Combined Immunodefiency in Arabians
A newborn Arabian foal is possibly one of the most beguiling creatures on Earth. Elegant of feature and blessed with beauty, he's full of promise for the future--and often descended of royal blood and worth many thousands of dollars. Imagine how crushing it is for a breeder to discover that a seemingly healthy foal has inexplicably died from what, for most young horses, would be a fairly routine and minor respiratory infection...or even from a wound with a bit of dirt in it. What went wrong?
In the case of the Arabian, there's a good possibility that what went wrong was CID--combined immunodeficiency. First described in 1973 by Travis McGuire, DVM, and Marinel Poppy, DVM, of Washington State University, CID is a genetic disorder that results in a defective immune system--specifically, the inability of the horse to produce functional T- and B-lymphocytes (specific types of white blood cells). Without the defense against disease that these lymphocytes provide, a foal is completely vulnerable to infection from the millions upon millions of disease-causing organisms he encounters every day in the soil, water, and air.
A Foal With No Defenses
A CID foal appears perfectly normal at birth, and might remain symptom-free for weeks or months. The reason for this is that he absorbs a temporary passive immunity from his dam's colostrum. But once this passive immunity has worn off, the foal has no chance of survival against the everyday onslaught of infectious agents in his environment. Respiratory disease is the most common killer, but practically any sort of infection eventually will be fatal--usually at an age ranging from 15 days to about four months (with three to four weeks being most common). Sometimes CID foals respond to antibiotic treatment at first, but eventually they succumb to an infection for which antibiotics are ineffective. Because CID foals are unable to launch a normal immune response, vaccination is of no benefit.
CID foals have an extremely low count of circulating white blood cells, and the defect can be confirmed in a newborn by testing for the presence of IgM antibodies in the blood. A normal foal will have low circulating levels of IgM in the blood, but a CID foal will have no IgM at all. If blood is drawn, however, it must be done before the foal nurses for the first time; after he ingests his dam's colos-trum, the test becomes meaningless, as IgM will be absorbed from the colostrum and the levels in the foal's blood normalized. In practice, of course, it's difficult to get the test done in this limited time frame.
CID also can be diagnosed at necroscopy, since affected foals have extremely underdeveloped thymus glands. Located in the chest area, the thymus ordinarily shrinks and disappears with maturity, so the younger the foal, the more significant a finding of an atrophied or abnormally small thymus. CID foals also have unusually small spleens, and the splenic tissue will show abnormal development when examined microscopically.
Circumstantial evidence is the most common method of diagnosing CID. Affected foals invariably sicken and die while quite young, and matings that repeatedly produce foals which die this way are immediately suspect. However, it's important to remember that more ordinary problems, such as a lack of absorption of passive immunity from the colostrum, also can be at fault for foal mortality--and that, overall, horses tend to suffer high losses of their young. Before making accusations as to pedigrees, these other possibilities should be considered.
Currently, there is no treatment for CID. A similar immune defect which exists in humans--the "bubble boy" syndrome that is reported periodically in the media--can be treated with bone marrow transplants. But in order for such a technique to work in a horse, the animal needs a functioning thymus gland to help the donor-derived lymphocytes mature normally, and that thymus gland is lacking in a CID foal. Thymus transplants have had limited success, sometimes prolonging life to the age of about eight months. The rejection of donor cells remains a problem without a solution.
Lance Perryman, DVM, PhD (formerly of Washington State University-Pullman, and now at the College of Veterinary Medicine at North Carolina State University), who has researched CID for some 20 years, says that his research team did achieve one successful bone marrow transplant on a CID foal. The horse lived to the age of five years, when he suddenly died from colic unrelated to his genetic disorder.
"It was a real heartbreaker," says Perryman, "but the bone marrow transplant wasn't really a viable solution in any case, since the horse was still genetically coded for CID and could never have been used for breeding."
The Root of the Problem
CID is caused by an inherited autosomal recessive gene--meaning it's carried by both sexes, and the foal must be homozygous (carry two copies of the defective gene, one from each parent) in order for it to be affected. Horses with only one copy of the CID gene (heterozygous) are called carriers, and outwardly, they're completely normal. Until very recently, the only way to detect whether a mare or stallion was a carrier of the gene was for them to produce a CID foal.
It was estimated in the 1970s that as much as 25-30% of the population of Arabian horses in the United States were carriers of one gene for the CID defect, although Ralph Clark, of the Arabian Horse Registry, notes that the number might have been generous, since cooperation in that early study was voluntary and most likely to be of interest to those with known CID horses in their herds. Even if that number is high, however, it's sobering to realize that each heterozygous horse with the CID defect has a 50-50 chance of passing the defect--and when bred to another carrier, has a one in four chance of producing a foal with no immunities. About 3% of Arabian foals born every year inherit the two recessive genes and never make it to weaning--a significant toll on the Arabian breeding industry, especially since the death of an eagerly awaited foal might also mean the end of a breeding career for a promising stallion and broodmare.
Forms of combined immunodeficiency have been identified in humans, mice, and dogs as well as horses, although they are not necessarily identical from a genetic standpoint. The equine form of CID is thought to occur only in Arabians and part-Arabians. What's not known is whether the defect is a result of a fairly recent mutation in the breed (as is HYPP in Quarter Horses), or whether it has existed, without a name or explanation, for many years. Katheryn Meek, DVM, a researcher at the University of Texas Southwestern medical center, notes that it is possible that CID does exist, at some very low level, in other breeds.
"So many other breeds derive from the Arabian that it's possible CID has made its way into these populations, too, but at a much lower gene frequency. If only one out of four is a lethal homozygote, and the gene frequency is very low, CID could be out there and not identified as such."
In other words, foals of other breeds might (rarely) succumb to CID, and because veterinarians associate the abnormality exclusively with Arabians, it might go undetected.
Because there is no cure for CID, the only way to prevent the recurrence of affected foals is to avoid breeding known carriers to known carriers. Certainly the owner of a mare who has produced a CID foal should never send that mare back to the same stallion; and the cautious course of action for the stallion owner would be to geld the horse. Screening carrier horses out of Arabian herds has been strictly voluntary, however, and the degree of cooperation has varied. Most conscientious breeders refuse to breed known carriers of the CID gene, but without a simple method of detecting the defect until after the fact, many "slip through the cracks" and could produce several healthy foals before a mating produces a fatal result.
Developing a Test
With funding from the Morris Animal Foundation and the International Arabian Horse Association, as well as the National Institute of Health, researchers have been working on developing a reliable test for the CID gene for almost 20 years. The experimentation often has taken its lead from human research, where similar immune system defects have yielded clues as to how CID might work. Comparisons with DNA sampled from mice with a form of CID also have been pivotal.
One form of human CID, in which the CID gene interferes with purine metabolism and causes an abnormally high accumulation of uric acid in the serum, looked promising, and Perryman did discover that the horses in the CID-carrier herd maintained at WSU also had elevated uric acid levels--but that the elevated levels were not consistent from horse to horse. Further work demonstrated that the enzyme ADA, which is defective in some cases of human SCID (severe combined immunodeficiency), was not defective in CID foals.
Information like this brought researchers significantly closer to identifying the particular genetic marker for CID. The breakthrough really came in 1995, when a team of researchers led by Katheryn Meek, DVM (in collaboration with Perryman), determined that the process which is defective in CID foals is VDJ rearrangement.
"My particular area of interest," says Meek, "is how DNA regulates the immune response for many different antigens. It happens through a process called VDJ rearrangement (V, D, and J being gene segments in the DNA)--a clever strategy which allows lymphocytes to produce specific antibodies to many different antigens. There are many more antibodies than there are genes, so it's impossible for a single gene to be responsible for producing a single antigen. Instead, as it turns out, the V, D, and J segments can be 'mixed and matched,' so that every different combination of V, D, and J gene segments makes a different antibody."
This, in essence, allows the immune system to come up with an infinite number of responses for any type of foreign invader it might encounter.
"If the VDJ rearrangement is impaired (through a mutation or deletion)," Meek continues, "the immune system does not develop. And we know that the VDJ doesn't work in these horses."
Because CID is an autosomal recessive trait, they hoped that they would find that a single gene was responsible for the problem...but there were no guarantees, and a large area to cover.
"It's like looking for a needle, not just in one haystack, but in many haystacks," explains Perryman. "At first, you don't even know where to begin. But as you gather more information, you can at least determine in which haystack you should be searching, and further research is like giving you a powerful magnet to help in the search. Eventually, once you have the location narrowed down, you're going to find that needle...but it's still going to be hard work."
Finally in 1995, Meek's team determined through a series of biochemical tests that the factor defective in CID foals is an enzyme called DNA-PKCS. With this important starting point in place, and through a labor-intensive process of cloning and "amplifying" DNA taken from fibroblast cells of a CID foal, and a technique called polymerase chain reaction (PCR), Euy Kyun Shin, a scientist in Meek's laboratory, was able to sequence roughly 12,000 nucleotides of DNA from both the normal and mutant DNA-PKCS genes. The process took about 11 months of hard lab work, but in the end, it bore fruit, when a five base-pair deletion was identified in the summer of 1996.
Meek confirms that every subsequent sample that was tested (taken from known CID foals and CID carriers in a herd of Arabians maintained by Washington State University) had the same defect. "The result has been the same--they all had the same deletion (of base pairs)."
The next step was to try to identify horses suspected of carrying the CID defect. Using a DNA sample from the "mystery" horse, the same PCR procedure was used to amplify (copy) the gene in question in a test tube. Once sufficient DNA was available, the DNA spanning the defective area was screened to determine whether or not the animal harbored the five base-pair deletion in either of its two copies of DNA-PKCS.
Thus a reliable test for CID was finally born, more than two decades after the syndrome was first described.
"Once you have the mutation," says Meek, "it's easy to develop the test."
Perryman notes, "It was very important to me to hand this solution to the Arabian industry, especially with all the support they have shown for the research. We've been working towards this for about 20 years, so I feel great about having accomplished this."
Testing Your Herd
Bidding for commercial production of the DNA test was vigorous, but it was VetGen Laboratories of Ann Arbor, Mich., which was awarded the contract.
VetGen, founded in 1995 by a team of geneticists from the University of Michigan and Michigan State University, until now has focused its attention on genetic disorders in dogs. (Among its many current research projects is the search for a reliable DNA test for canine hip dysplasia, a disorder which affects millions of large-breed dogs.) The impetus to bid on a test for horses came from staff members Jeff and Richard Sloan, who have been active in the Arabian horse industry for many years, and who recognized the importance of the CID test.
VetGen president and CEO John Duffendack says his company designed the CID test (available to the public since the beginning of June, 1997) to be simple for either the veterinarian or the breeder to perform. DNA can be tested either by isolating leukocytes from a blood sample, or from a cheek swab, the method used for many of VetGen's canine tests. A swab with a plastic handle and very small bristles on one end (provided in the test kit), is "twirled" between the horse's cheek and gum to pick up surface cells, then shipped to VetGen's labs (cheek cells are apparently very stable, which allows the genetic material to be shipped by regular mail, with no special requirements). Once the cellular material arrives at VetGen, it is analyzed through what Duffendack calls a "fairly labor-intensive procedure including DNA extraction, followed by PCR, and the use of restriction enzymes."
Gel electrophoresis is used to read the results of the test, which are "absolutely reliable," says Duffendack. "You get one of three results--affected (with CID), carrier, or clear (of the defect). There are controls built into the testing process so that you won't get false negatives or positives." The turnaround time on a single sample is approximately two weeks.
The cost of the CID test is not inconsequential--a single test runs approximately $180, although VetGen offers "herd discounts" that make it somewhat more economical for a breeder to test an entire herd. Two to six horses can be tested for $150 per horse; seven to 15 horses for $125 per horse; and more than 16 tests cost about $99 per horse.
"The idea," says Duffendack, "is to provide an encouragement for breeders to test as many horses as possible."
Although Ralph Clark at the Arabian Horse Registry expects that the price of the test will discourage some breeders, he also notes that for those who have suffered the loss of a foal, the cost might, by comparison, seem trivial.
The CID test can be ordered through your veterinarian or directly from VetGen. Call 1-800-4VetGen (1-800-483-8436) for ordering information, or check their Website at http://www.VetGen.com. Because the shipping of samples is relatively straightforward, the CID test is available internationally as well as in the United States.
Duffendack notes several advantages to routine CID testing.
"The most important thing is that you can selectively breed out the defective gene, gradually, without affecting the breed as a whole. And when you know the status of your horse, you don't have to fear the results of breeding. Carriers can now be bred with the confidence of never producing an affected foal. You can therefore make optimal use of their bloodlines."
In addition, he says, breeders might want to test foals which are "doing poorly," to see whether the problem stems from CID, or if, for example, the foal has contracted pneumonia, but is immunologically sound.
"It can help the breeder determine whether it's worth treating the infection aggressively." CID analysis could also become part of a routine pre-purchase examination for Arabians, with the certification of the test results becoming useful in establishing a fair purchase price for the horse.
Overall, Duffendack notes, "The Arab industry has been very positive (about the development of the test). I think it eliminates the uncertainty and the fear that has been lurking behind the scenes for a long time."
"The idea (of the test)," agrees Meek, "is to be able to check all the breeding stock--both mares and stallions--and eventually, with careful breeding based on the results, eliminate the CID trait from the herd."
She expects that some breeders might be hesitant, particularly with valuable animals with good potential in the breeding shed.
"I hope it's used to benefit the breed rather than wipe out an individual's breeding herd--that's not the intention."
Arabian breed associations still are wrestling with the implications of CID testing. Says Ralph Clark, "We still have to determine how to best introduce the test to the membership and come up with recommendations for how to best implement it. I would doubt that the test is ever going to be made mandatory...but our committee (to discuss the subject of CID testing) was only appointed in the middle of May, so nothing's been decided yet. If the price remains high, it's less likely that there will be widespread screening, especially as the breeding industry's a little depressed right now."
Perryman is quick to express his gratitude to the Arabian industry for its financial support of his research over the years--an indication, he says, of the importance breeders place on this test.
"I am hoping," he says, "that people will employ this as a useful tool in their management decisions, both in terms of breeding contracts and buying or selling. I don't think it has to be legislated to be useful.
"Of course, the solution is to prevent the production of CID foals--which we can now do. If every breeder tested every mare and stallion before breeding, we could conceivably never again produce a CID foal. The tool now exists to do that. And as a secondary goal, we can decrease the frequency of the defective gene in the gene pool, and eventually eliminate it."
Does development of this test give researchers a start in identifying other markers for genetic disorders in horses? Yes and no.
"In each case," says Perryman, "you need to identify the enzyme or protein that's defective. That's still a laborious biochemical process. Once you have the right protein, it's becoming straightforward to clone the sequence and locate the gene or genes that are responsible--it's hard work, but there is a protocol in place. The strategies are established now, but because there are so many haystacks, each disease represents a new challenge."
About the Author
Karen Briggs is the author of six books, including the recently updated Understanding Equine Nutrition as well as Understanding The Pony, both published by Eclipse Press. She's written a few thousand articles on subjects ranging from guttural pouch infections to how to compost your manure. She is also a Canadian certified riding coach, an equine nutritionist, and works in media relations for the harness racing industry. She lives with her band of off-the-track Thoroughbreds on a farm near Guelph, Ontario, and dabbles in eventing.
POLL: It's Derby Week!