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When does polyneuritis equi (PNE) begin in a horse? A polyneuropathy manifests in a horse that is susceptible to the disease.  Which horses are susceptible? Horses that are particularly “inflammatory” or a horse that is allergic to vaccines, even a horse with endocrine issues.  Some horses that contract a virus can succumb.  Once typical signs are present, the disease can be recognized.  But what about the pre-symptomatic phase, what does that look like in a horse with PNE? 

Biomarkers could identify the earliest stage of PNE, if one were looking. At this stage the horse doesn’t have any outward appearance that there is anything wrong, and if you could ask, the horse would agree, all is well.  At this phase it is entirely possible the horse clears the inflammatory issue, probably most of them do regain health and that is why this disease is so rare.

A few horses will start to show early signs of polyneuritis and these signs can be detected, although they are often attributed to other causes-like protozoal infections, EPM. The signs can be so subtle that an owner feels like there is something wrong but they can’t quite put a finger on it.  In human disease the period between possible symptoms/signs and definite symptoms/signs is called prodromal.  Once there is “phenoconversion” and the horse leaves the prodromal phase then enters the symptomatic phase, it isn’t hard to recognize the clinical presentation as PNE.

What is the duration of the sub-clinical phase of disease, the disease is present but there aren’t any outward signs? Polyneuritis equi is a rare neuromuscular disease that is difficult to recognize in the field even when clinical signs are present.  We think a field diagnosis is difficult because it isn’t a disease that is forefront on a clinicians differential diagnostic list.  As in other neurological disease syndromes, ones choice of descriptions has shaped concepts and paradigms in PNE-and that is that the disease is untreatable. Our work concentrates in early diagnosis and treatment options for horses with PNE because this is the meerkattime that damage isn’t permanent and the horse can be returned to normal. Veterinary training and the literature describe end-stage disease. Unfortunately, little can be done in the late stages, hence the grim prognosis and current recommendation of euthanasia.

It is our hope to change the way clinicians think about and diagnose PNE; that would change the paradigm.  It would change the outcome. Paradigm-changing is a step-wise process.  We spent years getting our ideas and research studies completed.  Now we will complete a clinical effectiveness trial on horses recognized with PNE, and many of these will be end-stage.  The study will allow us to collect effectiveness data and define biomarkers to detect treatable disease.  The goal is to use biomarkers in a second effectiveness study.  All the data will help us get answers to more questions. Can there be a genetic pre-disposition to disease?  Is disease caused by a specific virus or microbial agent?  Is it possible that environmental exposure to an “agent” is the inciting cause?  A useful strategy to study PNE is to define a population of horses that have a high likelihood of developing PNE.  can we define that population? You see, to change what’s ahead one has to continually look in a different direction. 

We will be sending out requests to veterinarians and owners that have cases suitable for our effectiveness field trial.  There are some criteria that make sure the horse can be included in the trial.  We can answer questions for you, if you think you have a horse with PNE. We will provide study medications at no cost and we will pay for some of the pre-entrance testing.  We need you to help us change the PNE paradigm.

 

blogger-image--213779556If you search the literature you will quickly realize that polyneuritis equi (PNE) has a grim prognosis. The prognosis is poor because there are no recognized treatments. Only palliative care and euthanasia are offered as the standard of care for the PNE horse.  It is our goal to change that with our Hope Initiative.

We began to recognize a pattern in horses with neuromuscular disease and a literature search led us to papers published in the 1980’s.   One paper authored by Fordyce gave specific signs used to evaluate horses with putative PNE and led to the Fordyce Score.  We found quite a bit of research done  in horses relating to PNE. The disease is rare and presents clinically with diffuse involvement of extradural nerve roots of multiple peripheral nerves. The pathology is a polyneuropathy characterized by  inflammation  and demyelination.  PNE has no known single cause.   The inflammatory process may initiate primary or secondary demyelination of nerves by a “bystander mechanism” where any antigen that reaches the peripheral nervous system attracts and activates lymphocytes and macrophages.  We’ve reviewed the topic for you in our eBook Polyneuritis explained, available on Amazon for $4.99. The disease may be reversed if it is caught early and remyelination of the nerves can restore function.

A spectrum of signs can be recognized in PNE-horses and careful case analysis gave us the heads-up that treatment may be initiated before chronic demyelination progressed to end-stage disease.  That’s when  we knew that a horse can recover from PNE.  We were surprised to find that the literature described a diagnostic test for myelin protein antibodies many years ago.  The presence of these antibodies were related to disease.  The molecular structure of myelin was determined and the immune-mediated process was determined experimentally in animals.  Some tweaks in the diagnostic protocol were needed, using a recombinant protein avoided non-specificity issues and folding  the protein into its native state was necessary to make the test useful.  Finally, epitope mapping was highly illuminating to understand the pathogenesis of disease.

We’re developing a  licensed treatment.  Our  next step  is a field effectiveness study.  A (any breed) horse that is between 2 and 30 years old , 605-2420 pounds and shows signs of PNE with a Fordyce score of 4 is considered eligible.  There are some other restrictions, and qualification to receive medication is made on a case-by-case basis. Some testing is required, horses need to be negative for sarcocystosis by serum analysis and the possibility of trauma is ruled out by examination, history, or radiography.  Your  veterinarian can use endoscopic exam or radiographs to rule out osteoarthropathy. Vaccinations must be current (within a year) and include rabies, EEE, WEE, and WNV. Of course, before starting any medications a serum chemistry and CBC should be done.

Some of the other qualifications are: the horse is expected to be manageable and cooperative with handling, examinations, and the owner is able to give medications daily.  The study involves a two week treatment and two clinical examinations made by your veterinarian.  The owner will need to fill out a  checklist each day the horse receives the medication and this checklist is how we evaluate the response to treatment.  If you are unsure that  your horse qualifies based on the clinical signs you are seeing, you can email us or use the following  links, answer some questions, and we’ll get back to you. https://forms.gle/cjTLUwMt4fqhovo77

You may already know, based on your experience, that your horse shows signs of PNE, or your veterinarian has made the diagnosis. This link is useful to determine the Fordyce score and eligibility for the study: https://forms.gle/jcuTjM4RoQUEip6q7

You or someone you know may love a horse suffering from PNE, tell them to contact us and join our Initiative to give hope to horses suffering with PNE.  blogger-image-702499765

We’ve been reading a lot about amyotrophic lateral sclerosis (ALS) lately.  One thing is abundantly clear and that is there will be no single treatment to cure this disease.  There are inherited forms of ALS, fALS, and then there are sporadic cases, sALS. Our take on the overall picture of ALS is that no matter the inciting event at some point there is a final common pathway…that is inflammation.

Dogs get late onset fALS.  We suspect horses get fALS as well.  Is someone looking for it?  It would be very rare and most likely, with no treatment options, the horse would be euthanized before a clinician would think of ALS. Horses get subclinical inflammation presenting as a peripheral neuropathy.  After some time passes the neuromuscular disease progresses to the lower motor neurons (polyneuritis equi) and then it affects the upper motor neurons if the horse lives long enough. Diagnosis is the big issue here, to recognize a case of equine ALS.

The sporadic form of ALS seems more insidious…the sub-clinical pathways probably take a long time to surface into clinical disease.  The dysregulated systems that are forefront in human fALS are being targeted with specific small molecules-it is unlikely they will target sALS patients.  Even after years of failed attempts to find the cure for ALS the approach is the same: one path-one drug. New molecules come along, and so far they failed, even if they showed promise in mice that develop ALS.

The newest platform is skipping the animal step and moving right into people with promising drugs or small molecules. The entry criteria for the studies using novel small molecules targeting fALS are not available to sALS.  The treatments are super expensive. These approaches will never translate into an equine therapy.

Back to our hypothesis that it will take multiple drugs to combat presumptive equine ALS (eALS).  And this is a huge problem.  If one decided on a cocktail that was effective, the licensing process for the therapy would be impossible to get through FDA.  The effectiveness trial is mind boggling…we are suggesting the cocktail may involve 5 drugs at a minimum. The safety trial alone would cost at least a million dollars, our one-drug safety trial was nearly a half-million alone.

How does one start to evaluate therapeutic cocktails for a rare disease such as ALS?   Initially an animal model of the disease is necessary.  And a non-subjective test to evaluate if the disease is present in the animal.  And then one needs to identify the drugs that could be beneficial based on a firm understanding of the disease process. After all this is complete, it would be possible to approach FDA.

After our experiences with a simple and direct treatment using two well known drugs with specific actions for a specific disease with a defined and useful animal model, we can absolutely say the task for licensing a putative five drug ALS cocktail is insurmountable.

Ah, but a man’s reach should exceed his grasp, Or what’s a heaven for? (Robert Browning)

We have an approach that just may be doable.  And that is finding single therapies that hit multiple targets.  Our idea will rest upon finding a useful diagnostic test to ascertain effectiveness.  We aren’t afraid to try our approach in ALS models and compare the results to multiple drugs that target dysfunctional ALS pathways. Of course, testing multiple drugs together is a huge step that is outside the box thinking.  Out of the box thinking is what it will take to tackle ALS.

Our reach is big.  We’ll let you know how it goes.

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.