Mare Reproductive Loss Syndrome Research Findings

"We can prevent mare reproductive loss syndrome (MRLS) as we experienced in 2001 and 2002 by keeping horses away from caterpillars," said Bruce Webb, PhD, a University of Kentucky researcher who has been studying the condition that terminated thousands of mare pregnancies in Central Kentucky and nearby states. The acute problems of MRLS might be behind us, but MRLS is present tense for Webb and other researchers who spend their laboratory hours studying the condition. The pervading theme at a Nov. 30 MRLS update meeting at Keeneland sale pavilion in Lexington, Ky., was that researchers have learned much about MRLS, but more needs to be understood before they can intervene in the MRLS disease process.

The scientists presented 14 months of Kentucky Thoroughbred Owners and Breeders Foundation-sponsored research material on novel ways to control Eastern Tent Caterpillars (ETC, which have been linked to MRLS) with species-specific viruses, laboratory models for MRLS, and other MRLS-related material. Possible future study avenues were also presented with the suggestion that we might better understand the abortion-causing mechanism of MRLS and other "natural" causes of foal loss if we are able to monitor mare and fetal health by using catheters and probes placed in a fetus and the dam's uterine artery to monitor blood flow between a mare and the fetus, among other things.

Tent Caterpillar Research

Eastern Tent Caterpillars have long been the major focus in MRLS studies, and researchers now know that these caterpillars cause tiny lesions in the mare's gastrointestinal (GI) tract and suspect that these lesions allow bacteria in the GI tract to enter and circulate in the mare's body to somehow reach the fetus, and ultimately cause abortion. Karen McDowell, PhD, David Horohov, PhD, and Neil Williams, DVM, PhD, of the University of Kentucky performed skin tests, much like human allergy tests, to find out if external ETC hairs could modify the mare's immunological response. Suppression of her natural immune response could prevent a mare from attacking bacteria that invade from her GI tract via those lesions. McDowell injected an irritant commonly used in allergy testing, PWM (Poke Weed Mitogen, which produces a predictable reaction) and setae alone and together, intradermally. They expected the welt reaction to the PWM/setae to be less than with the PWM alone, indicating immunosuppression by the setae. That wasn't the case, however, both in mares previously exposed to ETC and those with no prior exposure to ETC.

Additionally, McDowell took samples of the tested areas for Williams to examine under a microscope. He found when the setae were injected alone, there was an inflammatory reaction where the setae were injected, whereas the inflammation was more diffuse in the PWM-injected skin. There wasn't a notable difference in the inflammatory reaction between the PWM/setae samples and those with PWM alone. Both parts of the project indicated that ETC setae do not modify the immune response of the mare in this test.

In another experiment, Webb investigated when it is safe to put pregnant mares back in fields that likely contain dead caterpillars by placing dead ETC in a research field and following how the caterpillars decomposed over time. Tent caterpillar material was identifiable and recoverable in the field for seven weeks. "This most clearly mimicked killing with insect sprays," Webb said. "Insects not killed are likely a more transitory threat because they normally leave pastures to pupate and the cuticle that causes the gut lesions is broken down in the process". After trees are sprayed, Webb recommends keeping pregnant mares off areas of pastures that contain caterpillars for eight weeks.

Webb also sought to answer why we don't have high tent caterpillar numbers and MRLS abortions every year. In 2001 and 2002, there were unusually large numbers of ETC nests, but that changed dramatically between 2003 and 2005. "Our study indicates that an insect virus that kills tent caterpillars is likely the reason," he said. "A tent caterpillar baculovirus was isolated in 2003. This virus is rampant in central Kentucky.  Between 20-80% of the nests we collected in a six-county region were infected (the virus kills the ETC) and 90% of the insects in affected nests had the virus.

"This far outweighs any major virus outbreak  we've seen in the human population," Webb added. By introducing these viruses early in the ETC season via spraying trees, Webb contends the virus could be spread by the caterpillars themselves and dramatically decrease ETC numbers. Huge trees don't require a thorough spraying with the virus because the ETC will spread the virus themselves, and this could also help alleviate the problem of unsprayed ETC nests on neighboring properties. He said that based on work with similar viruses, a mere 2.2 lbs. of virus-infected caterpillars could be used to control ETC in 250 acres.

No MRLS in Mice

The size of mares, their management requirements, and their long gestational periods make them expensive and time-consuming to use in studies, so Willem De Villiers, MD, chief of Gastroenterology at the UK Hospital, Kyle Newman, PhD, of Venture laboratories and Webb investigated the possibility of making MRLS research more efficient by using pregnant mice instead. They investigated the response of research mice to the two major types of bacteria that have been isolated from MRLS aborted equine fetuses. The bacteria did not reduce the litter sizes or induce abortions in the mice, and Newman wasn't able to recover the bacteria in the few baby mice that died after birth from apparently unrelated causes. Mice, therefore, were deemed not useful for MRLS research at this time.

Bacterial Culprits

Newman tackled the challenge of identifying levels of "suspect" bacteria for the cause of MRLS in different parts of the mare's digestive tract. The highest levels of the types of Streptococci that are routinely seen in MRLS were found in the mouth, with about 50% of the total isolated bacteria in the mare's mouth being MRLS-associated Streptococcus.  Although this same bacterial species was present in the lower GI,  it was much less common. "In the GI tract, we're looking for a needle in a haystack that is about the size of Chicago," he said. "There are between 500 and 1,000 different  bacterial species with a total of between one and 100 trillion total bacteria in the GI tract, and we're looking for one." In an unexpected result of this study, the numbers of the MRLS-associated Streptococcus bacteria increased by about 10 times when mares were fed ETC.

Using a method called DNA fingerprinting, Newman assayed the bacteria from different parts of the mare's GI tract.  He determined that different strains of bacteria were found in different gut regions and discovered a 100% match between the suspect bacterial types found in the mouth of a mare and the bacteria that infected its fetus during MRLS. The bacteria causing MRLS "is literally coming straight from the horse's mouth and is part of the normal (gut) flora of that animal," he said.

The obvious questions raised by this research are why these particular bacteria are involved, how and why they get from the GI tract to the fetus, and why doesn't antibiotic therapy work in treating these infections in mares.

Studying the Fetus In Utero

Carrying on the suspect analogy, Cornell University's Don Schlafer, DVM, MS, PhD, Dipl. ACVP, ACVM, ACT, said, "I consider the crime scene (of MRLS) the fetus, which we, as researchers, have had a hard time accessing. It is possible to surgically assess fetal lambs and calves with some born after being studied in utero, but the horse has a reputation to be very sensitive." Schlafer has been doing fetal surgeries on cattle and sheep for years, and recently he has applied his research to equine fetuses, where he hopes he can unlock some of the mysteries of MRLS.

Schlafer has placed catheters in the blood vessels of unborn foals and has surgically implanted special doppler ultrasound blood flow probes around the mares' uterine arteries and others around umbilical arteries (vessels that send blood from the fetus to its placenta). These probes send signals through wires to external monitors that indicate exactly how much blood is flowing through each vessel. He has monitored the blood flow between the fetus and its placenta and also studied the amount of blood flowing from the dam to the gravid (pregnant) uterus as she rests and exercises (via a radio transmitter sutured to the outside of the mare), and when the umbilical cord is kinked or folded over, simulating cord tension, an important cause of abortion in mares.

His observations have been interesting. Data from one mare at about 9.3 months gestation showed that the fetus was sending approximately 4.2 liters per minute to its placenta (66 gallons per hour). He has also found that during periods of excitement and exercise, blood flow to the uterus drops significantly and in one instance when the mare bolted on the treadmill during exercise, flow to the uterus and to the placenta dropped by half. "We never expected mom to stop blood flow to the fetus as often and dramatically as she did," he said. "We can follow blood flow in utero under a number of different conditions."

When discussing ways fetal monitoring could be used to study the cause of MRLS-associated abortion and the role bacteria play in those abortions, Schlafer said that we are uncertain when the bacteria cross the placenta. "If we have catheters in the fluid compartments of the placenta and the fetal blood vessels, we can collect samples repeatedly over time to test for the initial entry of bacteria, and if there is evidence that the fetus is getting ill, or that placental function is abnormal before bacteria enter the uterus, then we should be looking for caterpillar-associated 'toxins' that might have an important role in why these mares abort after ingesting caterpillars." Using this technique, scientists could also take bacteria that are commonly isolated from fetuses aborted in MRLS, and inject them directly under the placenta, and monitor the progression of the disease from the perspective of the fetus." The application of this type of fetal monitoring has many implications. Beyond understanding MRLS, researchers could learn more about nocardioform placentitis (another devastating cause of abortion) and other infectious diseases. Additionally, he suggests we could eventually investigate fetal responses to viruses, bacteria, adn other microbes that infect the gravid uterus, and perhaps even investigate possible immunization of the fetus in utero.

The fetal research is only preliminary--Schlafer is currently seeking funding to continue his group's work and says he and colleagues need to gain more experience and expand these fetal monitoring approaches. "I'll admit we're at the very beginning," he said. "There are a lot of ramifications for fetal health and mare health and for better understanding diseases of pregnancy."

Looking Forward

McDowell finished up the conference by reviewing all of the MRLS research in a nutshell, reinforcing the recommendations of keeping ETC away from pregnant mares. 

"This isn't the first or the last reproductive problem we'll see," she concluded. "It's still a work in progress, but I think you can see progress is being made."

About the Author

Stephanie L. Church, Editor-in-Chief

Stephanie L. Church, Editor-in-Chief, received a B.A. in Journalism and Equestrian Studies from Averett College in Danville, Virginia. A Pony Club and 4-H graduate, her background is in eventing, and she is schooling her recently retired Thoroughbred racehorse, Happy, toward a career in that discipline. She also enjoys traveling, photography, cycling, and cooking in her free time.

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