MRLS outbreak in Washington raises new scientific questions

It was Saturday May 5, 2001. A usually festive time in the Bluegrass, as "My Old Kentucky Home" played at Churchill Downs, and 17 horses battled in the 127th running of the Kentucky Derby.

Down the road in Lexington, and on farms in surrounding counties, it was not a happy time.

A tragedy was unfolding.

Young foals were battling for their lives - a mysterious toxin had already claimed more than 300 foals , and before the carnage was over an estimated $ 225 million was lost in the Central Kentucky horse industry from a deadly disease , Mare Reproductive Loss Syndrome ( MRLS ).

MRLS, eventually linked to an overwhelming infestation of Eastern Tent Caterpillars (ETC), which occurred between April and June of 2001 and 2002 in Central Kentucky, produced both early and late fetal losses. Early fetal loss (EFL) occurred within 35 and 100 days after mares were bred. In mares that underwent ultrasound examination during this period, fetal death followed by expulsion of the fetus associated with the presence of abnormal echogenic fluid (cloudy and flocculent) around the fetus.

Late fetal loss (LFL) occurred as abortion during the last trimester of pregnancy. LFL was associated in many, but not all, cases with a swollen and engorged placenta (premature placental separation or "red-bag" syndrome). Foals born alive were sometimes weak and required intensive veterinary care. In addition to reproductive losses, a number of cases of pericarditis (inflammation within the sac surrounding the heart) and severe unilateral uveitis (inflammation of one eye) were associated with the occurrence of MRLS.

May 2003, in Kitsap County, Dr. Kenneth Feigner, DVM noticed a curious similarity to that previous period and raised the possibility of MRLS in Western Washington, thousands of miles from Kentucky.

Exploring the possible links

What links western Washington with Central Kentucky?

According to Feigner, in years past he would see an abortion every year or two. Half the time he can determine a cause. But spring of 2003 was very unique. "Last year we had four abortions and one foal that was born very compromised, and in each case in the pastures where these mares were kept were very heavily infested with Western Tent Caterpillars (WTC)," Feigner stated.

According to Gary Wolbert, Wolbert's Olympic Horticultural Spray Services, sometimes the Eastern Tent Caterpillar (ETC) can be found in western Washington, though the overwhelming predominance is of the Western species. Regardless, the WTC and ETC are probably not uniquely different from each other in their physiology and eating habits. Therefore, the WTC well might affect horses similarly, and the potential for their involvement in MRLS in Washington as seen with the ETC in Kentucky is probable. In all of the aborting mares there was no other abnormality that Feigner could find. He did a necropsy on a couple of them and sent samples to Washington State University for diagnostic workup.

"It was only after you see one or two, and you say it's just a matter of chance, but by the third one and more it seems too weird," Feigner says. Feigner also sent in some tissue samples and discussed the situation with veterinarians in Kentucky, those working on the follow-up to the MRLS problem there.

"They asked if we could send them large volumes of frozen and alcohol-preserved caterpillars for analysis. The frozen ones to be used in subsequent animal testing to see if they could induce abortion in mares, the alcohol-preserved ones for toxicity testing." Feigner has yet to hear back from Kentucky as to the fate of those bugs. "Kitsap County in 2003 was absolutely overrun with caterpillars, Feigner says. "I've never seen anything like it. The sprayers agreed they'd never seen anything like it either."

"One thing I did notice was that during the year before this outbreak, in the Fall 2002, there was a huge number of WTC moths. One could see millions of them broiling in the lights." The insects ranged from Silverdale to Hansville. Vast areas of alders looked like they were in the middle of winter, not summer, because they were totally stripped bare.

Last Fall 2003, he saw almost no WTC moths, and was hoping the sprayers would be wrong, and this year would be not nearly as bad.

"Another thing that I have noticed that would correlate well with MRLS in Kentucky, is that this year we've come up with an inordinate number of mares that were checked in-foal, but have come up open. That is something that we don't see very often, we've had quite a number of them compared to other years."

From those mares that aborted in 2003, and from the diagnostics, Feigner did not see echogenic material or evidence of similar infection to those foals noted as ill in Kentucky. "I did not see any of that, or such similarity. From the few samples submitted, the WSU Diagnostic Lab essentially came up with "nothing," which I understand is pretty typical of MRLS."

Caterpillars everywhere

"On a lot of these farms in Kitsap County, caterpillars were floating within the water troughs," says Feigner. "Even when I suspected that there was something going on (in relation to the caterpillars), I had these mares muzzled so they would not ingest the insects as they drank, that didn't seem to make a difference. They could leach out into the water, but what happened was hard to determine or to know."

Last year this infestation was so uniquely located in Kitsap County, that those people that bred their mares there and took there mares back home where they didn't have the problem, their mares had their babies normally.

It's a potential I guess, says Dr. Bud Hallowell, DVM, equine reproductive specialist, "but I haven't heard of anyone around here (Auburn) having any problems, any mares aborting this year."

"A former client of mine did have a few abortions last winter, at about 9 months or so, though they seemed to be random and unrelated, as much as we could tell.

Though she thought it wasn't MRLS, she did come up with an interesting observation. Some of those affected here in Washington were mares that were previously affected with MRLS in Kentucky, and had lost foals the previous year. Those affected were found in different fields, and it was curious that some of them were mares that were previously affected with MRLS, even though some of them had not gone back to Kentucky and were therefore not exposed to that environment.

She thought it might be a residual affect from the previous exposure and resultant MRLS that might be lingering. It was total speculation that they might be related to their previous bout of MRLS."

Worm in the ointment

Back in the Bluegrass that fateful May, the University of Kentucky initiated a strategic plan within the entire equine industry- veterinarians, horse farm managers and scientists from several disciplines were brought together to determine what had plundered Thoroughbred and other horse farms throughout the region. Since that time with dedication and determination, this stalwart group made incredible progress.

During the past three-year investigation several potential suspects were called into question including the possible pasture toxins - Fusarium mycotoxins, zearalenone mycotoxin, ergot and loline alkaloids in the general pasture foliage and in tall fescue specifically, phytoestrogens and cyanide in white clover, nitrates in representative forage samples, poison Hemlock and cyanide from the black cherry tree leaves. Even ethylene glycol dropped from airliners flying overhead and emptying their bathrooms was considered.

Enemy # 1

The true culprit was not found in the growth of the pastures, but turned out to be a small invader to the trees surrounding them. The Eastern Tent Caterpillar became known to be horse enemy #1 in relation to MRLS.

The first inkling of the involvement of the caterpillars was their heavy infestation, where astride many of the pastures was the wild back cherry tree, the ETC's primary host tree.

Looking back at the calendar, it was noted that the caterpillars had stripped the trees by April 25th . This contamination date correlated well with the first episodes of fetal loss.

Only the delivery to the mare remained unclear. The working theory was that the horses consumed the caterpillars and their excrement, but how MRLS developed from consumed insects was still a mystery. That began a more thorough look at the role these little pesky creatures play in the disease. During 2002 and 2003 experiments were conducted. From selective mare pastures seeded with caterpillars and their excrement, and mares fed various caterpillar body parts, fresh and preserved, a cause and effect relationship between the tiny bugs and lost foals was determined.

More investigation

In February 2002, Bruce Webb, Ph.D., entomologist, and Karen McDowell, Ph.D., Equine Reproduction, University of Kentucky, began to investigate the involvement of Eastern Tent Caterpillars and MRLS.

He began a study to manipulate exposure to ETC and environmental conditions as much as possible to reproduce the syndrome under experimental conditions and take measures that would identify one of more proximate causal agents. To mimic conditions that occurred on the farms in Spring 2001, 29 pregnant mares were placed in each of three adjacent paddocks, 20 feet from each other. One pasture was seeded with caterpillars, the second with caterpillar frass (waste), the third was the control, not contaminated. A physical barrier was placed around the field with the caterpillars to keep the mobile little creatures contained.

In addition to the experiment, sentinel farms were monitored for the emergence of caterpillars. As of mid-March 2002, not only did the Easter bunny begin delivering eggs, the Eastern Tent Caterpillar egg-hatch began. This also began the University of Kentucky's close watch on the effect of ETC's on horses in the central Kentucky, and the farms' diligent program to eradicated the bugs.

Friday, April 26, 2002, similar in time to the previous year, the University of Kentucky Equine Management Advisory noted the beginning of the movement of Eastern Tent Caterpillars. The "worms were beginning to turn," as ETC's were crawling about looking for a sight to pupate. This meant that for two to three weeks the caterpillars would stray as far as 100 feet or more from their nesting trees, depositing waste as they migrated, and accumulating in large numbers on adjacent trees, fence posts and barn walls. University of Kentucky entomologists noted the importance of eradication at this time to limit their potential damage, and recommended several insecticide programs and physical barriers to halt the movement of the insects.

By May 1, 2002, just five days prior to the 128th running of the Derby, results came in from the LDDC-Veterinary Science experimental caterpillar field study. The results were consistent with caterpillars playing a role in the cause of MRLS. Fifteen mares on two treated and one control pasture showed evidence of MRLS. Foals were lost to six of 10 mares on the caterpillar seeded pasture, six of 9 mares on the caterpillar frass seeded pasture, and three of 10 in the control, unseeded pasture. The study was quite conclusive to the relationship of caterpillars to MRLS, though a few mares in the control pasture lost their foals to MRLS. A breach in the physical barrier, which resulted in caterpillar contamination of that field was determined. As a result, the University of Kentucky Department of Agriculture continued to recommend that exposure of pregnant mares to ETC be controlled.

The other bugs

Another piece to the puzzle was noted by Dr. Stuart Brown, DVM, Hagyard-Davidson-McGee Associates, PLLC. "The one thing that I found to be particularly striking in a lot of the mares was these alpha-strep bacteria," Brown explains. Nothing in particular caused him to look for them. It's a routine part of a protocol for working up a mare that has aborted at anytime. They do cell cultures and cytology on them. About 65 percent of the mares Brown examined (of about 300 mares that were affected with MRLS) had grown alpha strep organisms, probably of three to seven different species. Dr. Mike Donahue (University of Kentucky Livestock Disease Diagnostic Center) was growing a large number of alpha streps and also

Actinobacilli

species. "I find it to be a very striking correlation, that he (Donahue) comes up with 70 percent in late fetal losses from approximately 500 cases he had, and I came up with 65 percent in the 300 or so cases I had," Brown says. He would have initially thought that they were primarily opportunistic organisms. That was very much the feeling, Day 1, when they had the problem, because they found these alpha strep organisms in a lot of fetuses at that time. People thought they were probably just part of the mare's normal flora and they were just secondary invaders.

Brown questioned that, but was not sure.

Thinking about how pathophysiologically that organism would have gotten into those mares, it didn't follow too well for him that this was an ascending infection, from outside the reproductive tract down from the vulva. Brown pondered how they got there. How did they come to be grown in such prominence in these mares? It wasn't that they were super pathogens, but if they didn't know what they were, or where they normally resided in the horse, it drove them to identify the alpha streps and the actinobacilli as a prominent finding of the clinical syndrome. In a paper presented at the 1st Workshop on Mare Reproductive Loss Syndrome (2002), Dr. Mike Donahue concluded that non-beta hemolytic streptococci (alpha-streps) and actinobacilli, two groups of bacteria not normally considered to be important causes of abortion in mares, were recovered from most of the fetuses associated with MRLS.

Two bugs together

As of May 2004, "as far as we're concerned caterpillars caused MRLS, from our research and the research of others," Webb states. "The efforts we have taken to control caterpillars have prevented MRLS in the last year or two. We have not seen any this year. We don't know if we are going to see any."

The caterpillars and the bacteria are related parts of the same syndrome. "The results from our last summer's work suggest that the most likely cause of the disease was the physical structure associated with the hairs of the caterpillar," Webb suggests. They dissected thousands of insects into three fractions - the gut, the internal tissues and the cuticle. Only the cuticle causes abortions. They made changes to the structure of the cuticle. They've attempted to extract compounds off of the cuticle. Organic and aqueous extractions have essentially no effect on the abortionagenic activity. If you pulverize the cuticle, you see reduced activity.

Co-investigators McDowell and Dr. Neil Williams, led a study showing that similar abortions can be caused in pigs by feeding them tent caterpillars. They have necropsied pigs that were fed tent caterpillars, and have identified a lesion that occurs throughout the alimentary canal (gut). They call these microgranuloma lesions. At the center of each of those lesions is found a tent caterpillar hair or setae. "We think that the setae are punching holes in the alimentary canal, to allow bacteria to reach the bloodstream," Webb says. The bacteria establish a septicemic infection. They pass through the bloodstream to the reproductive tissue, and you get bacterial replication as the most likely cause of the disease. We think that these alpha streps (as seen by Brown and Donahue) are coming from the mare's gut via this mechanism he adds. "They are normal commensal organisms. We think that the hairs are actively 'opening the door' to let the infection proceed."

"We haven't nailed it down," Webb says. "It still is a hypothesis, but certainly we know that the cuticle is involved. We know that the microgranuloma lesions occur, and we know that the bacteria are in the gut and are somehow passing through the bloodstream to establish infection in the fetal fluids and placenta. We haven't shown formally that those infections cause abortions, and that's where are work is headed over the next six months or so," he says.

The initial cyanide theory has been discounted. It has been discounted both by the equine science department, where they tried to introduce cyanide to try to cause abortions, and in experiments done on the tent caterpillars to show that the cyanide is actually metabolized and rendered harmless.

"It is curious what you are seeing in western Washington, I don't know the Western Tent Caterpillar, but I understand that they are sibling species, so that they would have a lot of similarities," Webb explains. It is possible that in western Washington infestation of WTC's may potentially cause MRLS.

Farm management

After the devastation of spring 2001, several drastic measures were taken by Kentucky farm managers to control the caterpillars and mares exposure to them in 2002. These measures included spraying trees with pesticides, eliminating cherry and crab-apple trees, manual removal of ETC eggs and tents, tree injection to make the leaves toxic to the ETC, keeping mares off pastures adjacent to cherry trees (the primary ETC host tree), keeping mares stalled at night, and limiting mares pasture exposure during the daytime.

According to Wolbert, for western Washington, the best control method is a bacterial insecticide, Bacillus thuringiensis , a non-toxic spray to be used around the immediate area of small or juvenile tents.

In Kitsap County, especially in 2003, though there was a tremendous population of caterpillars inhabiting several trees (alders, birch, apple and ornamental cherry), there was a significant reduction in numbers somewhere between the caterpillar and metamorphosis to the moth. Certain wasps are the natural predator of the WTC. The eggs by wasps of the Eulophidae family and the mature larvae are parasitized by wasps in the Braconidae family. These have seemed to help in the control of the WTC.

Wolbert advises farm managers to prune alders and other trees that overhang the pastures, have trees sprayed when they initially see the tents, remove and burn tents from trees, and monitor areas within their pastures to remove accumulating numbers of caterpillars, especially from water troughs and buckets. Another caterpillar control method is to paint the trunks of trees that border pastures with Tree Tanglefoot® Pest Barrier (The Tanglefoot Co., Grand Rapids MI), a sticky substance that entraps migrating caterpillars (once caterpillars strip foliage from a tree they migrate significant distances searching for alternative food sources).

According to Wolbert, most area veterinarians believed the contamination of caterpillars to horses occurred from those floating at the surface of water troughs. Though not the natural choice of grazing horses to eat caterpillars, though where they were extremely plentiful, consumption was almost inevitable. Similar explanation was offered in Kentucky. It would be prudent of farm managers to remove accumulations of caterpillars that would be in the areas where horses would be forced to consume them with food or water.

According to Wolbert, infestation in 2003 and 2004 in parts of Pierce County and Kitsap County was extreme. Elaine Anderson, Washington State University, Master Gardner Program Coordinator for King County, confirmed that 2003 was an exceptionally bad year for caterpillars, and predicted that 2004 would be a similar occurrence. By June, the movement and infestation of the caterpillars is finished.

Epilogue

It is really amazing how quickly this MRLS puzzle has been solved. Looking into the future the scientists are hoping that this is going to tell them something more about equine immunology. It may be relative in other contexts. The question is certainly whether the other insects, other similar insults to the digestive tract may cause similar problems. These are questions worth thinking about.

Whether this occurrence in western Washington is a duplicate of what occurred in Central Kentucky is open to speculation, but with a significant infestation of Western Tent Caterpillars I would suggest that the possibility is a relatively good one.

In Kentucky they are also looking at gypsy moths, which are basically on the borders of Kentucky at this point. If it is the physical structure of the hairs that is causing the abortions, then you have to suspect other hairy caterpillars to have a similar effect. They have done a few things with gypsy moths, but up to this time do not have any evidence that there is an exactly parallel problem.

It is certainly curious that horses eat things that have greater texture and coarseness than Tent Caterpillar hairs, but they do not cause such problems. Is there something unique about those hairs or cuticle that creates the problem? As far as anything that would be an irritant to the gut wall, it is curious that the fiber of hay and grain does not cause a similar problem. It is obvious that the horse's diet is of such material but it begs the question of something peculiar about the caterpillar setae that causes the lesions.