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It will soon be our 20th Anniversary at Pathogenes!  We’ve paved new ground and published most of our work because I grew up in the “publish or perish” era.  Now days science is all about technology transfer, it’s “patent or perish”! You may have participated in some things we invented that didn’t get off the ground, looking at down-regulation of molecules in response to S. neurona infection. I never cease to be amazed that this assay predicted the degree of illness a horse would have in a few months. It was expensive and cumbersome to run and ultimately discarded.  How about our horse-side antibody assay?  Who could predict that would strike out?  In a mere 15 minutes you knew if antibodies were present in serum or CSF.
I loved that one. Here is what it looked like:Dipstick assay

We took some side trips, identifying a cell that could support the replication of millions of PRRS virus in vitro, we grew 7 species of Eimeria in cell cultures resulting in a vaccine that would eliminate all those hen houses, and developed monoclonal antibodies, one  against S. suis and provided a local University with a much needed pig-herd vaccine. We collaborated with folks in Germany (looking at pigeon sarcocystosis), Norway (an acquired polyneuritis in Norwegian horses) and Canada. Sarcocystosis and Toxoplasmosis in dogs and cats didn’t escape our interest. But our heart belongs to people dealing with neuromuscular diseases in horses. We remember Amy and Ty from years ago, their picture shows the bond we have with our horses.

We have a paper, in press, that blazes a new trail for others to follow.  The paper explains the rationale for detecting S. fayeri toxin in horses, if you want the punch line--we detect cysts in live horses! We aren’t giving up our quest for new treatments or determining how disease progresses in horses, mouse or man. Horses have always led the field in neuromuscular research, spinal nerves were an important source of myelin for research in Myasthenia Gravis and Guillain Barre syndrome.  As most of you know, we got our ideas and direction from the work done in the 1980’s.  Wish we could say it was our idea and our hard work, but really, we  just pieced together a logical story from published literature.

Our current  directions are identifying the inherited genes that predict late onset ataxia in horses and defining the expressed protein environment in response to specific treatments given to animals with neurologic disease.  We are interested in the molecular targets of drugs in the nervous system and why they act for weeks after a dose.  We are surprisingly close to figuring that one out! You'll need special tubes to participate in these studies, contact us to find out if you can be a part of this research.

What stands out in our work is our ability to give clinicians information about their case and how it compares to hundreds of similar cases across the country.  We document all the information derived from our assays and connect the data to the feedback we get from you.  It’s time to catch up and let us know about your horse.  Even if our last contact was years and years ago, you are still in our system and gentle on our minds. Why, we may even have a picture of your horse if you sent one! We’d like to hear from you.  Here are some useful links:

To give us your update please use this form:

A veterinarian can give us information about a horse with neurological disease:

A horse owner can give us information about a horse with neurological disease:

If your horse was treated we’d like a post-treatment update:

If you suspect polyneuritis equi this form is appropriate:

We have been a bit quiet lately and that’s because a friend is very sick.  He has amyotrophic lateral sclerosis (ALS). In order to help him, the Pathogenes team needed to catch up on the particulars of this most horrible disease.  There is no cure for ALS and quite frankly, no useful treatments.  It has taken us a few months to read stacks of papers and gather a team of experts. And form a plan.  Our plan isn’t simple and it isn’t easy.  It’s complicated and can change.  As we formulate and fine tune our approach to stopping the progression of ALS our experts review our work for scientific accuracy and feasibility. We don’t mind that no one has heard of what we propose.  We will test our hypothesis and march forward. 

You can find a lot of information on ALS on the Web and we won’t repeat it here.  You can check out our ALS tab for some links we found useful. It is worth pointing out that there is familial ALS (fALS) in which genetics plays a big part.  Only 10-15% of patients with ALS (PALS)…these folks are acronym heavy…get fALS.  Most people get spontaneous ALS (sALS), close to 90%.  There are different camps and controversies concerning the pathways involved in disease and how to approach reversing the progress of motor neuron (MN) death, but an overriding theme of those that investigate ALS is the compassion and sharing of information.  Basically, ALS is associated with an enzyme mutation (superoxide dismutase, SOD) and/or other mutations that cause MN death.  Motor neurons make muscles work. You need motor neurons to breathe.

What stirred us to take a break from our bench work and communicate with our EPM-centric following is learning that dogs get spontaneous ALS!   Dog-ALS is associated with the enzyme superoxide dismutase, the mutation is in SOD1.  The onset of dog disease is late in life.  That means there are ALS cases in people, dogs, and genetically engineered mice.  Horsey people realize there are unknown causes of spontaneous neuromuscular disease in horses that cause them to be wobbly.  These horses can progress until they can’t get around and are euthanized.  In some cases there is no diagnosis and no treatment.  We reviewed two of these diseases.

Equine Motor Neuron Disease (EMD) is an acquired neurodegenerative disease in horses affecting the lower motor neurons of adult horses. The disease is characterized by the onset of abnormal nerve function and muscle wasting resulting from the deterioration of motor neurons and myopathy. Horses from 15 months to 25 years old can get EMD. EMD is considered to be a multifactorial disease, however a dietary deficiency in vitamin E is considered to be a major predisposing factor in its development. This is largely related to when horses have a decreased antioxidant capacity leading  to accumulation of free radials and that results in oxidative damage to the ventral motor neuron cells. Could a decreased function of horse-SOD be a factor?

No one knows what causes Equine degenerative myeloencephalopathy (EDM) that is a diffuse, degenerative disease which primarily causes damage to the horse’s spinal cord. EDM is considered to be an advanced form of neuron-axon dystrophy. EDM may have a genetic basis. Horses can develop EDM and equine motor neuron disease (EMD) at the same time and in association with an underlying vitamin E deficiency. Horses with EDM show clinical signs of a general proprioceptive  ataxia-“I don’t know where my feet are” and an abnormal base-wide stance while at rest. Horses will usually start to show signs of EDM when they are 6 to 12 months old. Horses with mild cases of EDM may present as performance-related problems. At first the condition produces subtle signs, being nothing more than  "clumsy" but ataxia slowly progresses as clinical signs are usually slow and insidious. Ataxia signs will become more apparent and worsen over time. Paralysis and spastic muscular movements will become more evident, until late stages where the horse is unable to get up from laying down without assistance. The only way to get a definite diagnosis that a horse has EDM is by conducting post-mortem examination shortly after death.

We’d like to test horses for EMD and EDM for antibody against neurofilaments.  It’s a serum test.   Once we have some results we will share them with everyone.  If we can demonstrate that horses also have a form of ALS, and why shouldn’t they?, we can start looking at treatments in this species.  If you have a horse with a diagnosis of EMD or EDM send us serum and we will test it.  Be sure and have a firm diagnosis.  Not just a “This is on the differential” or “It’s nothing I’ve seen before!”. Testing is expensive and we’re proposing to pay for it.  We need to know and have proof that several diseases have been ruled out.  You can email us for the form to send in a sample for this specific testing until we put a submission form up on our site. We want late cases as well as cases that are early.

The funny thing is that the deeper we delve into ALS and our approach to treatment, some paths seem to converge. As the ALS community gets closer to understanding the pathophysiology of disease we are finding common roads.  All roads seem to be leading to Rome after all. 

At some later date when we have some good news for your ALS friends we will ask you to share what we find.  Until then, we are going back to work.



Muscle fasciculations are visible, fine and fast contractions of fine muscle fibers that occur spontaneously and intermittently. Injury to central or peripheral nerves can result in muscle fasciculations. The pathophysiology is different for different sites of the injury. It is thought that most fasciculations have a location away from (distal origin) the motor nerve in normal animals as well as patients with motor neuron diseases.

Fasciculations are known to be associated with hypersensitivity of denervated muscles and they are observed in some diseases such as amyotrophic lateral sclerosis in people. In horses the fine tremor of the face, muzzle, or lips is best associated with West Nile Virus infection.

Other triggers of fasciculations are progressive spinal muscular atrophy, neuromuscular junction disorders, electrolyte disorders, systemic diseases and some medications. Even healthy animals can have fasciculations-these are usually located in the forearm or the eye-lids. Fasciculations were thought to be a prelude to the onset of a progressive or lethal disease that involved the lower motor neuron. However, benign fascicular syndrome has been described in young healthcare professionals.

In normal individuals physical exercise, stress, fatigue, and caffeine abuse can cause or aggravate twitchy muscles. A diagnosis of benign fascicular syndrome can be diagnosed after five years. A benign diagnosis is made when there isn’t a progression to motor neuron disease and that takes 5 years.

Muscle tremors are abnormal and are motor disorders, although fasciculations are not classified as motor disorders. Some genetic diseases of the cerebellum associated with motor disorders are accompanied by fasciculations. A specific type of fasciculation with cramping occurs in peripheral axonal excitability. This occurs when adjacent neurons (to the damaged neuron) begin to re innervate partially denervated muscles. Sometimes in genetic disease, fasciculations affect the tongue as well as the trunk and limb muscles. The lower motor neurons are involved in these cases.

Rare cases of fasciculations occur when muscles fail to relax. Failure of muscle fiber relaxation can occur with neoplasia, immune-mediated disease, heredity, and degenerative disease. In these cases, the pathophysiology is hyperexcitability of the peripheral nerves and consequent continuous muscle fiber activity. Continuous activity occurs when potassium channels are dysfunctional. Potassium channels can be damaged when there is an antibody response against proteins in this structure. Cramps, stiffness, delayed muscle relaxation and excessive sweating can be seen clinically. Motor neuron disease is associated with fasciculations in people. These diseases are detected using EMG’s, electromyography, this machine measures the muscle response or electrical activity in response to nerve stimulation of a specific muscle. During the test, one or more small needles (also called electrodes) are inserted through the skin into the muscle.

Systemic disease, drugs and heavy metal toxicity (like lead) can also induce fasciculations. Low blood levels of phosphate (hypophosphatemia) and calcium disorders (secondary to hyperparathyroidism) sometimes result in fasciculations. Calcium disorders occur with some renal diseases. Neostigmine, a drug, can increase fasciculations in cats by increasing the concentration of acetylcholine (a direct effect) in the concentration of acetylcholine at the neuromuscular junction. Some anesthetic agents work using the same pathway at the neuromuscular junction and have the same results. Mercury toxicity should be considered in peripheral neuropathies of unknown origin that are also accompanied by tremor, ataxia, and depression.

There is no specific treatment for the muscle contractions because fasciculations are a symptom of an underlying condition. It is necessary to identify the origin of the underlying condition and that condition is the therapeutic target.

Scoring systems are useful to evaluate humans and animals.  The well known APGAR score is a test given to newborns soon after birth.  This test checks a baby’s heart rate, muscle tone, and other signs to see if extra medical care is needed.  The test is given at one minute after birth and again five minutes after birth. Another well known scoring system is the Mayhew Score, or more often the “Modified Mayhew Score”, that is intended to evaluate horses for equine protozoal myeloencephalitis.  The Mayhew Score differentiates upper motor neuron diseases from lower motor neuron diseases using an extensive neurological examination.  The scoring system allows the clinician to form a differential diagnosis list. Scoring systems are generally named for the author of the system.

The Fordyce Score is a system described by Fordyce, Edington, Bridges, Wright, and Edwards in 1987 that was designed to evaluate horses with cauda equina neuritis (polyneuritis equi, PNE) and differentiate PNE from other equine neuropathies by ELISA.

Polyneuritis equi is a condition with some specific characteristics that are paralysis of the tail, bladder, rectum and the anal and urethra sphincters, accompanied by an area of analgesia (loss of response to stimulus)  around the perineal region.  Muscle wasting is common over the hindquarters and the horse can have an uneven gait. Cranial nerves can be involved and characterized by a drooping lip and ears, inability to blink and atrophy of chewing muscles, although signs can involve other cranial nerves as well.

Because the pathology of PNE is inflammation of the nerve roots that form the cauda equina and any other peripheral nerves that are involved, it was common to examine affected nerves by histopathology.  Histopathology was used because horses were not diagnosed until late stage disease and euthanasia was the recommendation. The Fordyce team recognized that antibody against the animals own myelin protein could be measured pre-mortem using an ELISA test.  The first scientists to recognize that there were circulating antimyelin protein antibodies in experimental allergic neuritis in rats, was the Kadlubowski Group in 1980.  And in 1981, they recognized the same condition in horses with PNE.

Molecular techniques in the 1990’s allowed researchers to refine the antigens used to analyze serum samples.  This was important because laboratories that used a crude mix of myelin protein from horse spinal nerve tissues in their assays got varied results. The P2 myelin protein is the required  molecule and then it needs to be presented in its native form.  Rostami and Gregorian mapped the myelin P2 protein epitopes (short amino acid sequences that are reactive to the immune system) and showed that a small piece (peptide) of the protein caused an autoimmune reaction that appeared later in the course of disease.  The difference between the whole P2 protein antibody reaction and the peptide antibody reaction was that animals became refractory; they stopped responding to the epitopes on the whole P2 protein.  They stopped responding even when clinical disease was apparent.  Yet if the peptide was stimulating the animals’ immune system, they did not become refractory.

The animal isn’t a bystander in this disease.  The horse will heal the peripheral nerves because the cells that lay down more myelin (Schwann cells) are not compromised.  It is the degree of inflammation that can get out of control in late, irreversible disease.  In chronic disease the horse will heal (scar) the damaged nerves using fibrosis or calcium.  These healed nerves can’t conduct messages to the muscles that they innervate.  As the myelin is sequestered from the immune system the anti-myelin protein antibodies fade from disuse. This leaves the horse with end stage disease.  Thus there is a progression of anti P2 antibodies in the serum.  There will be no antibodies early in disease, as disease progresses antibodies are present, and finally, antibodies are absent in late disease due to the healing process.

Another IMG_0550reason for inconsistent assay results from some researchers was the selection of cases.  Because cases selected for study were end-stage it would not be expected that all horses would be seropositive on their ELISA assay.  A more uniform selection of cases can lend validity to the anti-myelin P2 serum assay and Fordyce was the first researcher to do this.  Fordyce clinically assessed animals for PNE giving one point to each of the following signs: tail paralysis, urine drippling, rectal dysfunction, perineal analgesia, muscle wastage over the hindquarters and any sign associated with a cranial nerve neuropathy, ear droop, inability to blink and masticatory muscle wastage (see picture). Fordyce considered a score of 4 or greater was consistent with PNE. Fordyce correlated 12 of 14 seropositive cases  (titer at 1 to 8) that were considered to have PNE based on clinical score and/or post mortem criteria .  These were gold standard cases. One horse had shivers and seroconverted to negative after 5 months. Thirteen horses with non-PNE neuropathies and 20 control negative sera were seronegative on the assay. Fordyce concluded that the presence of antibody to P2 in horses suffering from PNE is useful as a diagnostic for PNE.

We agree with Fordyce and have fine-tuned the anti-myelin protein P2 assay (MP2)  to include that small peptide (myelin protein 2 peptide, MPP) discovered a few years after his work was published. We believe that the value of a combined assay (MP2 and MPP) will allow us to measure the duration of the condition, if not the severity. The advantage to a diagnostic test is recognizing disease before it is late stage and irreversible. A diagnostic test will enable researchers to find effective treatments.  Let us know how you find the utility of the Fordyce Score system in your evaluation of equine neuropathies.  Call us to find out more about serum testing in these cases.

Infection with Sarcocystis seems inevitable.  Sarcocystis are niche organisms, they’ve adapted to infect the intestine of limited hosts and complete their destiny as a muscle cyst.  Generally, the host is unharmed.  Occasionally, things run amuck and the host becomes diseased.  It is estimated that 80% of horses in the US get infected with Sarcocystis .  Most of the time the horse-adapted fayeri infects muscles where it makes a sarcocyst, this disease is equine muscular sarcocystosis or EMS.  Less than 10% of horses with EMS show signs of disease, but some infected horses do get sick.

Most horses in the United States also encounter S. neurona and most infected horses are none the worse for it. Horses don’t get sick because the immune system eliminates the organism. Sarcocystis neurona-infected horses develop antibodies and bountiful cytokines that are effective in killing the protozoa. A most important cytokine is interferon-gamma. But occasionally, the most recent estimate is 14 of 10,000 horses, suffer the devastating disease EPM.

It is believed that the organism invades the central nervous system (CNS) and causes physical damage. In nearly all cases of experimental  (natural challenge) S. neurona infections in horses, no organisms were found in spite of producing clinical signs.  Histologists noted plenty of inflammation present in CNS tissues of the infected horses. Were the organisms there and removed quickly?  Could the organisms hide out in other tissues waiting for the right moment to manifest? Were the samples taken at an inopportune time? Was inflammation the culprit and no organisms ever got into the CNS?  Those are unanswered questions that are being investigated.

There is circumstantial evidence that some horses don’t develop the right kind of immune response to eliminate the parasite, maybe these are the horses that get organisms in CNS tissues. It was surprising when horses with deficient immune systems, Arabian foals that show severe combined immune deficiency syndrome (SCID), were infected with S. neurona and surprisingly, they got plenty of organisms in their blood stream, none in the CNS, and no clinical signs.  It looks like an inflammatory response (the SCID foal can’t produce) is responsible for transporting organisms to the CNS and producing clinical signs. When a population of normal horses were likewise challenged they got clinical signs, but no organisms were found.

Some expect that  horses will only improve by 1 grade (on a scale of 0-5) with treatment.  Also, 10-25% of those horses that do respond to treatment are expected to relapse after treatment begging the questions:  Are horses relapsing because they are re-exposed and have a new infection or is the initial infection latent-ready to manifest at any opportunity? How long can a protozoa hide before re-emerging? Can the horse make protective immunity?

As scientists ponder the best way to prove what is happening (and don’t forget it could be more than one mechanism!) you need to know some things to effectively evaluate new information.

It is important to understand how protozoal parasites mature.  I will use the term synchronous to mean they all mature at the same time.  Protozoa progress through their life-cycle stages on a one-way path to complete their destiny as a sarcocyst. Parasites (merozoites) go through a couple of replications, move to the next stage, and finally when they reach the muscle they encyst as slow metabolizing bradyzoites.  Bradyzoites aren’t expected to move from a muscle fiber to another muscle to make new cysts.  Cysts degenerate, unless they are passed to the definitive host where the parasite can complete the sexual part of the life-cycle and begin a new generation of infectious organisms.  After the initial gut infection (sporozoite) and a few rounds of replication the resulting merozoites can’t go back to an earlier stage.

In 2001 we showed that S. neurona could be released from some cells, but not others, using an ionophore. The optimum time of release was 10-20 days after infection and this could be repeated once in another 30 days.  When we examined the stages of the parasites by electron microscopy it was apparent that the parasites were not synchronized because there were multiple stages in the cells.  We didn’t find a method that satisfactorily synchronized the cultures. Even cloning a culture from one cell resulted in asynchronous maturation of the colony.

In one particular cell line parasites grew very slowly, it took 120 days for them to establish a colony.  The slow growing culture was infectious when transferred to a more traditionally used cell line and when transferred, they matured quickly.  The colony was still asynchronous. In horses we expect the gut infection is asynchronous.

Sarcocystis are generally restricted to the hosts they infect. For example, S. muris can infect mice but not horses. S. canis infects dogs but not horses. One point of interest is that opossums can be a definitive host for many sarcocystis-ones that infect skunks, cats, birds, mice, and horses. True hosts are ones that support the life-cycle. Aberrant hosts are hosts that don’t support the completion of the life-cycle.

Dr. David Lindsay is a master of growing Sarcocystis and published papers on chemicals that delay, but don’t kill specific protozoa. He published an interesting experiment that treated Toxoplasma-susceptible mice and then infected them with Toxoplasma.  The take-home-message from his work was that mice that were allowed to produce an immune response faired better with later challenge when they were compared to animals that were treated during the infection process. When mice were treated they didn’t produce a protective immune response and subsequently succumbed to toxoplasmosis. He also published work that showed diclazuril fails to eliminate S. neurona from laboratory cultures and showed the ability of decoquinate to render the cultures sterile.

In a recent experiment it was shown that the interferon gamma-defective mouse could be infected with a mouse-opossum strain of S. neurona.  Untreated mice were diseased and the organisms could be recovered from CNS tissues. The experiment further showed that diclazuril could inhibit S. neurona activity, but not eliminate the parasite, providing evidence that recurrent disease could be a result of persistent infection and treatment failure rather than simple reinfection in this mouse model. The take home message was that S. neurona can resume its activity after cessation of diclazuril in a live interferon-gamma deficient mouse.

One must be careful when interpreting study data from one animal to another.  In the mouse experiment, the mice were injected with cultured organisms that did not allow stimulation of a natural immune response in the gut.  A similar experiment in 2001 used mice that ingested sporocysts of the same Sarcocystis strain as the above experiment (a natural infection) and also administered diclazuril in the diet.  After discontinuing treatment the mice did not have organisms in the CNS.  The discordant results may be the method of administration of the protozoa or even when tissues were examined. after the discontinuation of therapy  Obviously, there is more work to be done.

All the above considered, we have an issue with diclazuril used for our non-inferiority study.  It isn’t a fear of persistent infection and relapse after treatment because that has not been shown in the horse. We did test several hundred horses with clinical EPM for a lack of interferon-gamma and didn’t find one.  Our insurmountable task is showing that in our study a comparison drug, diclazuril, is as effective as it was when licensed.  We have the daunting task of showing that diclazuril is 67% effective in treating EPM. If the statistics don’t support 67% of diclazuril-treated horses clinically improve when diagnosed with EPM (the horses must have CSF tap confirming disease before treatment) the study is not acceptable.  When diclazuril was licensed to treat EPM clinical improvement was seen 59% when based on clinical signs.  Because diclazuril was considered successful when antibody declined the CSF when there was no clinical change. the reported stats are 67% effectiveness. That didn’t fit our criteria of success and we won’t be asking for a post-treatment CSF sample.  Other factors that render the data unacceptable are concomitant drugs with diclazuril, like DMSO, levamisole, steroids, phenylbutazone, flunixin , or firocoxib.  If the attending clinician administers these treatments while waiting for CSF analysis, the case is not useable.

While we put on our thinking-cap, please fill out our survey if you treated your horse with diclazuril for 28 days (no other treatments within 6 months of treatment) and let us know the outcome of treatment.  We can use the data to know if 67% effectiveness is an attainable goal.