Piroplasmosis Update
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by Heather Smith Thomas
Equine Piroplasmosis (EP), also called babesiosis, is a blood-borne parasitic infection in horses transmitted primarily by ticks. It is caused by two protozoa that live in red blood cells. It affects horses, donkeys, mules and zebras. The protozoa that can cause EP are Theileria equi and Babesia caballi.
Infected animals can be carriers of the disease agent for long periods of time, probably for life, and thus act as a source of infection to other equines. The protozoa in the blood of an infected animal can be transmitted by competent ticks or by transfer of blood to a non-infected animal.
This disease is endemic in many regions around the world, such as the Caribbean, South and Central America, Mexico, the Middle East, Africa, Europe and Asia. At the present time it is not endemic in Canada, Australia, Japan, England, Ireland, Iceland or the U.S. so efforts have been made to try to keep it out of these countries. Horses imported into the U.S. go through quarantine and testing to determine their infection status (since they might appear healthy but still be carriers of the disease agents that cause piroplasmosis) before allowing them to enter our country.
EP was recognized in the southeastern portion of the U.S. many decades ago. In 1962 a joint Veterinary Services and State of Florida control program for B. caballi was begun in south Florida to try to eradicate this disease. The program included quarantine and treatment of infected animals, spray treatment (to kill ticks) for infected and exposed animals, along with movement controls to prevent spread of this disease. As a result of this diligent program, the U.S. was declared free of EP in 1988.
To reduce the risk of reintroducing this disease, all imported horses had to be (and still are) tested for the presence of antibodies to T. equi and B. caballi before entering the U.S. The import testing is conducted by the National Veterinary Services Laboratories. In August 2005 the official import test was changed from the CFT to the c-ELISA test, which is more sensitive in detecting chronically infected animals.
Sporadic cases in which animals were found serologically positive for EP disease agents (but without clinical signs of the disease) have been found in the U.S. Most of these infected animals have been linked to previous imports from countries where EP is endemic—animals imported prior to the test change in 2005.
RECENT OUTBREAKS
Then in late 2008 and early 2009 two outbreaks occurred—in Florida and Missouri, respectively. These included horses with clinical signs due to infection with T. equi. The disease agent was found to have been transmitted through less than optimal hygiene practices, including re-use of the same needles on multiple horses, and blood doping among horses involved in unsanctioned racing. Regulatory actions resolved both outbreaks.
Dr. Mike Short, Equine Program Manager, Florida Department of Agriculture and Consumer Services, says that currently Florida is trying to educate horse owners about the risks of spreading EP by re-use of needles and other management practices. “The USDA produced a good one-page (front side English, back side Spanish) document to help educate horse owners. We tried to distribute this to folks we thought were higher risk—the horse owners who might do some racing or international movement of horses,” he says.
“In light of some of the things that have happened in recent years, after the outbreak here in Florida in 2008 (with subsequent euthanizing of apparently healthy horses), we are trying to educate horse owners. We’ve learned a lot about this disease—since the 2008 incident—regarding testing, and whether or not we can successfully treat horses, and how to know if they are cleared of the organism,” he says.
“Our outbreak here in 2008 was really the first incident where officials confirmed that we had some positive EP horses here in the U.S. We thought there might be a few chronic positives, but we didn’t know for sure. Even if there were some infected horses here, we didn’t know if they were transferring the disease to other horses,” says Short.
“Florida had a problem many years ago, in the 1960s, due to spread via a specific tick, but we haven’t found that tick here for quite awhile. During and after the 2008 incident, we did extensive tick surveillance with assistance from the University of Georgia Southeastern Cooperative Wildlife Disease Study. We are confident that it wasn’t a tick issue. As the investigation progressed and we learned more about the practices of these horse owners and trainers, we realized it was being spread by humans via ill-advised management practices,” he says.
“Unfortunately, there are horses racing on illegal tracks, doing weekend racing and community betting, with no oversight. We were amazed at some of the things they were doing to try to get their horses to win. Many of these practices, such as blood doping, were a perfect mechanism to transmit this organism. This is how we infect horses experimentally—take blood from an infected horse and give it to another horse,” he explains.
“The trainers who were doing these things had the highest number of positive horses, so we were confident that this was how EP was being spread. This was eye-opening for all of us, that this type of management was such a common practice and that the disease could spread so readily,” says Short.
“We think the start of the outbreak was one or possibly two infected horses, and it spread from there. It made us feel better, to know that the transmission wasn’t vector-borne, since it isn’t very easy to control ticks. It certainly made the equine community feel better, because they realized that if they were not doing those kinds of things (re-using needles between horses, etc.) their horses would be safe. This was much better than thinking their horses were at risk from native ticks,” he says.
The 2008 outbreak was finally resolved after much time and effort. “We tested more than 200 horses, some of them multiple times, and quarantined 25 premises. It took several months to follow all the leads and all the horses. Unfortunately for these horses, however, all the owners chose to euthanize the positive horses because it’s so difficult to maintain a permanent quarantine,” says Short.
Another group of EP-positive horses was discovered in October 2009 on the King Ranch in south Texas. In that particular situation, however, the diesease was spread by ticks. Due to certain weather conditions a few years previous, the tick population in southern Texas had proliferated. Ticks enabled the disease to spread amongst this group of horses, some of which probably brought the parasite from Brazil.
The good news in this situation is that a group involving state and federal agencies, researchers and veterinarians, and the ranch owners themselves, worked together in an experimental research treatment program. They eventually came up with a treatment that works to clear the parasite from carrier animals. This treatment involves multiple high doses of imidocarb diproponate.
At the time this group of horses was discovered, the U.S. regulatory agencies did not think this disease could be successfully treated to eliminate the organism. “We were telling horse owners that maybe someday this would be possible, but it might be years or maybe never,” says Short.
BREAKTHROUGH ON TREATMENT
After the 2009 outbreak in Texas, where a large number of horses were put into a treatment research program, a successful treatment was found. “This gave us the ability to do extensive research on EP. Dr. Don Knowles at ARS in Pullman, Washington, worked with those horses and a few others around the country. The lab at Pullman, in cooperation with the National Services Laboratories, did most of the recent research on this disease in the U.S. coming up with the current official c-ELISA test, and research with the confirmatory testing and treatment,” says Short.
“The c-ELISA test is doing a very good job now of screening imported horses and finding any positives. If we don’t have a competent vector (a tick that can readily spread it) here in the U.S. and people are educated and aware of the problem and stop doing unhealthy practices, there won’t be much risk. This is what we are trying to do now in Florida—educate horse owners who are racing or importing horses from different countries,” he says.
Dr. Don Knowles (Research Leader of the Animal Disease Research Unit, ARS-USDA, and Professor of Veterinary Microbiology and Pathology, Washington State University) was one of the researchers involved. “The late Dr. Ralph Knowles, who was a Chief Staff Veterinarian at USDA, was instrumental in the progress we’ve made. He worked for years trying to learn more about this disease. It is caused by two different parasites and he deserves the credit for coming up with the first use of imidocarb for treatment. Ralph had done a lot of work on this, but it still wasn’t clear (especially for the particular parasite that infected horses at the King Ranch) whether or not it actually rid them of the parasite. The data was inconsistent. The drug seemed to clear some horses, and not others,” says Knowles.
We needed a way to completely eliminate the parasite from a carrier animal so this pathogen could never be passed to another horse. “This is very important, for any exotic disease. We can’t take any chances of having EP spread from one horse to another by ticks or by needles,” says Knowles.
“Ralph worked diligently on this, and it was his research that we built on. Our advantage was that we had modern diagnostics and better technology for tracing infection when we went to Texas to deal with these horses. There was much discussion regarding whether imidocarb worked or not. There was no doubt that it worked for reducing clinical signs of the disease; the treated horses would recover and feel better because it killed the parasites.” But we didn’t know whether or not it would eliminate these parasites completely in the individual horse.
“The diagnostics available to Ralph at the time he was working on this were inadequate. But with his limited detection tools he did a great job. So we worked off his data, and with our modern detection tools–PCR tests, immunoblots and laboratory procedures–we could see that these horses were actually clearing the infection,” says Knowles.
Under natural conditions there are always some animals with weaker immune systems and some that resist treatment. “There are also some pathogens that resist treatment. So we were happy to see that this treatment actually worked, and that we could tell whether or not it did work,” he says.
“We have a publication describing the treatment and the first 25 horses in our project—and how we showed that they truly eliminated the infection. These horses are no longer a threat to other horses, and could probably come out of quarantine,” he says.
“The U.S. is considered free of this infection/disease so when it reappeared this was a new thing for the regulatory agencies, to figure out how to deal with horses that are already in the country and infected, as opposed to screening incoming horses to keep the disease out.” This treatment program is revolutionary because it proves that the horses can be cleared of EP, and would no longer need to be quarantined.
“When this new outbreak occurred and the decision was made to treat these horses instead of euthanize them, thanks to Ralph Knowles’ earlier work we didn’t have to reinvent the wheel. We just needed to fine-tune it a little,” he says.
“I’ve been with ARS my entire research career, and the reason I like working for ARS and collaboratively with Washington State University is that we get to work on practical problems that have an impact on society. We get to go right to where the rubber hits the road—things that matter for agriculture and animal health.” This breakthrough on EP will make a big difference for horse owners who have an infected animal.
THE TEXAS RANCH HORSES
Earlier, finding a horse positive for EP was a devastating situation, because the horse would have to be euthanized or quarantined for the rest of its life. The fact that horses can now be successfully treated is good news. “It’s a big deal emotionally for the owners, and for the genetics that won’t have to be sacrificed,” says Knowles. “The King Ranch is one of the largest cattle ranches in this country and they depend on their horses to run the ranch.” Their horses’ bloodlines are also highly valued by horse breeders around the country and around the world.
“This was a huge deal for the ranch, and they stepped up to the plate to help with our research. They enrolled many of their horses in the treatment program,” he says.
“In a way it was a ‘perfect storm’ for the King Ranch. They are located in an area that is a good habitat for ticks. In the years leading up to this discovery of infected horses, weather conditions were perfect for tick expansion, and for those ticks to be spreading the disease. Before this happened, we didn’t know that this particular tick was competent for transmitting the infection. Glen Scoles was the entomologist who figured it all out. It was a major detective effort,” says Knowles.
Dr. Angela Pelzel-McCluskey (Epidemiologist, USDA-APHIS Veterinary Services, Western Region) says that currently out of the 163 Texas ranch horses that were treated, all but 53 have gone c-ELISA negative. “They were treated in groups at different times. The length of time it takes a horse to go negative seems to be related to how long it has been infected. If we were to experimentally infect a horse and let him stay infected for several months and then treat him, that horse would probably go c-ELISA negative within 3 to 6 months. By comparison, a horse that’s been infected for years would take a lot longer to go negative after treatment,” she explains. It also depends on the immune status of the horse, and how well the body can assist the drug in getting rid of the organism.
“The younger horses on that ranch, that were only 2 or 3 years old when we treated them, had a limited time in which they could have been infected, compared to older horses. The young horses took anywhere from 4 to 11 months to go negative. By contrast, some of the horses on the ranch were in their twenties. I haven’t yet put all the data together to see which ones are still positive and what age they are,” she says.
“We’ve proven that there was infection on this ranch as far back as 1990 or longer. We have anecdotal information that there may have been some positives there in the 1980s, from some export testing. These horses were from specific bloodlines. Some people really like those particular bloodlines from this ranch and bought more horses every few years. So when we went looking for horses that were sold recently, I also found horses that were sold many years ago. This is how EP was dispersed, via movement of these horses to other locations,” says Pelzel-McCluskey.
“The King ranch has other ranches around the world, and at one time had a huge ranch in Brazil. They moved horses around their various ranches in the 1970s and 1980s, and this may be where the EP originated on their Texas ranch. We can’t prove this, but all the evidence points that direction, since EP is endemic in Brazil.” The disease may have been spread via horses moving back and forth.
“We have a couple tick species in south Texas that can propagate the organism. After many years, in this sort of environment, the ticks helped add to the infected horse population—and then it spreads faster and faster. In the early part of the past decade, we had some big tick explosions in south Texas because of climate and weather conditions.”
Horses that left the ranch before that big expansion in tick population were less likely to be EP positive. “We found very few positives in horses that left many years ago, compared with the horses that left the ranch between 2007 and 2009. A large percentage of those horses were infected, due to the role of ticks. We had a big problem with cattle fever ticks in south Texas at the same time,” she explains.
The Texas EP-positive horses posed a serious problem. “When it’s a population of high dollar Quarter Horses with unique bloodlines that are irreplaceable, you can’t just kill them all. You have to create another plan. This is why we went to long-term quarantine and enrollment in the treatment research program. This program expanded our knowledge of the disease and led to an effective treatment,” she says.
“A lot of credit goes to that ranch for investing hundreds of thousands of dollars into treating these horses. They didn’t treat all of them, however. They had to cull back their herd, making selection decisions regarding which ones to spend the money on, and euthanized more than 100 horses,” says Pelzel-McCluskey.
“One of the ways we knew the treatment was working was after we took 25 of the 170-plus that had been treated (and were PCR-negative and looking good) and transfused some of their blood into splenectomized ponies.” These were research animals that had their spleens removed and thus incapable of mounting any immune defense. They would certainly develop the disease if exposed. This was one way to determine that there was absolutely no risk of the treated horses spreading piroplasmosis.
“The receiving pony had no immunity, and even a single organism would have caused the disease. As part of that process, while we were researching this and doing the blood transfusions, we did have one horse out of the first group of 25 that we were going to transfuse that did not go PCR-negative. We thought the treatment didn’t work on that one. So to prove it, we transfused its blood into a pony and it did transmit the disease. That showed that the PCR test worked,” she says.
“Now that we’ve done all the horses, we’ve had a total of six that failed treatment. Their blood stayed PCR-positive and was capable of transmitting the disease. So we knew that our monitoring worked and that we could catch it if a horse did not respond to treatment. But we were wondering what happened with these six horses. Was the organism resistant to the drug, or did the horse’s body not help fight the disease? We don’t know what happened, but in the meantime while we were wondering whether we needed to try a new drug, or a larger dose, the private practitioner who was working with these horses re-treated them with the same dose, and cleared them. That’s when we discovered that multiple treatments could be effective.”
It was trial and error, but it worked. “We were really frustrated because there are only a handful of chemotherapeutic type drugs that might have some action on this organism, but getting enough of the drug into a horse without killing the horse is a hard challenge. In our research effort, we weren’t even considering repeating the same drug, but in the meantime the private practitioner got desperate and he treated them again with the same protocol and they cleared. We’ve followed them all after their second round of treatments and they’ve all cleared,” she says.
“Maybe their failure to clear the first time had something to do with the level of infection—the dose of that organism. We have a quantitative PCR that can tell us how many organisms per ml of blood are in that horse at any given time. When we take blood from infected horses, they can have different levels at any given time. Some might have almost undetectable levels; in some horses that we know are infected, one PCR test will be negative. It may depend on where we are drawing the tiny sample out of the blood tube at the lab, or how much organism the horse has, what the immune status is that day, and so on. This is why we use a series of PCR tests.”
The spleen is constantly trying to eliminate any red cells that have this organism attached. “In some cases the horse is knocking it back and keeping the disease under control and in other cases the organism is proliferating everywhere in the body. If we have horses with a high load of parasites and hit them with this imidocarb therapy, we wonder if we got them all or not,” she says. But by trial and error, the research came up with something that works.
It took a group effort from federal and state agencies, including the USDA-ARS (Agricultural Research Services, the research arm of the USDA) that had done many years of research on piroplasmosis, the State and Federal animal health officials drawing the samples and managing the quarantines, the NVSL (National Veterinary Services Laboratory) running the official testing, private industry requiring ongoing surveillance testing, and the ranch putting up money for the treatment. This is a tremendous success story. Without this unique situation in which the ranch chose to treat the horses, we would never have been able to make this progress.
“We also found a new competent tick vector (Amblyomma cajennense), and the Brazilians were very excited about that because they have this tick everywhere and didn’t think it was involved in transmission of EP. It was definitely involved in our situation in Texas. What the Brazilians didn’t realize is that only the adult male ticks moving between horses are the ones spreading the disease. This particular tick does not transmit the parasite to its eggs or larva, so this is a very different form of transmission than what we generally see with ticks,” she explains.
The Brazilians first thought this discovery was impossible because they had tested that tick many times. But they were looking at the disease being passed along through the life cycle of the tick. “They didn’t try to take an infected adult male tick to see if it would transmit the disease. The males are the ones that move between animals; the females simply attach and stay on the same animal. So in our experience with the Texas ranch horses, we found a new vector, and a new treatment that works,” she says.
