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ureaA chemist will tell you that molecules changed the world.  The book, Molecules That Changed The World, by KC Nikolaou and T Montagnon will have you believing it by page 333. The synthesis of urea is credited as not only the earliest contribution to organic synthesis, “but as the single most important blow to the vestigial theory” held in the 1700’s. “Despite using the terms daily, chemists often forget that the classifications of inorganic and organic compounds originally arose from the theory of vitalism, which divided matter into two classes based upon the response of the material to the application of heat.” Plants burn, rocks don’t.

What was apparent, and explained by Nobel prize winner (1902) Emil Fischer, is that principles such as  asymmetry, the intrinsic importance of organic chemistry in understanding biological mechanisms, and the art of extracting, identifying, and synthesizing naturally occurring compounds and their analogues for medicinal purposes are important. These concepts have been the foundation of our understanding how to treat horses with equine protozoal myeloencephalitis and polyneuritis equi.  Our story is similar to the story of Aspirin, the most successful medication in history, because treatments we use are steeped in history, synthesis, and hormone chemistry.

Aspirin’s story is using the medicinal properties of a natural product that is optimized through subtle chemical manipulations, and began 3500 years ago.  Egyptian physicians advocated salicin, in herbal preparations of myrtle bark, as a remedy for rheumatism and back pain. Humans are not the only ones that seek this chemical from the bark of trees such as willow.  Supposedly, forest monkeys and apes nibble on the bark of these trees for pain relief.

The first ever clinical trial used pulverized, dried willow bark that was given in a tea or beer, to 50 patients, and published by Edward Stone in 1763. Advances came in 1828 when Johann Andreas Buchner removed tannins and other impurities obtaining a relatively pure sample which he called salicin.  Ten years passed until Raffaele Piria made the next advance by hydrolyzing salicin, splitting it into its sugar and phenolic components.  Later, he succeeded in oxidizing the hydroxymethyl group of the phenolic fragment to make salicylic acid.  In 1853 another chemist, Charles Frederic Gerhardt prepared acetylsalicylic acid and with that step, Aspirin was born. The best was yet to come.

In 1859 Hermann Kolbe synthesized Aspirin by heating the sodium salt of phenol in the presence of carbon dioxide under pressure and commercialized the process, laying the foundation for todays pharmaceutical industry. What came next is the realization that salicylic acid wasn’t a panacea because there were unpleasant side effects.  The effort was on to modify the chemical structure in order to obtain a derivative that might be devoid of the undesirable side effects such as foul taste, mucosal membrane irritation, vomiting, and ulcerations.aspirin

The breakthrough came in 1897 when a chemist at Bayer, Felix Hoffmann, synthesized acetylsalicylic acid. Bayer now owned the miracle drug they trademarked as Aspirin. Aspirin was used in experiments to determine the mode of action  increasing the knowledge of pain and inflammatory mechanisms. Prostaglandins were identified in 1935 and the cascade of biochemical reactions associated with inflammation were revealed. Finally, in 1971 three scientists linked the ability of Aspirin to inhibit a critical enzyme step in the prostaglandin pathway.

The protagonist Aspirin is heroic.  However, the story isn’t complete without considering the antagonist.  New research spawned the new generation of ‘super-analgesic’ drugs such as Celebrex and Vioxx that didn’t have the side effects of Aspirin.  Yet, unexpected side effects were noticed, like heart attacks and strokes with long term use.  The popular drug Vioxx, with $2.5 billion dollar sales, was removed from the market in 2003.  Isn't it ironic that Aspirin is frequently used to reduce the incidence of myocardial infarction and stroke? The positive effects of Aspirin on heart disease isn’t through the prostaglandin pathway, but by inhibition of another product from the arachidonic acid cascade, thromboxane A2, a hormone discovered in 1975.

Aspirin is a synthetic chemical derived from natures molecule that has profound effects on prostaglandin mediated inflammatory pathways and in an alternate pathway, inhibits the clotting cascade driven by a hormone. Our path, clinical trials using hormones and synthetic chemical-mimics to modulate pathologic inflammatory pathways (non-prostaglandin mediated) are similar to Aspirins story.  Presently, we are concerned with chemical modifications of molecules. As you can see from the work on Aspirin, there are unexpected effects with the slightest modification of a molecules structure.  Structural changes can be non-enzymatic (on the shelf) and enzymatic.  Non-enzymatic modifications are made by facilitating chemical breakdown products, this is done by manufacturing processes, storage conditions, or compounding.  Non-enzymatic modifications of a molecule can effect anti-inflammatory, inflammatory, and in some cases, no effect that depends on the modification. We know that a simple amino acid change, such as a valine, in a hormone can elicit profound effects on enzyme binding to its receptor and affect outcome of a treatment.  We are also looking a the effects of chemical modifications on hormone receptor binding. We will put all this together in a story and should have quite a story to tell.

vitamineThere are some things to consider when supplementing and testing  vitamin E levels in horses.  Why do it?   The need for supplementation probably stems from deficiencies that are associated with degenerative myeloencephalopathy, equine motor neuron disease, vitamin E-deficient myopathy, and nutritional myodegeneration—or a diagnosis of EPM (unproven).

You decided to test instead of supplementing because conditions that warrant supplementation are rare, supplements are  expensive, and over supplementation may not be benign.  There are high-performance liquid chromatography tests that are as expensive as they sound. There are also enzyme linked immunosorbent assays that are used to capture vitamin E (alpha-tocopherol) from body fluids.  The when and how samples are collected and handled are important to obtain accurate values from ELISA testing. There is a study funded by Kentucky Performance Products, KPP--the makers of Elevate, that determined the effects of feeding different formulations of their products on serum, CSF (cerebrospinal fluid) and muscle tissues.  They used liquid chromatography methods.

It is necessary to test to find out if the horse has a deficiency. How do you test for vitamin E in serum? When using the more common ELISA test on serum, the collected blood should be allowed to clot for 10-20 minutes at room temperature and then removed from the tube carefully without transferring any sediments.  The serum should be protected from light during storage or transport to the lab. When plasma is used, the sample should be collected in an EDTA or sodium citrate tube.  The plasma should be removed from the cells within 30 minutes and no sediment should be removed with the plasma. Samples should be tested within 5 days, stored cold., and protected from light.  Samples stored frozen can be tested within one month.  If samples are stored at –80 the test can be used within 60 days.  Hemolysis will change the results of the vitamin E test.   The time of feeding vitamin E, the horses diet, and other supplements that are added to the diet can affect the bioavailability of  vitamin E and its detection in body fluids.

Horses get natural vitamin E from green forage.  Horses that are confined to a stall or they are required to abstain from fresh grass, are at an increased risk to develop deficiencies.  The normal value of vitamin E in the horse is > 2 micrograms/ml.  A value less than 2 micrograms/ml would mean the horse was deficient in vitamin E. There is no information available for over supplementation and the effects of vitamin E toxicity in horses. Horses show individual variation in the ability to absorb vitamin E from supplements.

The bioavailability of vitamin E when supplemented is important.  Natural vitamin E is composed of one stereoisomer while synthetic vitamin E has several isomers, of these isomers only one is readily available to the horse. If the neurological disease is due to a deficiency of vitamin E in the central nervous system the synthetic acetate form of vitamin E has no impact on the CSF levels according to KPP. The KPP study claims the micellized water-dispensable form, a liquid, is as much as 6 times more bioavailable than other synthetic forms and allows a rapid rise in serum concentrations within 12 hours.  Levels of 10,000 IU/day can increase CSF levels within 2 weeks.  Likewise, serum levels decline rapidly after discontinuing supplementation.

The bottom line for supplementation is that horses without clinical signs of deficiency can be supplemented with a synthetic form at 10 IU/kg body weight per day and serum levels are expected to  increase after 47 days or so.

If neurological disease is present, a diagnosis of equine motor neuron disease or vitamin E deficient myopathy (diseases that are responsive to supplementation), are expected to respond to vitamin E.  A regime of supplementation of 5000 to 10,000 IU vitamin E given daily is the standard protocol.  Remember, the water dispersible form is more appropriate for these conditions when it is desirable to increase levels quickly.  There is a rapid decline in serum values when horses are removed from some forms of vitamin E supplementation and a tapering regime may be appropriate. Another point is that there is no correlation between serum and muscle levels of vitamin E. If the disease is vitamin E-deficient myopathy an alternate protocol may be required.

To summarize, if a horse is suspected of a low vitamin E level due to disease or diet, the serum and/or CSF should be tested.  The normal vitamin E value is >2 micrograms per ml of serum.  Abnormal values will be returned if the sample wasn’t collected and handled properly or if the sample was held too long, even if it was frozen. Light is detrimental to vitamin E in the serum and in supplements, samples need to be protected from inactivation as soon as they are collected.  Samples that contain microparticles or are hemolyzed will give an abnormal value.  It is important to select the most appropriate dose and formulation to achieve normal and sustained values when supplementing.  The rate of decline in the serum may depend on the form of the supplement that is given. And finally, there is an individual variation in the response to supplements so individual protocol should be designed to achieve a therapeutic response.

 

funny rat We predict that in the year 2050 there will be no animal experimentation.  That includes animal testing for drug licensing.  Today, 100 million animals are used in experiments, about 13% of those are involved in studies that are required to license drugs.

Happily, a large step toward the goal of providing alternatives to terminal animal studies was published recently.  The FDA commissioner Scott Gottlieb, MD, stated that the FDA is trying to reduce, replace, or refine the use of animals in research. This is heartening news. We hate the current standards for new drug development that require euthanizing animals in studies. Even if a drug is commonly used in other species—if a new indication is under investigation, terminal studies are required. We’ve repeatedly argued against this position.  We lost the debates. But FDA is not insensitive to the issue.

On the horizon are simulation models that include in vitro (non-animal) dissolution tests and computer modeling that will replace animals in some studies.   A first step to animal-alternative testing is the validation of simulation methods designed to show the equivalence of a proposed generic formulation to an approved drug. We can predict a future that allows these same models for new drug development.  Currently, FDA requires data to understand how a drug performs in a live animal.  In our case, mountains of data obtained from goats and dogs and cats won’t substitute for horses.

A part of the generic licensing process involves an animal drug developer to perform “bioequivalence studies”.  These live animal studies compare the originally approved product and a proposed generic version to see if they are similar enough to link them in terms of safety and effectiveness. These are expensive studies and these studies are ignored by compounders.  Another subject for another day.

Back to the good news. There are studies afoot at FDA that will create a physical model to simulate properties of a specific drug when tested in live animals. The proposed studies use dogs. The live animal data will validate the simulations for anti-parasite formulations that are currently used in dogs.  If the studies are successful, animal drug sponsors may use these data to aid in designing in vitro studies.  And that will save the lives of animals.  The initial studies used to equate the simulation to the live animal are designed to use a small number of anima.  The animals are subjected to minimally invasive procedures, perhaps as simple as a blood draw. And more good news, the lab-dogs will be adopted as pets.

The FDA is encouraging drug companies to develop and validate alternatives to animal testing, including in vitro dissolution tests and computer modeling. For now some drugs may still require live animal studies, and these studies may require euthanizing the test subjects to support a new animal drug. We’d like to get back to the future.  We developed a simulation method that is designed to test the equivalence of a proposed generic anti-protozoal medication against approved drugs.  As licensed EPM drugs age out of patents there will be an opportunity for licensed generics. We are refining an animal model that can prevent excessive horse use and will design and test the validation parameters.  I suspect that it will take a year or so to get the rubber stamp on a validated model.  We would like to be instrumental in the movement to decrease animal experimentation.

In January of 2017 CBS Evening News (Lee Cowan) reviewed a book by Doug Preston…The Lost City of the Monkey God.  I won’t spoil the story for you--it’s a must read if you are obsessed with parasitic protozoa as we are.

The Lost City is a captivating true story and reveals a curse bestowed on man.  Legend has it that this ancient and magnificent Lost City, located in the rain-forested mountains of Mosquita was cursed.  Mosquita is a 20,000 square mile section of Honduras and Nicaragua.  The inhabitants lived in the area between 2600 B.C. and 1800 B.C and somehow incurred the ire of the Gods.  The angry Gods brought on a series of catastrophes.  Diseased and devastated, defeated inhabitants melted into the jungle, leaving everything behind, perhaps to appease the Gods.  The Lost City was not Mayan, yet it existed at the same time as the Mayans. These prosperous civilizations vanished at the apex of their existence.

Since the Lost City was first reported by Cortés in 1526, explorers and charlatans searched for them in vain.  That is, until the invention of LIDR in 2010.  The modern three-dimensional radar technology enabled mapping of the ruins beneath the dense canopy. It wasn’t the ruins that revealed the curse--the explorers may be cursed.

If you ask bestselling author Doug Preston, he’ll say he doesn’t believe in curses. And yet, here he is, being treated for leishmaniasis that he contracted while on expedition to The City. Half of the expedition party have contracted leishmaniasis--I believe at least one member died and some are not able to get treatment. The parasite is transmitted by a sand fly, the bite releases the protozoa into the skin and causes lesions that may take years to heal--cutaneous leishmania. Effective treatment at this stage may be life-saving but there are no current satisfactory treatments for cutaneous leishmaniasis.

As disease progresses, the parasite migrates to the mucous membranes of the mouth and the nose, and eats them away; this is mucosal leishmaniasis. The nose falls off, the lips fall off, and eventually the face becomes a gigantic open sore.  Organisms move on to the the organs, and visceral leishmaniasis is often fatal. Visceral leishmania is the second leading cause of parasitic death worldwide. The treatment is rough because it poisons the patients organs.  Patients die from treatment complications of renal or liver failure. Dogs get leishmania and are often euthanized when the diagnosis is made. Horses get leishmania, two cases were reported in Florida. When you read the book, you will realize why the curse reached Western Civilizations. We lead lives that prevent us developing immunity to this developing 20th century threat.

We are invigorated because there are some novel studies to report, and they parallel our investigations.  A promising combination therapy showed a complete clinical cure in 75% of the patients (human) with cutaneous leishmaniasis! The study also reported that 10% of the patients had a partial improvement and alas, the remaining 15% had an underlying chronic diseases and they had no response to the treatment. There were no cytotoxic effects associated with the drugs in the range of the experiments.  The mechanism of action of the drugs predicted promotion of some cytokine gene expression levels and reduced others.  The experiments supported the anticipated changes in cytokines. This is great news because researchers are understanding how to target parasitic protozoal diseases by modulating the response to infection. This has been our mantra for years.

What gives us satisfaction is revealing that the patients were unresponsive to the anti-leishmania drug, Glucantime, but responded when when Glucantime was combined with the immune modulating drug levamisole. Here is some evidence that end-stage unresponsive parasitic protozoal disease in patients is treatable when combined with levamisole.  The absence of cytotoxicity in the treated patients when given the combination of drug/levamisole is also highly noteworthy.

In the last 100 pages of The Lost City, Doug Preston suggests the Curse may be the downfall of Western Civilization--a result of invading parties driving civilizations to extinction by not so unintended consequences and bringing back malady-in-kind.  While we doubt the impact of our studies with levamisole (in horses with EPM and polyneuritis equi) will save the world, we think our efforts are worthy. Support us in our research.  You never know where it will lead.

leishmanialeishmania 2

Leishmania parasites are shown in a host cell and in tissues.

Photos were taken from goedleven.be.

 

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.