Thought de jour: Preventing EPM

 

Shoestring, loved by everyone that knew him.

Can EPM be prevented with drugs? What are the unintended consequences of continued antiprotozoal therapy?  These are complex questions without simple answers. Equine protozoal myeloencephalitis is a syndrome that involves infection with pathogenic protozoa and, in some horses, the infection can cause neuroinflammation.  It is the inflamed neural tissue that causes  clinical signs.  How is it that few horses that are infected with S. neurona get EPM when the parasites can infect organs quickly?  The susceptible horses have an inflammatory response that goes to the central nervous system.

We were taught (Elitsur, 2007) that the lymph nodes of normal horses harbor parasites one day after ingesting infectious stages of S. neurona! The parasites move to the liver (day 2), lungs (day 5), and by day 7 neuroinflammation (but not parasites) can be detected in the brain and spinal cord of susceptible horses. Most horses have antibodies against S. neurona.  It takes a couple of weeks after infection to detect antibodies. A longstanding conundrum with S. neurona infections is that infection rarely results in EPM when EPM is defined as parasites in the brain or spinal cord.  The majority of horse studies established neuroinflammation associated with S. neurona infection as the cause of clinical disease. The presence of antibodies at the time of clinical signs tie the signs with the immune response to the parasites.  Infecting immunodeficient horses (SCID foals) can lead to a parasitemia and no clinical signs because these horses lack specific inflammation producing cells. Good evidence that the immune cells can transport parasites and produce molecules that result in clinical disease.  Immunocompetent horses quickly clear parasites but continue to have clinical signs.  Good evidence indicating that inflammation doesn’t resolve when parasites are removed.

Can preventing infection prevent neuroinflammation in the EPM susceptible horse?  Perhaps the first question relating to preventive therapy is how one decides if a horse is at risk for EPM?

Which horse do you select for prevention treatment? Very few horses get S. neurona in the brain and spinal cord.  Yet serological surveys show over 80% of horses in the US have been infected.  Our evidence that antibody against S. neurona does not indicate  protozoa made it into the central nervous system.  It is probably early immune responses that prevent parasites migrating into the central nervous system.  Antibodies indicate an immune response to S. neurona infection, and once produced antibodies can remain for many months after parasites are gone.

The immune responses initiated by infection thwart parasite migration and infection of the CNS.  However, in some horses the immune system causes clinical signs because inflammatory molecules enter the central nervous system.  Anti-protozoals won’t affect the clinical course of immune mediated neuroinflammation.  That said, the overwhelming scenario is that horses are clinically normal after infection.  Therefore, one must assume that the majority of horses produce immune reactions, in addition to antibodies, that are protective against disease.  A horse should have clinical signs as well as antibodies against protozoa to justify treatment with an antiprotozoal.  It only makes sense to treat prophylactically if an animal has a history of multiple infections.  It would not make sense to treat seropositive, clinically normal animals or an animal that has resolved EPM.

Some horses exhibit signs attributed to EPM but have other conditions.  Many of these horses are treated for EPM and are being treated for the wrong disease.  The unintended consequences of antiprotozoal treatment in a horse with another condition is worsening of the underlying condition and no response to treatment.

The horse that needs prevention therapy is one that has chronic relapsing disease, is seropositive after becoming seronegative post-treatment, and responded to anti-protozoal therapy.  The horse that needs prevention is one that other causes of disease have been ruled out.  There are several serum tests that can point out other neuroinflammatory conditions.  We find the CRP, Lyme, S. fayeri, MPP, and MP2 (anti-myelin protein antibody) tests useful.

There may be unintended consequences of prophylaxis.  Treating all S. neurona exposed horses may thwart protective immunity. In vivo experiments have shown that mice treated with 10 mg diclazuril/kg, before and continuing for 10 days after infection, did not develop protective immunity whereas mice treated with 1 mg of diclazuril/kg survived challenge exposure. Perhaps there is inefficient and incomplete removal of parasites. A chronic infection stimulates protective immunity.  The authors showed in vitro treatment of host cells with 10,000 nanograms of diclazuril,  used for 24 hours, did not kill all the parasites.  One thousand nanograms of diclazuril in similarly treated host cells resulted in killing only 31% of the parasites.  There were plenty of parasites left to infect an animal.

Ponazuril experiments had the same results. The dose required for protection and no relapse of infection in mice was 10 and 20 mg/kg per day before and for 10 days after exposure.  Although death was prevented in mice using 10 and 20 mg/kg ponazuril given 3 and 6 days after exposure, parasite DNA was found in the brains of these mice. You could expect these results because horse experiments showed that parasites were already in the lymph nodes, liver, and lungs by day 6!  Based on the mouse and horse experiments  treating a horse once every seven days makes no sense.  Also, take into account how long it takes to get effective drug levels into the animal.  It only took 1 day for the parasite to enter a cell willing to transport it to the lymph nodes.  Prophylaxis will require daily treatment and then it must be instituted before infection.

What about horses?  It was convincingly shown ten years ago that horses “treated with ponazuril at 5 mg/kg minimized, but does not eliminate infection and clinical signs of EPM in horses”.  These horses were treated 7 days before infection and continuing until 28 days after infection. However, seroconversion was significantly decreased in the treated horses.

Reducing antibodies against S. neurona does not equate to preventing EPM. A recent publication cites a reduction in antibodies to S. neurona in foals treated daily with diclazuril.  The foals were born to mares that had antibodies to S. neurona.  This is evidence that S. neurona is in the environment.  There was no correlation between the serum antibodies and disease on the farm.  Foals nor mares were subjected to CSF analysis.  The take home message was that daily treatment of foals (daily for 11 months) reduced serum antibody production by the foals.  This study doesn’t answer the question of antibody reduction and disease.

The studies that show the effectiveness of EPM-prevention by delaying or reducing antibodies against S. neurona requires giving anti-protozoal drugs to horses followed by S. neurona challenge. There have not been, and probably won’t be, published studies showing that diclazuril or ponazuril prevents EPM in a challenge model. The challenge studies show just the opposite, disease was not prevented.

What happens when triazine agents are given to infected horses, ones with EPM?  A paper in 2013 states “the results of in vitro and in vivo studies of triazine agents with various apicomplexan parasites clearly indicate that removal of triazines after appropriate treatment time results in regrowth of the parasites.  This suggests that although some stages are killed, other stages are inhibited and retain the ability to begin development again once the drug is removed.”  The author speculated that “the intact immune responses can likely remove most of the inhibited stages in cases of successful treatment.  In unsuccessful cases, relapse can occur because of failure of the removal of some of these stages by the drug or by (failure of) the immune system (to remove parasites).”

If antibodies are an indication of immune response to infection, what other protective immune responses are inhibited?  Is it possible to make horses susceptible by trying to prevent infections because a protective immune response is never initiated?

Another Prophylaxis study design is available.  It is possible to document recurrent disease in horses.  We look for antibodies to S. neurona in horses that also have clinical signs due to neuroinflammation.  The chronic cases are recognized by observing several “relapses”, usually over several years.  These horses may respond to antiprotozoal treatment.  In this study each animal must serve as its own control.  This was the approach used in studies conducted to support licensing Marquis and Protazil (FOI for each product) for the treatment of EPM so there should be no issues with study design.

In our Prophylaxis study the horses are identified by clinical exam, antibody testing, and response to treatment.  The main criteria for entrance to the Prophylaxis study is chronic relapsing disease due to EPM.  The selected animals receive  prophylaxis and then they are monitored over the course of a year.  The outcome variable, the measured response, is by veterinary exam.  The history of relapse and treatment responses are critical to make this study meaningful.  Our Prophylaxis study is ongoing and conducted by veterinarians that have these cases.  If you have an interest in this study contact us at Pathogenes.  We can tell you if your case qualifies.

Can overuse of antiprotozoal drugs lead to resistance? Treating horses empirically makes no sense. Drug resistance is a huge issue.  Is it a reasonable to assume that drug resistance will not be induced in an intermediate host?  The horse harbors the asexual stage of the parasite and selection of an organism due to resistance should not be possible.  Harboring a resistant strain is only an issue for that horse because the horse is a dead end host. However, when enough drug is in the environment, or inadvertently ingested by a definitive host, drug resistance becomes possible.  Wherever the definitive host sheds virulent drug-resistant strains of oocysts the susceptible horse will have no treatment options.

The methods used to select drug resistant strains in vitro are easily accomplished.  A small amount of drug is added to the culture.  Increasing amounts of drug are added until a resistant population remains. This sounds eerily similar to some treatment protocols used in the field for EPM.

Alternatively, drug resistance is identified by growth of the parasite in the face of treatment.  Using triazine drugs parasites regrow when the drug is removed.  This is due to stage related susceptibility to the drug, most likely not drug resistance.  To find a triazine resistant strain the organism would be isolated while the horse is being treated.  This is accomplished in a laboratory setting.

Thoughts de jourPreventing EPM makes sense in horses that have chronic relapsing disease due to protozoa and the disease is responsive to anti-protozoal therapy.  It is critical to identify these horses and limit treatment to this group.

It is counterproductive and expensive to treat all horses with drugs.  It boarders on harmful because this will foster resistance. It is important to monitor serum antibodies in horses with chronic, relapsing EPM.  It’s also important to monitor inflammation.

Can EPM be prevented?  We prevented EPM in a merozoite challenge model and related the protective response by a serum antibody titer (Ellison 2009). Yes, it can be done with a vaccine.  What we found was that vaccine effectiveness was strain specific.  Using very common antigens for vaccine is a way around strain specificity but opens the door for initiating inflammation.

Preventing disease with vaccine was based on inducing protective immunity. Can the same be done with drugs?  A study that first identifies horses with chronic relapsing EPM differentiating disease from other conditions that mimic EPM is critical.  Measuring disease over time and response to treatment will answer the questions in susceptible horses.  To us prevention only makes sense on a case-by-case basis.

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