bojesen: Normally streptococci grow readily, for example on blood agar plates, from one day to the next. The dormant fraction shuts down its metabolic rate, so it does almost nothing. Most clinical microbiology diagnostics rely on bacteria dividing and growing. When they do not grow, you take a sample, culture it as usual, and often see no result at all, so you conclude there is nothing there.

Martin Hinz: So you get a false negative.

Prof. Bojesen: Exactly. And there is more. In our early studies we found the bacteria can get inside the mare's own cells and hide there, not only in the upper layer of the mucosa but as deep as about 10 millimeters into the uterine lining. When streptococci approach the surface of an equine cell, there is a kind of crosstalk that makes the cell form a cup-like structure, the bacteria enter it, and it closes over them. The bacteria then sit in a vacuole inside the cell, hidden from the horse's immune system for prolonged periods. No wonder they are difficult to find.

Dr. Petersen: And they sit in pockets. You can have infection in the right horn, in the left horn, with very few clinical symptoms, no fluid accumulation, no discharge. If you do not look for it, you will not find it.

How activation works

Martin Hinz: So how do you fix the problem?

Dr. Petersen: What we discovered was a resuscitating factor that stimulates these bacteria to grow. You add our product, bActivate, which allows and stimulates the dormant bacteria to multiply. Where they were located deep in the tissue in pockets, once they start to multiply they come to the surface of the uterus and spread throughout it. Now it is simple to sample the uterus and recover metabolically active bacteria, culture them in the lab, identify them, and choose the best treatment.

How common, and the risk factors

Martin Hinz: Within that 10 percent of problem mares, how many have a dormant infection you can activate?

Dr. Petersen: When we compile large datasets, about 80 percent of problem mares are infected with dormant streptococci, this latent endometritis, across all breeds. It is a wear-and-tear issue. You see it in mares that have had more than three foals, mares with a history of endometritis, and mares more than 12 years of age. When we looked at the statistics, it is enough to have just one of these risk factors.

Martin Hinz: So if a mare is difficult to get in foal and she is 12, the chance of a hidden infection is as high as 80 percent?

Prof. Bojesen: Yes. Age 12 or older, three foals or more, or a previous episode of endometritis or abortion. Any one of them puts the mare in the high probability category. Several studies document that around 12 years of age is a turning point.

Why infection builds up over time

Prof. Bojesen: A mare can get infected with her first pregnancy or first foaling, when the cervix is open and she is exposed to the lower reproductive tract microbiota. Streptococci are part of that. At that point the mare is doing well on all parameters, so she can still get pregnant and carry a foal, even with a low-grade dormant infection. Each time she foals she is exposed again and the load builds up. By the time a mare has had three foals or more, you reach the threshold where the infection starts to compromise the uterine lining, and the quality of the lining declines as the mare gets older and has more foals.

The Hagyard study

Martin Hinz: Tell me about the early study at Hagyard Equine Medical Institute, led by Dr. Kristina Lu.

Dr. Petersen: In the early days I did a sabbatical at the University of Kentucky and introduced the vets at Hagyard to our findings. That led them to try it in a selected group of broodmares that had been barren, some of them for years. We started around 2010. About 70 percent of those problem mares became pregnant, and the majority that we followed produced a foal. The fascinating thing is that these mares had looked dry, with no clinical symptoms, and after activation they would show purulent discharge and fluid, and you could recover a substantial amount of bacteria.

Martin Hinz: How many mares were in the study?

Dr. Petersen: Around 64 problem mares, all of them mares Hagyard had been unable to get in foal. These were high-value broodmares that had already had intense treatment from a very good repro team.

Martin Hinz: And how many live foals out of those 64?

Prof. Bojesen: At least 32 that we know delivered a live foal. Some were lost to follow-up, and some were sold already five or six months pregnant, when the risk of losing the foal is very low.

Why flushing and kerosene fall short

Martin Hinz: Many vets flush with different things, including kerosene. Why is that not a good long-term method?

Prof. Bojesen: These flush agents work in very different ways, if they work at all. Even saline cleans the inside of the uterus and can help a little. Kerosene strips the mucosal lining completely, so it removes infected cells in the upper lining, but it does not reach the deeper parts of the uterine wall where bacteria still sit. So it is harsh and does something, but it is not a guarantee you get rid of the dormant bacteria deeper in the tissue.

Dr. Petersen: We did studies in New Zealand, where kerosene is very common in problem mares, and despite its frequent use we found a prevalence of chronic dormant infection similar to elsewhere. Kerosene may help in some cases, but it is not the cure. I have seen cases with adhesions and scar tissue suspected from previous kerosene use. It is petrol, extremely harsh to the tissue, with a risk of forming scar tissue.

Biofilm and acetylcysteine

Martin Hinz: When I talk to vets, studs and owners, I often hear about biofilm: the mare has a problem with the biofilm. Is that related to the dormant infection?

Prof. Bojesen: It is somewhat related, but not fully. A biofilm, to define it, is when bacteria make a micro-colony on the mucosal surface, a superficial formation that grows. The bacterial community establishes on the surface and secretes an extracellular matrix, a slimy substance with a lot of proteins. Some of the bacteria inside a biofilm also become dormant. What people often want to do is break up the biofilm to get rid of it, and many use acetylcysteine, which is known to break up mucus, like when you have a cold and a lot of mucus in your airways.

Dr. Petersen: Mucolytic.

Prof. Bojesen: Yes. Acetylcysteine has some effect there. But the important thing to remember is that dormant infections are not just on the surface. They can be in the biofilm, but they also sit in the tissue, and acetylcysteine does not get into the tissue. It does not bring the dormant bacteria back into the active state. So you can use acetylcysteine to break up the biofilm to some extent, but it does not fix the dormant infection.

Martin Hinz: Would it help in some cases, get the mare in foal, and then the problem returns the next year?

Prof. Bojesen: Since much of this problem is actually in the tissue, fixing it on the surface is not even halfway. It is an easy, inexpensive treatment, and you feel like you are doing something, which is probably the feeling both the vet and the owner have after using acetylcysteine.

Martin Hinz: If you have a biofilm, do you also have a dormant infection?

Prof. Bojesen: Not necessarily, but they can easily be present at the same time. You can have dormant bacteria within the biofilm, but that is still on the surface, a fairly superficial problem, different from the real dormant infection deeper in the tissue.

Scar tissue and the immune system

Prof. Bojesen: Scar tissue is a generic reaction. A chronic infection causes a low-grade inflammation all the time, which can cause scar tissue to form. If a uterus has a lot of scar tissue, it cannot support the fetus through the entire pregnancy, and that is one of the things that prevents a mare from producing a foal.

Dr. Petersen: The uterus also has to create an environment suitable for pregnancy, where the immune system is downregulated so the embryo can grow. That is a beautiful place for a bacterium to hide. In chronic cases there is a battle between the low-grade inflammation in the endometrium and the environment needed for pregnancy, and that prevents the pregnancy from establishing.

Prof. Bojesen: Some treatments simply dampen the immune system, which makes sense to a point. But if you have a dormant infection, you do not get rid of the bacteria, you only hold down the immune system, maybe too much. When we activate with bActivate we provoke a controlled, short inflammation, get the bacteria to grow, and then treat. You provoke an infection, but you fix it quickly, so the inflammatory reaction is kept to a minimum.

The treatment protocol

Martin Hinz: Morten, what is needed to carry out a bActivate treatment?

Dr. Petersen: It is very straightforward, all procedures done in a regular equine reproductive practice. The mare is in heat. You sample the uterus, for example with a low-volume lavage. You infuse 10 ml of bActivate, just as you would infuse antibiotics. You come back two days later. During those two days any dormant streptococci are activated and multiply, so a second sample recovers them. The day after, you have culture results from both samples, giving a full picture of the bacteria present. Then you treat as you would any endometritis: uterine lavage followed by antibiotics the bacteria are susceptible to. If streptococci grew, especially more in the second sample than the first, you know it was activated, and you also treat systemically with penicillin intramuscularly for three days. Short, high-concentration, and very effective.

Prof. Bojesen: You treat both in the muscle and locally, because you need to hit the bacteria from both the outside and the inside of the uterine wall. Streptococci are, from very large studies over many years, almost always sensitive to ordinary penicillin, so you do not need broad-spectrum antibiotics.

Martin Hinz: Does it have to be exactly 48 hours?

Prof. Bojesen: We also sampled after 24 hours, and a large number of mares are already positive at one day. There is a slight increase, maybe around 10 percent more, after two days. Most are positive already after 24 hours.

The Kildangan / Godolphin study

Martin Hinz: Tell me about the study in Ireland.

Dr. Petersen: This was at a big stud farm called Kildangan, part of the Godolphin group, with Meta Osborne, a very experienced reproductive vet. She was open to trying it but a little skeptical. That first season she pulled out 19 problem mares, real problem mares, high-value thoroughbreds, some barren for years. They were activated, and the positives were treated. Out of the 19, 16 were positive for streptococci and treated with penicillin. With natural cover, 14 of the 19 got in foal and carried a foal the following year, at an average of 1.1 cycles per mare, basically one cycle. These were some of the worst performing mares they had, so it was pretty amazing.

Re-activation study and diagnostic sensitivity

Dr. Petersen: We also wanted research data on how effective the treatment is. We identified 19 activation-positive mares, treated them, then came back the following cycle and activated them again to see the status of the uterus. Of those 19, 18 had no growth, they were sterile and clean. The single one that was activation positive again had been treated only locally in the uterus, not systemically, which shows how important it is to treat both locally and systemically.

Martin Hinz: And how good are the standard sampling methods?

Prof. Bojesen: There is a lot of data showing the swab is not very effective. If you have 100 mares that are positive and you swab them, you catch only around a third. Flush or biopsy are better, catching roughly 60 to 70 percent, about twice as good. But even with flush or biopsy you will very rarely find the dormant streptococci, because all three methods depend on the bacteria growing. If they do not grow, you miss them, which is why you need an activation medium like bActivate.

Martin Hinz: Why do people still use the swab?

Prof. Bojesen: It is easy, cheap, and familiar to owners and vets. A biopsy means puncturing the mucosal lining, which some people are wary of, although in horses the lining is thick and it is very hard to do harm. We highly recommend biopsy or flush over the swab, but for dormant streptococci you still need activation.

Prevention and closing

Martin Hinz: Would it be a good idea to keep a mare clean before resting her for several racing years?

Prof. Bojesen: We have not made a formal study of when to prevent, but since dormant infection clearly increases after three foals, it makes good sense to start thinking about activation as a preventive tool after two foals. An activation treatment does not harm the uterine lining, so you can prevent the dormant infection building up earlier than most people do now.

Dr. Petersen: Practically, a mare that has had several foals might end up foaling late, in May or June, and you rest her over autumn. That is an excellent time, on the northern hemisphere in August or September, to activate and treat if you see bacterial growth, using that spare time constructively.

Martin Hinz: You can find more on YouTube by searching for bActivate and endometritis, on the bActivate Facebook page, and on the bActivate website. Thank you for listening, and we will be back with a new episode.