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IMG_0329What does it take to consult with a veterinarian about a neurologic horse?  Is a consult only an opinion?  And how does the consultant form an opinion?

We adhere first to the Veterinarians Oath https://www.avma.org/KB/Policies/Pages/veterinarians-oath.aspx and the veterinarians client-patient relationship https://www.avma.org/KB/Resources/Reference/Pages/VCPR.aspx. These are part of our assessment of a case and documented on our submission form.  Because, in the consulting/research business if it isn’t recorded and validated then it didn’t happen! 

An opinion is based on education and experience.  Our opinion continually evolves by our research and the research published by others.  And then very carefully analyzing what we can apply for each case. Our evaluations are a lengthy process resulting in information that is relayed back to veterinarians, one-on-one.

Initially, our opinion is based on evaluation of a blood or cerebrospinal fluid sample. There is a lengthy process of evaluation for each sample we receive.  We review and validate the data (and data entry) that we get from the veterinarian, a minimum of 5 reviews , at least one documented by the QA  officer, and this is before the samples are tested.  Each sample is tested against known values and the results of testing are documented. The results are first captured by computer,  these data are evaluated, recorded in the consultation record, and then interpreted. The validation process for each result involves another five reviews starting with the equipment measuring the reactions to the last step, result interpretation. The interpretation draws information from the case history that was recorded on the submission form by the veterinarian and a review of the prior data.  Did the gait score change? Was the horse treated and with what? Is there other information that is useful?

At this point, we have a basis for the consult.  The consulting report is sent to the veterinarian.  If comments are needed-- something is obvious to us but may not be to the busy veterinarian, we send an email.  We use the data that was entered for each parameter into many algorithms that are programed into our computer.  These are the algorithms that spit out reports for field studies, FDA forms, or new research.  In house, we tag some samples for more testing or more analysis.  When the veterinarian calls we quickly call up the complete consultation record on the animal and discuss our view of the case and listen to what questions they may have.  This may result in more testing, sending peer reviewed literature, or sending summary tables of data under review by our statistician.  We may have more discussion after the information is reviewed by the attending veterinarian.

Another review of the data for each horse is made by Dr. Benedetti. She selects cases that are more complex and would benefit from more testing, or at least a more detailed discussion with the clinician.  At each point notes are made to the file if there are any significant values for the data that we track.  One horse may change our algorithm, that’s how new ideas happen.

We find interesting things. For example, unexpected results prompt new queries of our data base.  We build in more parameters into our new query as appropriate. That is the power of a data base.  Here is a recent case, a 20 year old untreated horse presented acutely with an abnormal gait.  The veterinarian requested testing for auto-reactive antibodies against myelin and a CRP. The anti-myelin protein antibodies were present but the CRP was 0 micrograms per ml of serum.  That’s unexpected!  Generally we expect untreated horses with MP2/MPP antibodies to have elevated CRP levels.

Dr. Benedetti queried 19,161 records and found 487 records that were similarly tested for a minimum of MP2/MPP.  Of those, 403 had detected MP2/MPP antibodies and an elevated CRP while 26 had a CRP of 0.  Our result is unexpected, only 5% of samples we tested are similar! Other parameters that we captured from submission forms are age and treatment history, the start of our understanding of this case. The group of samples with this result (antimyelin antibody positive and tested for CRP, CRP=0) come from horses that are teenagers, and 12 of them had no history treatment.   We can do more evaluations for region of the country,  time of year, or how the samples were shipped. We may take the 12 horse data set and design a questionnaire that would go to many veterinarians, targeted to a specific case to get more data for our query.  The case may be one they saw years ago, we would be looking for outcome, or a recent submission that would spur additional testing. Based on the results of the investigation we evolve our algorithms.  We expect veterinarians to call us and ask and discuss the case.  More discussion will most likely lead us to the correct interpretation or lead us to another investigation.

If we find it appropriate, we may design a survey.  For horse owners we may make the survey a quiz. Our consultation program is designed to analyze a unique case against a large data base giving veterinarians the wisdom of the crowd. 

1Recently we quizzed horse owners about their interest in equine protozoal myeloencephalitis. Eighty-three percent of horse owners look to their veterinarian first for information about EPM! Ninety-seven percent knew that EPM was caused by a protozoa and 92% knew the opossum transmits the disease to horses. Eighty-five percent thought the infection was treatable and 10% felt the disease was very difficult to treat.  An accurate diagnosis only rated with 33% of owners.  There are a couple of areas veterinarians have some work to do, for example 13% of owners think that vitamin E will treat EPM and 7% think vitamin C is involved.  The majority of owners recognize the importance of inflammation in disease.

Veterinarians learned the basics about sarcocystosis in school.  And as they developed the art of practicing veterinary medicine they found new information provided by drug companies that paid for the, research.  The resulting  papers described aspects of infections.  Most papers concentrated on antibody detected following infections.  The disease syndrome (infection plus inflammation) wasn’t explored because horses didn’t develop EPM from the induced infections.  Information was dispersed at continuing education meetings and peer reviewed reports.  Controlled studies using horses are useful however, these studies are limited by expense and the interests of those funding the investigations. 

Published field case reports provide an overview of disease pathologies interpreted through the lens of academia because these reports describe particularly difficult cases referred to a university. Typically, horses suspected of EPM are seen by veterinarians and these cases are not referred.  A veterinarian may form opinions referenced on just a handful of cases. 

We offer consulting to veterinarians.  Our reference comes from statistical data from thousands of cases of suspected sarcocystosis seen by veterinarians across the country.  Our statistician evaluates the data we provide and renders an opinion.  Some things are obvious.  Some things we think are true, but he tells us our data is biased by the samples we get.  It is unlikely that veterinarians send us samples on horses without suspect EPM.  We are able to condense the information and tailor it to each case.  And just in case a vet is too busy, if a case starts to drop through the cracks we send an email with suggestions.  As we discover new areas of interest we will test samples and we provide that information back to the veterinarian, just in case it will make a difference.  The power in our observations are the data base.  We make the single field case relevant to statistical data from the crowd.

Pretty soon we will resend a survey out to veterinarians.  It is designed to gather data that we will forward to our statistician.  Our goal is to define some unusual presentations of neurological disease and determine the prevalence of those diseases.  When we are ready, we’ll give you the heads up so you can fill out the survey for us.  And we will also ask horse owners to prod their veterinarians to complete the survey.  Numbers are important because the decision to produce new treatments is based on need.  Common disease are interesting to large pharmaceutical companies.  Rare diseases rely on grass roots companies.  Micro companies work on rare diseases because there is a need and there is a wealth of information to be exposed. We have inquiring minds.  And when something is obvious to us we begin to let you know.  And here is where we start, a survey. Thank-you in advance.

tangle-of-nervesThe disease progression of polyneuritis equi (PE) includes an immunological response to an infection or allergic neuritis (in response to proteins released by trauma or infection).  The initial infection stimulates harmful responses that persist after the infection resolves and is no longer detected.  Damage is to the myelin covering nerves, specifically the P2 protein. A similar disease in people is Guillain-Barre syndrome.

A French physician described a variant of Guillain-Barre called Landry’s paralysis in 1859.  Landry described a wider group of peripheral neuropathies.  Polyneuritis equi may be similar to Landry’s paralysis. Most of the research in human neurological disease that depended on spinal tissue began to get material from horses when Mad Cow disease hit Europe.  The scientists didn’t want to continue to use nerve tissue from European cows.

We define polyneuritis equi by the presence of anti-myelin protein antibodies in serum of horses showing neurological disease.  Six published references link serum antibodies to anti-P2 protein with clinical signs in horses. There is also useful unpublished data. One researcher at CA Davis attempted to develop a P2 ELISA but failed to get a relationship between several cases and a positive ELISA test.  Another clinician at OSU tried to produce experimental polyneuritis equi by injecting P2 into a few horses, but his model failed. Why can we detect antibodies in serum when others didn’t make the connection? The negative data from these scientists are explained by the processes that produce disease. This is a boon for our work.

An antecedent infection sets off a harmful immune reaction.  The nerves are repaired by remyelination, but sometimes the process begins a damage/repair cycle. The repair process deposits microscopic calcium on the damaged nerve, calcification is a common result of chronic inflammation.  At this point the disease is terminal in horses. The disease is called cauda equina neuropathy (CEN) and is characterized by recognizable histological lesions that are a progressive granulomatous inflammation of peripheral nerves. CEN is chronic and has an apparent predisposition for involving the extradural nerve roots of the cauda equina.

There is an experimental model that was developed in rats. Rats are given P2 protein injections that results in EAN (experimental allergic neuritis). EAN can be induced in other animals. Rat-EAN can be induced by peripheral nerve myelin, myelin basic proteins, P2 protein, or small pieces of P2 protein, Neuritogenic Peptides. There is a positive correlation between the severity of the clinical signs and the dose of Neuritogenic Peptides from P2.  The disease in rats is characterized by demyelination of the roots of sacral and sciatic nerves given “high” doses of P2. 

There is mild disease accompanied by mild signs in 40% of a low dose, P2-immunized group.  Neither group showed signs attributed to the central nervous system indicating the disease involves the peripheral nerves.  And ponder this, repeated stimulation using the P2 antigen resulted in refractory disease, the rats stopped getting sick! Compare that with results when giving repeated doses of the Neuritogenic Peptide—rats continued to suffer disease.

In a field case antibody is made anywhere on myelin protein.  If there is continued damage to one area of a nerve and on a smaller scale, the myelin P2 protein, then anti-P2 antibodies are produced.  These antibodies are measured by ELISA.  After several rounds of the inflammatory cycle it is probable that the nerves calcify.  Its possible that is the refractory stage of disease and no antibody is made, the damage is now due to the calcified nerves.  However, in disease many areas of P2 elicit antibody production and that includes the Neuritogenic Peptide.  The rat studies indicate there will be no refractory period and antibodies will continue to be measureable against the this peptide. 

Based on these data we developed our ELISA to measure both P2 and the Neuritogenic Peptide, hopefully to gain enough data to stage the disease process.  The CA Davis team didn’t look at the neuritogenic peptide in their assay and, in late disease, it is probable P2 antibody wasn’t present.  In the OSU experimental model it would be expected that 40% of the horses would show mild disease; it would take at least 20 animals to come up with definitive disease using whole P2 protein.  However, if the Neuritogenic Peptide was employed, all the animals could be expected to show signs and measurable antibodies.

Why do horses get this disease?  You have to look at the structure of myelin P2 protein to understand and treat polyneuritis equi. 

Todays challenge is showing that the uncommon disease, polyneuritis equi, PE, is a recognizable condition that veterinarians should consider as a diagnosis in horses with neuromuscular disease.  There are only a few published papers and manuscripts report the cause is unknown.  The cause is probably due to an infection that stimulates immunity.  Once turned on the horses immune reaction attacks the insulation on nerves, myelin proteins, to cause disease. There are two presentations of PE, classical and atypical.  The atypical presentation involves the (cranial) nerves, these are nerves that exit the skull above the cervical vertebrae. The classical presentation involves the sacral and coccygeal nerves leading to paralysis of the tail. rectum, and bladder with loss of sensation in the area controlled by these nerves.

A horse can have both classical signs with atypical components that include behavior changes, ataxia, proprioceptive deficits and hemi-paresis (sidewinder gait). Horses of any age, breed, or sex can be affected, ages of reported cases range from 1 to 35 years old. The amazing thing is that there is a specific marker,  antibody, anti myelin P2  antibodies, that are found in the serum of affected horses.  Identification of the marker was first published in 1981.  The ELISA test for detecting antibodies was published in 1987. We published our test in 2015 after looking a  statistically significant number of horses that presented with unusual, unresponsive disease.  These horses were “diagnosed” as equine protozoal myeloencephalitis.  These were chronic, relapsing cases often receiving multiple treatments for EPM.

Signs of polyneuritis equi are diffuse because the inflammation affects multiple nerves in the body.  A similar rare condition is cauda equina neuritis, this is a chronic untreatable disease that has an apparent predisposition to involve the extradural nerve roots of the “cauda equina”. The cauda equina nerves exit the spine after the terminal part of the spinal cord (see picture). Cauda equina neuritis (CEN) has a specific microscopic lesion, granulomatous inflammation of the peripheral nerves.  It is most likely that polyneuritis equi is a spectrum of disease that terminates in CEN.

Polyneuritis equi may be the result of the bodies response to several different organisms, perhaps virus, parasites, or even some bacteria.  Most veterinarians at referral clinics that suspect CEN look for the classic histopathology or perhaps ultrasound/radiographic evidence to the classic lesions. I spoke to 7 veterinarians at 5 university clinics and one veterinarian from Kentucky.   They don’t see or look for early PE. They are biased to look for chronic disease because end stage cases are referred to them, not the early ones.  It is logical they wouldn’t look for a rare disease, especially early in the course of the process. They overlook these cases.  It is probable that the peripheral neuropathy is reversible in polyneuritis equi, if it is detected and controlled early in the disease process.

If you think your horse has signs of polyneuritis equi discuss it with your veterinarian.  Have your veterinarian read our paper on the proposed mechanism of disease, especially the part on why the disease is overlooked.  Send a serum sample using the ELISA submission form, mark the Sidewinder MP2/MPP and CRP box.  We can discuss the most appropriate plan for the horse.

cauda equina anatomy

Equine protozoal myeloencephalitis caused by Sarcocystis neurona is rare, even more uncommon is EPM caused by Neospora hughesi.  Two organisms, N caninum and N hughesi are associated with disease in horses, N hughesi being identified as the cause of neurological disease in most cases. Healthy horses have Neospora antibodies in their serum (IFAT).  A recent survey reported 34% of over 5000 samples from horses in 18 states were positive.

Natural disease

Neospora hughesi, isolated from an infected horse, was proposed as a new species in 1998 and then it was reported in a Canadian horse in 2009. The protozoa  are associated with the viscera, the fetus, early abortions and a few cases of EPM.  The clinical signs are similar to those caused by S neurona.  EPM due to Neospora may be more of a concern in Canada than the US, that is because opossums aren't found in the western part of the country. Most clinical disease has been found  in animals suffering from another concurrent infection.  Disease is possibly related to horses with compromised immune systems. The definitive host and life-cycle for for N hughesi is unknown, the definitive host for N caninum include the dog and coyote. Isolation of the organisms from horses is rare.  Because there are only 3 characterized isolates of N hughesi a “gold standard” serum panel, needed to develop diagnostic tests, seems elusive.

Experimental disease

There is a report using experimentally infected horses to define immune parameters for diagnosis by IFAT.  The infected horses showed no clinical signs of disease, no gross or microscopic lesions, nor did they find the organisms in the horses.  Results for whole parasite ELISA and recombinant ELISA failed to discriminate between the noninfected and the experimentally infected animals. There was no correlation between antibody values and clinical progression, a similar result that they reported from samples obtained from the naturally infected Canadian horse. The IFAT validation paper also reported that sera from 2 of 3 naturally infected horses were comparable with values of noninfected (control) horses. Modified direct agglutination test (valuable in N caninum testing from numerous species) results are a bit hard to interpret because one infected horse was false negative at all time  points after challenge. And 3 of the 7 experimentally infected horses were false positive before infection.  Four of the 7 noninfected  horses were false-positive at multiple time intervals.

The IFAT using Neospora was negative for horses with proven S neurona. However, the IFAT detected a difference in serum antibody responses between N hughesi-infected and non infected horses. The CSF titers for the noninfected horses were negative, along with 2 of the 7 infected horses. Based on the IFAT titer results, 1:640 was selected as a cut-off value.  Puzzling and interesting, they reported that frozen-thawed samples had higher values than when tested prior to freezing. That's a tough one to figure out.

Also interesting, and puzzling, is 6 of 7  “infected” horses had low CSF titers (one was negative) and all were disease free.  That contrasts to a naturally diseased horse (gold standard positive) with a CSF titer considered “very high”, 5  to 10 serial dilutions higher than considered positive for the experimental infections. Whatever the source of the antibodies in the CSF in the experimentally infected horses, it wasn’t parasites in the CNS. The results of the IFAT infection study allows for detecting exposure and determining seroprevalence, but not disease in horses.

Cross-reactivity with other EPM organisms

The above IFAT study and a review of cross-reactivity studies indicates that testing format is important in distinguishing between Sarcocystis and Neospora. In 2005 a specific N hughesi ELISA test was developed.  ELISA was used for detecting N hughesi antibodies using recombinant antigens suggesting the potential for use of serodiagnosis of N hughesi infection in horses.  These tests do not unambiguously differentiate infections caused by N hughesi or N caninum but they do differentiate between Neospora and Sarcocystis.  Therefore, currently available Neospora ELISA  tests for serum are genus rather than species-specific for these protozoa.

What’s unknown

The important unknown is the relationship between N hughesi infected horses and clinical disease due to N hughesi.  How long are N hughesi antibodies detected after exposure?  That's not known. What is the life-cycle of the parasite? We don't know yet. What is the definitive host?  Probably a good guess is a dog or a coyote.  How about the relationship between S fayeri and Neospora? Is there a relationship between abortion or reproductive difficulty and Neospora in horses as in cattle? Yes, most likely yet unproven. It is suggested in the literature that horses have persistent infections, despite the inability to demonstrate parasites, is this true?  Does the parasite form cysts in horses? Horses are big, cysts are small.  Disease is rare.  It will be a long time to figure this one out.  There are no published effectiveness treatment studies. There are no licensed treatments for horses.

Why do you care?

A horse that is suspicious for EPM and with no antibodies against S neurona, perhaps testing for N hughesi is reasonable. It is more likely that S fayeri is the culprit. When using these tests, recognize the limits of testing and the validation that was used for tests. coyote-W.M.-Giuliano