Toward Solving the Medical
Mystery of MRLS

From elementary school through graduate school, students are inculcated with the scientific method as the preferred way of discovery. To many, the scientific method seems contemplative, systematic, thorough, and exhaustive, which indeed it usually is. For scientists, the method is rigorous, logical, and fascinating, and maybe even comforting because of its measured pace.

Every now and then, however, all those qualities of the scientific method must be put to work fast, at breakneck speed, because scientific discovery cannot wait for the niceties of time. In such urgent times, experiments succeed and fail in rapid fashion; ideas are proposed, tested, and pursued—and discarded in favor of more compelling theories—in days and weeks instead of years. In these times of urgency, the scientists themselves renew their understanding of their passion and excitement for the process of science, and their appreciation of it becomes even more keen.

Such was the case in May 2001, when the number of stillborn, weak, and dying foals and early aborted foals in Central Kentucky mares grew alarmingly high. During the weekend of the Kentucky Derby, Lenn Harrison, the director of the UK Livestock Diagnostic Center, knew that his life in the near future would be directed toward research at machine gun pace until the cause—and hopefully a prevention strategy—was discovered. On Derby Saturday alone, 73 dead foals and fetuses—10 times the normal number—had been delivered to the back door of the facility; by Monday, that number had reached 276. It was becoming abundantly clear with each new foal or fetus that whatever was causing problems was doing so with a vengeance.

At the same time, veterinarians Roberta Dwyer and David Powell of the Maxwell H. Gluck Equine Research Center were fielding questions from Central Kentucky horse breeders and local equine veterinary practitioners who also had noticed an increase in stillborn, weak, and dying foals as well as aborted fetuses during the last days of April.

More than a few times that weekend and throughout the next few weeks, when weekdays, weekends, and workdays were indistinguishable, Harrison thought about other scientists—epidemiologists who worked on medical mysteries such as Legionnaire's disease, the Hanta virus—as he organized a strategy to deal with the mystery. Not only would the scientists have to be good, they would have to be fast, because every foal or fetus lost due to the mysterious illness represented a monetary loss of great magnitude. With each ticktock of the clock, the cost of the disease, as well as fear among horse breeders, grew.

Pieces of the Puzzle
The pieces of the puzzle were intriguing. The epidemic seemed to be restricted for the most part to Central Kentucky, with few similar cases being reported in other states, and the disease affected all breeds of horses from ponies to Thoroughbreds to Morgans—horses that would most likely never have contact with each other—discounting a bit the idea that a communicable contagion was causing the problems. Further, the onset of the disease was sudden, with a sharp escalation in cases. Research had to find an answer—a control and prevention—if not an explanation. Harrison and the scientists from the Livestock Disease Diagnostic Center couldn't do it alone; they needed help.

Within days of the Derby, a team of more than 100 scientists from all over the College of Agriculture was assembled to investigate the mysterious disease. In some ways, organizing research to find answers as fast as possible is like playing the parlor game of 20 Questions. Instead of asking about whether it was animal, vegetable, or mineral, team members asked whether the cause of the weak foal and fetal losses was viral, bacterial, or environmental. If the cause were bacterial or viral, it might well be contagious; because of the great number of deaths and abortions, that possibility couldn't be ruled out entirely. And if it were environmental, what was the reason that it seemingly was confined to a very narrow band, nearly exclusively in the Central Bluegrass? Known viral agents, Harrison knew, would show up on necropsy and subsequent tissue testing. (Necropsy is a term used when a postmortem examination is performed on an animal, roughly equivalent to an autopsy in humans.) The pathologists found no indication of known viruses.

Bacterial agents, too, would show up at necropsy as damage to particular organs and could be cultured using petri dishes, a growing medium, and a warm environment. Unlike viruses, however, bacteria are found often on necropsy, and some bacteria are considered routine invaders of dead animals—sort of the coffin flies of bacteria. Early necropsies found rare bacterial infections in many aborted fetuses, but the scientists concluded that while they were rare—and even perhaps related to the syndrome—they likely were not the sole culprit, the smoking gun, that caused death.

And with either viruses or bacteria, the findings from necropsy should be pretty much consistent from accession (a fancy term for dead animals logged in for examination for cause of death) to accession. If, for example, the virus damaged the liver—and caused death by that damage—then the damage to the liver should be found in every necropsy performed in theory and most in practice.

Environmental Links
By May 14, just a hair over two weeks after the staff at the Livestock Disease Diagnostic Center first noticed the unusual number of foals and aborted fetuses being sent for necropsy, results of a survey of Central Kentucky horse farms indicated that the syndrome was widespread in the area, and it was continuing.

By this time, the mystery was occupying most of the pathologists at the Livestock Disease Diagnostic Center as well as scientists at Gluck Equine Research Center, the Department of Animal Sciences, and local equine practitioners. Several faculty members at Gluck remembered that in 1980 and again in 1981, early fetal losses were unusually high—but not as high as the current losses. No cause had ever been found. In both years, the mysterious syndrome started and stopped rather abruptly.

The unexpected starting and stopping of fetal and foal losses suggested strongly, but not absolutely, that something in the environment was related to the syndrome, by now named the Mare Reproductive Loss Syndrome (MRLS).

What was common about 1980, 1981, and 2001 that could be related to outbreaks of early fetal losses and weak and dying foals?

"A similar pattern of weather conditions just prior to the outbreak in all three years occurred," Harrison said. "March temperatures were below normal in all three years, followed by above-normal temperatures in April. Such a pattern could be related to an explosive biological activity in both plants and insects." All three years also were noted for having a frost or freeze in the third week of April followed by a warm-up just days later.

The similarity in weather patterns was strongly suggestive of something weather-related occurring that had an effect on pregnant mares and their fetuses, Harrison said.

Biological Factors
The avenue of the search now turned to biological factors related to weather—-either through plants or insects.

Organization of the research teams within and outside the College of Agriculture was facilitated by veterinarian David Powell of the Gluck Equine Research Center and included scientists from practically every discipline whose knowledge could define the scope of the inquiry.

Weather could be linked to a variety of possible agents of the fetal deaths and weak foal births, including mycotoxins, poisons given off by fungi that can explode when the weather is just right, ergot-type alkaloids (also given off by fungi), and phytoestrogens, which are plant hormones that mimic naturally produced estrogens. At first blush, all of these seemed among the possible suspects for the losses. If any of these were the cause of the syndrome, they should show up on testing. None did.

If the common culprits appeared unrelated to the losses, the question became more intriguing. Weather and what else?

"Early on, some people had suggested that eastern tent caterpillars might be the culprit, but it didn't seem plausible," Harrison said. "No one had ever reported that tent caterpillars were anything but fairly benign creatures that created inconsequential webs in trees in early spring."

But with time ticking away and no other obvious cause on the horizon, the scientists turned their attention to the eastern tent caterpillar. The literature on the caterpillars did, however, indicate that their favorite plant to feast on was the wild cherry tree. Wild cherry leaves can release cyanide, a poison, when ingested and were known to be deadly to livestock eating them in large amounts.

Agronomist Jimmy Henning, now a principal investigator on the problem, noticed on visits to farms where the outbreaks had been most severe that wild cherry trees—and eastern tent caterpillars—were also abundant. A thorough survey of farm managers with and without losses to MRLS was led by veterinarian Roberta Dwyer and confirmed the associations. But, as any scientist knows, simple presence of phenomena does not necessarily imply a cause and effect relationship. Nonetheless, the idea was intriguing and worth further exploration.

By May 20, 2001—less than a month after the first known cases—the deaths of newborn foals and spontaneous abortions of early fetuses dropped off precipitously, just as they began. This coincided with the drop in eastern tent caterpillar numbers. And although losses were down to normal levels, the scientists continued their search to prevent and explain the syndrome that reared up in 1980, 1981, and 2001. What about future outbreaks?

Applying Research
With eastern tent caterpillars coincidently being linked to the losses, the next step was to try to replicate the losses in a controlled experiment. Up to now, the scientists had to rely on survey techniques that helped narrow the field of causes, but they were less than conclusive in a scientific sense.

Entomologist Bruce Webb, whose specialty is insect molecular virology, and equine reproductive researcher Karen McDowell designed an experiment to see if eastern tent caterpillars were linked to the losses.

The experiment was straightforward: expose 10 pregnant mares to high levels of tent caterpillars and their frass (excrement), expose nine mares only to the frass, and try to minimize exposure of 10 mares to both caterpillars and frass in a control treatment.

Wow! Within a week, the first losses were noted, and ultimately 70 percent of the mares exposed to the eastern tent caterpillars or their frass aborted their foals. Mares in the no-treatment group also had abortions, but significantly fewer. Webb explains that those abortions were likely related to the fact that the experimental facilities failed to keep all the caterpillars in their assigned pens. Because of logistical problems, caterpillar fences—made of plastic pipe cut into a half-round shape so that the caterpillars would fall back to the ground if they tried to crawl outside the pen—could not be completed until the experiment was well under way. Webb’s data confirmed that some of the caterpillars escaped to other treatment groups. Indeed, he found out caterpillars are harder to keep in a herd than horses.

"From this experiment, we were pretty sure that caterpillars were associated with the fetal losses. And following our initial experiment, we worked with local equine veterinary practitioners to verify our findings with another experiment," Webb said.

In that experiment 15 mares were put into one of three treatments. Those in one group were administered ground-up caterpillars directly into the stomach in a saline solution; a second group received frass in a saline solution; the third set of mares received the saline only.

"Eighty percent of the mares treated with the ground-up caterpillars aborted their fetuses, while none of the other mares did," Webb said. "With that, we were pretty confident that whole caterpillars—and not just their frass—were associated with MRLS." So if horse producers wanted to minimize their losses to MRLS, they should try to minimize exposure of pregnant mares to the eastern tent caterpillars.

The 'How' Is Still Unanswered
Okay, if caterpillars are linked to the outbreak of the syndrome, then how? That's still under investigation by scientists.

Currently, they are pursuing three leads on the precise mechanism by which eastern tent caterpillars cause such destruction:

• First, it might be that eastern tent caterpillars are the vector for some heretofore unidentified pathogen.
• Second, eastern tent caterpillars could carry an unidentified toxin, such as the blister beetle carries canthradin poison, so lethal to horses but not other animals. Scientists are studying the eastern tent caterpillar further to determine if this is possible.
• Finally, something about eastern tent caterpillars themselves may cause internal injuries to horses, which would result in secondary pathogens getting a foothold in pregnant mares and foals.

"From the first MRLS cases in 2001 to the last in 2002, scientists have been perplexed by the many features of this syndrome," Harrison said. "Now that a strong, scientifically-based association has been made between MRLS and the eastern tent caterpillar, scientists can focus their research efforts on projects that will provide a thorough understanding of the mechanisms that cause this disease syndrome."

The Power of Teamwork
While the logic of science helped the scientists ferret out the association between eastern tent caterpillars and Mare Reproductive Loss Syndrome, it wouldn't have happened without the complete dedication and cooperation of more than 100 scientists in the College of Agriculture, who worked together with equine veterinary practitioners, and Kentucky farm managers. Every person involved provided yeoman's service to the cause.

Scientists vigilantly tramped through pastures throughout Central Kentucky, assessing them for a plethora of factors that might even distantly be related to the syndrome. In addition, they spent days and weeks investigating potential causes and some factors that were, quite frankly, long shots. Equine veterinary practitioners monitored their cases of MRLS and provided rich data to the UK scientists; without their help the association between tent caterpillars and the syndrome might still be obscure. Vigilant farm managers kept tabs on the situation and provided complete access to their farm records and situations, helping speed up the investigation enormously.

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