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Neurologic deficits are seen in horses with abnormal gaits, changes in behavior or signs that are limited to the cranial nerves.  These horses are difficult to diagnose because the list of etiologies that result in neurologic deficits is long. The list of disease-causing possibilities is long because animals have a short list of responses to infection and injury.

Long ago lowly slime molds and paramecia selected and perfected a group of chemicals that allowed them to achieve movement and the ability to communicate with other organisms. The chemical signaling mechanism that parasitic protozoa and animals use today is based on these evolutionary successes. Redundancy is built into the chemical structures and the way signals reach the target tissues. Redundant molecules also are used to turn off these systems. The basics of the pathways are common to all living things and they are primal.

Signaling pathways are also common to many tissues and they are ubiquitous throughout the body. An infectious agent will set off  a protective primal response in an animal. That response is called “innate immunity,” and this immunity results in signs that are non-specific to a single agent.  Vertebrates have many checks-and-balances regulatory pathways that control these common systems most of the time.  Sometimes the pathways become unregulated. As animals evolved to efficiently use the chemicals (cytokines) involved in protection against infections, parasitic microbes exploited the same systems. These highly successful disease-causing organisms use some pretty nifty  methods to evade or even hijack the innate immune signaling cascade.

The result is that an animal with neurologic disease has a limited repertoire of responses to several insults and they all look the same to the clinician.  We have accrued a vast amount of scientific knowledge about some diseases.  The diagnosis of these diseases is verified by  objective methods.  Some diseases have classically presenting signs that allow a veterinarian to easily rule them in or out. Of course, field experience is valuable in recognizing some classic signs. The diagnostician thins the list of possible causes of disease to the short list.

The short list for neurologic diseases that are found in horses often don’t have definitive diagnostics and that leads to using exclusion to help diagnose the cause. A diagnosis of exclusion  is a diagnosis of a medical condition reached by a process of elimination, which may be necessary if the presence cannot be established with complete confidence from the history, examination or testing. Such elimination of other reasonable possibilities is a major component in performing a differential diagnosis.

Diagnosis by exclusion tends to occur where scientific knowledge is scarce, specifically where the means to verify a diagnosis by an objective method is absent. As a specific diagnosis cannot be confirmed, a fall back position is to exclude that group of known causes that may cause a similar clinical presentation.  Polyneuritis equi (PE) is such a disease.  We have, along with our regulatory partners, put together a list of definitive tests and some that are exclusionary to pinpoint the tests most likely to give us the target population for our polyneuritis equi study.
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-1/
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-2/

The PE horse has evidence of neurologic disease that can be identified by neurological exam and those that are treatable have serum antibody against some specific proteins.  These are inclusionary criteria for our study.  The horse may or may not have antibodies against parasitic protozoa. To keep our study uncomplicated, we exclude horses with antibody against S. neurona, a parasitic protozoa that causes equine protozoal myeloencephalitis (EPM). Other exclusionary criteria are no recent history of trauma, no  recent respiratory infection (or a current vaccination for EHV-1 can satisfy this one).  Vaccination for rabies will exclude rabies as a cause.  Of course,  a horse would need to have a normal vitamin E level ( > 1.5 micrograms/ml serum).

Some treatments will exclude horses from our study.  They include anti-inflammatory agents within 3 days and anti-protozoal agents within a time frame that the anti-protozoal is expected to exert its effect.  Each expected effect from licensed anti-protozoal medication is different, some 30 days and some beyond 90 days.

Diagnostics are a major frustration for owners of horses with suspected EPM.  Owners spend many dollars on exclusionary diagnostics and don’t have an answer at the end of it.  Unfortunately, that is the nature of EPM. It is also the nature of Sarcocystis infections to trigger innate immune inflammatory cytokine responses that produce the signs associated with neurological diseases.  That means the horse can have two active pathological processes!  Because the nature of these diseases and the repair mechanisms that are associated with them use similar pathways, it is unlikely that there will be definitive diagnostics.  A good neurological examination by a veterinarian familiar with neurological disease and judicious use of diagnostics to form a short list  is the most successful path to health.
See: http://pathogenes.com/w/the-biology-of-sarcocystis/

crystal ballThe orbuculum, or crystal ball, was invented about 3000 BC according to Wikipedia.  Mystical orbs were used in numerous cultures to communicate with the gods or learn of future threats. Wouldn’t it be nice to identify horses that are genetically pre-disposed to get sarcocystosis?  A genetic EPM-crystal ball.

Thirteen years ago, we participated in studies that sought to identify cell markers unique to horses with equine protozoal myeloencephalitis, EPM. The idea was that cell markers or the “gene expression signature” unique to EPM would be found in immune cells circulating in the blood. The differences in gene expression between animals with and without clinical evidence of EPM would be analyzed using several statistical measures. Genes that showed statistically significant differences in clinically positive horses were compared to those that were clinically negative, and the genes that showed a significant difference (those significantly up-regulated) would constitute the EPM-gene signature.

Controlled laboratory studies testing the hypothesis that a gene signature could be found and  be useful in the diagnosis and treatment of EPM were undertaken. A controlled infection (induced stress) study was used to accurately know the day of exposure. Sarcocystis neurona oocysts were administered to 20 stressed horses to elicit disease. Blood samples were taken 10 times over 28 days to collect RNA, the measure of a turned on gene. The up-regulated genes (identified by the RNA analysis) were assayed on a custom microarray for determining gene expression (the specific array was patented, but not by us). In this blinded study (veterinarians didn’t know which horses were infected) clinical exams were performed,serum and CSF were tested, and post-mortem exams were conducted to ensure that clinically ill horses did get EPM.

This experiment was eventually published with the infection data, but the gene analysis data was not reported. Remarkably, the horses that showed signs didn’t have organisms that could be demonstrated in the brain tissues. Inflammation was considered diagnostic of successful infection. Scientists conducting this study identified a gene signature. Success! There were differences between the up-regulated genes in the clinically ill horses that were infected and those that were not infected, control horses.

Time to test the gene signature. Field cases of suspected EPM were used in a second study. The gene expression from horses with suspected EPM, those that had serum and CSF analysis to be as sure as possible the horses fit the diagnostic criteria at the time, came from clinical cases. It took 6 weeks to process the samples and get a result because the assay is technical. An obvious down side of the endeavor was cost. The hundreds of dollars that the eventual assay would cost, and the six-week turnaround time, made it a clinical non-starter. More importantly, the assay didn’t diagnose chronic disease (disease that was present after 28 days from the initial infection). The cases presented to veterinarians are chronic. The acute gene signature did not identify field cases that the veterinarians diagnosed.

In a third study, 13 animals were used in a merozoite challenge model that did not use stress. The horses were randomly assigned to a group, 8 were challenged while 5 were sham challenged. This study ran 90 days to detect an acute and chronic gene signature. Cells were assayed for gene expression at 28 days (acute) and 90 days (chronic). If the acute gene signature was the same in both models, an accurate marker between the two studies could be identified to identify acute, possibly current, disease. Likewise, the chronic markers, significant expression of genes at 90 days should match the field study and identify horses with long term disease even if the organisms were eliminated.

The cumulative results of the controlled studies identified 31 genes that were highly statistically different at day 28 between animals that developed clinical EPM and those that did not. An EPM index score calculated for the gene signature, identified in the first controlled study, was successfully used to identify some, but not all, of the horses with acute disease in the third, controlled study.

Horses with chronic EPM, day 90 of the third study, were not identified using the signature developed in the first study. Further, chronic EPM could not be identified in clinical field samples using the gene signature developed from acute disease, day 28, in either the stress model or the merozoite model. Because many horses present with suspect EPM after having had clinical signs for weeks or months, the value of a genetic signature was doubtful.

We identified drugs that selectively reduce the expression of some of the upregulated genes that were stimulated during acute and chronic disease. Some drugs returned horses to normal, removal of the drugs allowed the horse to again show signs of disease. Surprisingly, some drugs we tested made horses worse! We found that the innate immune response and the genetic signature of host cells are the key to disease associated with sarcocystosis.

The data was useful. The upregulated genes included MHC Class II receptors, chemokine receptors, IgG molecules, natural killer cells, several interferon-induced proteins and a handful of others.

More studies are undoubtedly in the pipeline. As those studies are completed and in a few years published, they may be compared to the work done in 2005. Or perhaps the EPM-gene signature is already relegated to the cutting room floor, the genes frozen in time, yielding space in the freezer for newer endeavors and lost to analysis. Our experience showed us there are disease-signatures present in the blood samples and these are markers that can effectively direct treatment for horses with disease and identify horses that are resistant to disease. We don’t think there will be a crystal ball that predicts which healthy horse will be come sick when exposed to Sarcocystis, sadly it isn’t that simple.

Horses with clinical signs of equine motor neuronprzewalski-1972728__340 disease (EMD) or vitamin E deficient myopathy require supplementation with vitamin E.  Horses that graze green grass should be fine without supplementation.  Supplements are expensive, here is a primer to guide you.

The critical factors associated with vitamin E are: determining that your horse is deficient (solution: test the serum levels); deciding what supplement is most appropriate (solution: determine what are you treating); delivering the dose efficiently to the horse (formulation and dose); and when to discontinue treatment (test the serum levels!)

Supplementing with the intention to increase vitamin E in the central nervous system (CNS) of a horse with neurologic disease requires a different protocol than supplementing for diet deficiency in a normal horse (a horse that has no access to green grass).  Studies show that some supplements do not increase the levels of vitamin E in brain tissue.  Studies show that vitamin E supplements do not increase the levels of the vitamin in muscle tissue.  And some supplements are active at five to six times other formulations!

There are no studies describing toxicity in horses from too much supplementation.  Vitamin E can be toxic because it is stored in fat (lipid) and is not excreted like water soluble vitamins are. It is possible that vitamin E inhibits vitamin A, another fat soluble vitamin although there are no published studies. In other animals, including humans, neurologic complications result from overdosing vitamin E!  There is no reason to suggest toxicity won’t occur in horses.

Horses showing no clinical signs of vitamin E deficiency

Supplementing with vitamin E can be expensive and can put a horse at risk for toxicity. If you suspect a deficiency you can easily test the serum concentration.  Pathogenes offers a discounted program for testing vitamin E in association with our clinical trials.  Call us for more information and be sure to send the sample with our Test submission form.

Is it time to stop supplementing?  A simple test will tell you.  Because there is a rapid decline of serum levels after discontinuing some forms of vitamin E, it is best to wait a week after stopping vitamin E dosing before sending a serum sample for testing a long time supplemented horse.

Clinically ill horses

Horses showing clinical signs of equine motor neuron (EMD) disease or vitamin E-deficient myopathy can benefit from treatment.  Often suggested, but not proven, vitamin E is supplemented in  cases of active equine protozoal myeloencephalitis (EPM), with or without measured low levels.

Determine the base line levels of serum vitamin E before supplementing.  In diseased horses requiring supplementation, 5000 IU/day of a soluble, natural form is useful.  Not all horses respond the same way to supplements- there is individual variation!  After two weeks of supplementing the serum level should be assessed and adjust the dose accordingly.  A tapered regime with a gradual transition to a natural powder form of vitamin E is  appropriate.  The natural power form of vitamin E will return a horse to a normal serum value in 7 weeks but  normal CSF levels are not achieved with this supplement-form.

Levels of vitamin E

Normal serum levels of vitamin E in horses are greater than 2.5 µg/ml.  A level that is considered adequate is a range between 1.5-2.4 µg/ml.  Horses with serum levels less than 1.5 µg/ml are deficient.

Normal levels decline significantly in just 18 days in horses that are not allowed access to grass and are fed a pelleted ration that is not supplemented.  Considerations here are horses stalled due to colic surgery, metabolic syndrome, or other similar conditions.  Horses don’t have green grass in northern climates during the winter, something to consider.

Vitamin E levels in serum
Normal 2.5 µg/ml
Adequate 1.5-2.4 µg/ml
Deficient less than 1.5 µg/ml

It is interesting to note that in a controlled study there were no differences in the mean concentration of CSF vitamin E in un-supplemented (normal levels were present) or supplemented horses (all supplements).  Or in deficient horses, before and after supplementation!  There was a significant (linear) correlation between serum and CSF concentrations, the higher the serum level the higher the vitamin E in the CSF of most horses.  It is possible there is a limit to the amount of vitamin E that can be measured in the CSF (does it all go to the cell membranes) or it is a fault in the testing protocol (little correlation of values with test results).

Types of supplements

Vitamin E is available as an injection, usually in conjunction with selenium, and is a form that is by prescription for use by a licensed veterinarian.  Some serious and life threatening reactions can occur with intravenous or intramuscular injections of vitamin E-selenium.  The injectable form bypasses the inhibition seen in some oral formulations. Oral synthetic and natural vitamin E preparations are available.  The synthetic has eight stereoisomers (the molecular shape of the molecule and its rotation).  Animals have a preference for only one.  Natural vitamin E comes in only one isomer, the one preferred by the liver.  There are two synthetic forms of acetate, the powder is twice as available to the animal as the pelleted form, the powder increasing serum concentrations in about two months.  The water soluble, liquid form, is five to six times as available for uptake by the horse and increased concentrations are accomplished in 12 hours.  Thus the acetate forms elicit a gradual increase when supplemented.

What?  Who said sulfur inhibits vitamin E?

Vitamin E and selenium are intertwined with sulfur metabolism.  There is a relationship between selenium and vitamin E overcoming sulfur-induced depletion in the body.

What the vitamin E Guru’s suggest

Horses that have no clinical signs of deficiency can be supplemented with the less expensive acetate forms at 10 IU/kg body weight per day over months to achieve normal serum vitamin E levels.

The acetate form isn’t a good choice in horses with clinical signs of EMD or vitamin E deficient myopathy.  These horses require an immediate increase in serum and CSF vitamin E concentrations.  The veterinarian can use an injection to rapidly increase levels and the treatment can be repeated at 5-10 day intervals.  This form is labeled for selenium-tocopherol deficiency syndrome that presents clinically as rapid respiration, profuse sweating, muscle spasms and stiffness accompanied by an increased SGOT (liver enzyme).

Levels can be restored to normal by giving 5000 IU/day of the soluble vitamin E and then tapering the regime to transition to 5000 IU/day of the oral acetate. This protocol resulted in horses with a prolonged increase in CSF concentrations 8 weeks after beginning supplementation.

Sulfur in the digestive track can inhibit vitamin E uptake, in sulfur-inhibition resulting in deficiency, an injectable form is preferable.

What we suggest

Test the serum  vitamin E levels before supplementing this essential nutrient and again after 7 weeks of supplementation. After 7 or more weeks, discontinue the supplement for 7 days and then test.  It may be wise to re-evaluate serum vitamin E levels after several weeks on the acetate form to ensure concentrations remain within a normal range. It may be of value to determine vitamin E levels in horses suspected of EPM.  A diagnosis of the neurologic disease EMD can be supported by measuring a low serum vitamin E concentration.  Test these horses when they are tested for suspected EPM and once on therapy, 7 weeks later.  Horses with equine degenerative myeloencephalopathy (EDM) will not respond to vitamin E.  EDM is an inherited condition that prevents uptake of vitamin E early in life.  Once neurological signs are present they usually don’t get worse…or better in these EDM horses.

Vit EFat soluble vitamins are important in equine nutrition, vitamins in this group are M, A, D, and E.  Fat soluble vitamins are important in cell membrane functions and immunity.  Vitamin E, also called alpha-tocopherol,  is unique because it isn’t involved in specific metabolic functions but it is the body’s major fat soluble antioxidant.  The action of the anti-oxidant vitamin is to prevent free radicals from destroying (though oxidation) fats in the body.

Vitamin E  is found in green grass, horses that are on pasture will get plenty of vitamin E in their diet. Because horses don’t make this vitamin it is called an essential daily nutrient.  Horses that may need supplementation include pregnant mares, mares with foals, young growing horses, and performance horses that do not have access to green pastures. Horses that are restricted from eating lush grass, horses with metabolic syndrome, are also at risk for inadequate levels. Vitamin E is a treatment for Equine Motor Neuron Disease (EMD) but has no effect in horses with Equine Degenerative Myeloencephalopathy (EDM).  Vitamin E is often suggested to enhance recovery of horses with Equine Protozoal Myeloencephalitis (EPM) although there are no studies to support this recommendation. You can read a little more about these rare diseases, EMD/EDM/EPM at http://pathogenes.com/w/horse-owner-information/

There are no cost-effective means of rapidly increasing serum and cerebrospinal fluid levels of alpha-tocopherol and then sustaining the concentrations; a recent report recommends a water-dispersible formulation followed by a gradual transition to the acetate form of Vitamin E over a 7 week period. This study also highlights the need for periodic evaluation of Vitamin E concentrations because responses vary among individuals.  There are plenty of reviews of supplements and amounts to use in each type of deficiency; the need for supplementation should be discussed with your veterinarian. Remember, supplements are not without risk, especially supplementing fat soluble vitamins. How much is too much and what does Vitamin E toxicity look like? In people an overdose can cause muscular weakness, fatigue, diarrhea and bleeding.  The possibility of bleeding is of the most concern in supplemented horses.

The good news is that Vitamin E is easily  measured in the blood.  The alpha-tocopherol levels are reported as micrograms per ml (µg/ml).  If a horse has more than 2 µg/ml the level is adequate.  If the range is between 1.5 –2 µg/ml the levels are marginal and if the levels are less than 1.5 µg/ml the horse is deficient. A blood level should be taken before supplementation, after 30 days and prior to discontinuing therapy.  If desired levels aren’t attained after 30 days a different formulation may work. Testing is less expensive that maintaining a horse on a needless supplement or overdosing a horse.

2 µg/ml Adequate, no supplementation needed
1.5 –2 µg/ml Marginal
<1.5 µg/ml Deficient

Pathogenes offers testing Vitamin E levels in serum and CSF.  The cost of the test is $40 for a sample run in conjunction with other laboratory testing.  The cost is $50 for Vitamin E only. Test Submission Form

Animals that respond to health challenges activate both innate and acquired immunity.  The innate immune system is not dependent on specific responses (as is antibody response) but stimulates many aspects of the host’s processes. In addition to many responses (such as fever, increased white blood cell count and more) the liver is recruited to produce acute-phase proteins.   Acute phase reactions are highly regulated.  There is a maximum serum concentration within one to two days and a decline with recovery from the infection.  Feedback regulatory pathways limit the response and these proteins decline in 4-7 days after the stimulus is gone.  When the receptors that trigger the production of acute phase reactions continues, the cycle becomes chronic.

C-reactive protein is an acute phase protein that is used as a diagnostic tool. Measuring and charting CRP values can prove useful in determining disease progress of the effectiveness of treatments. There is little published information about CRP values in horses and relating these values to disease.  We would like to determine why some horses that we follow are clinically well but continue to have a high CRP.  One possibility is intestinal parasites.  Recently we looked at the presence of parasites in  a group of horses, all were given a single dose of moxidectin gel. The horses were examined 42 days after deworming.  All the horses had intraluminal and encysted parasites,bots and tapeworms.  Is it possible that these parasites are associated with a chronically elevated CRP in some horses?

bots-gasterophilusgasterophilus

tapeworms-in-horses__mediumTapeworm uf

The CRP values in these horses ranged from 0 to 59 micrograms/ml.

We would like to associate parasite load (measured by fecal egg counts or FEC) with CRP levels.  If we look at fecal egg count reduction as a measure of treatment effectiveness and CRP we may generate some good data. This is where you come in.  If you have a horse that has high fecal egg counts (greater than 500 eggs/gram) we would like to treat the horse and then measure the FEC after treatment.  To determine the feasibility of running a GLP study (the expensive one) we want to survey some horses, 10 or so.  We would do fecal egg counts, treat, and run the count again after treatment.  We will run the FEC at no charge for the 10 horses that can participate in this program.  To qualify the initial determination that the horse has a high FEC must be known, you’ll have some of that data recorded already.  We will confirm that and then enroll the horse.  Contact us by email to determine if the horse will qualify and we can let you know how the study will be conducted. This study will be randomized and placebo controlled.  You need to understand what a placebo is and how it may affect your decision to participate in our program.