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We are conducting an EPM-treatment effectiveness study that will compare the treatment response between two drugs. The new drug is compared to a drug selected from those that are available commercially. The commercially available drug is called the Active, we will compare our treatment to an Active. The FDA’s Freedom of Information (FOI) Summary reports important information that was used to license a drug, this information is publicly available. The FOI’s found for EPM treatments show between 15 and 59% effectiveness. Studies were done in less than fifty evaluable horses. It is important to replicate the parameters used to determine effectiveness in the Active in order to compare the results between the studies.

The effectiveness of two products (ReBalance® and Protazil®) defined a successful treatment when the Western Blot test on CSF, compared before and after treatment, were negative. That means if a horse did not improve clinically, but antibodies weren’t detected by CSF-immunoblot after treatment, the horse was considered successfully treated. The ReBalance study used a total of 26 horses to determine effectiveness. Rebalance® was successful in 15% of the horses, that means 4.2 horses showed an improvement in clinical exam after 90 days. A few more ReBalance-treated horses, 42%, improved after 210 days. And, if clinical exam was evaluated with western blot conversion to negative as the criteria for success…at 210 days, a few more horses were considered successes. One interpretation is that if no antibodies were detected then the parasites are gone; the horse didn’t improve because there was irreversible neurological damage. Although commonly believed, that is not our working model of EPM. We believe that neuroinflammation can be reversed, if diagnosed and treated properly.

The Protazil® FOI shows 59% of horses are better at 48 days, unless a negative western blot test is included in the effectiveness analysis. In that case, effectiveness is 67%. An advantage to comparing our drug to Protazil® is the duration to an expected response. The FOI reports an improvement 20 days after the end of treatment, day 48. That is a darn site better than 90-210 days.

The folks testing Marquis® reported in their FOI that “Western Blot of the CSF did not appear to be a major factor in determining treatment success nor a reliable measure of treatment success”. They used gait exam as their assessment parameter and showed 59% improvement based on that exam.

Remember it was shown (Furr et. al. 2006) that prophylactic treatment with anti-protozoals delay antibody production in horses given oocysts as challenge, the challenge is similar to how horses are naturally infected. It was Dr. Furr and his co-workers that showed a delay in antibody production did not indicate that clinical signs would be prevented when horses were given ponazuril as a preventive treatment. The down side to Marquis® is 110 days to show an improvement, 82 days after the end of treatment, and that is a bit too long for our ideal comparison.

There is one notable reference in the new Marquis® flyer we just received and worth a digression from our current topic. The flier cites a paper published years before S. neurona was isolated from an EPM horse! This paper is offered as evidence that ponazuril “kills the parasite that causes EPM to stop it from inflicting further damage to the central nervous system (CNS)”! The paper really reports the effects of trianzinones on developmental stages of Eimeria in chickens, no mention of S. neurona, EPM or CNS stages of sarcocystis that cause EPM. Eimeria are coccidian parasites, don’t develop muscle cysts, and does not cause EPM. Eimeria is found and stays in the gut of a chicken. The flyer also cites coccidia in calves, lambs, and pigs as references. We side with Dr. Dirikolu (JAVMA, Vol 242, 2013) that reviews in vitro and in vivo studies to “clearly indicate the removal of triazines after appropriate treatment time results in regrowth of parasites…suggesting that stages are inhibited and retain the ability to begin development again once the drug is removed”. That means the action of the drug, in horses with S. neurona infections, is static, it doesn’t kill (cidal) all the stages. That is one explanation why horses relapse on this treatment. That is why it is important to test the drug against the species of organism in the animal species for which it was intended.

We are conducting the study, in horses, to show the field effectiveness against suspected S. neurona in horses. From the forgoing information our logical selection is Protazil® as our active placebo for our study. We will evaluate the clinical response to the treatment, not an antibody difference measured on a test before and after treatment.

We proposed a study using two treatments. Veterinarians call this kind of study an active control effectiveness study design, there is no placebo because the active control serves as the placebo. The advantage with this type of study is that a client feels more secure that a horse with EPM is getting a treatment and not a “sugar pill”. A veterinarian can enroll a horse and we have to have some assurance that there will be 4 cases/site over a couple of years. The site is the veterinarians practice.

Orogin® is a drug that is designed to treat Equine Protozoal Myeloencephalitis (EPM) due to S. neurona in horses. The Orogin® effectiveness study uses an active controlled parallel arm effectiveness design with animals randomly assigned to treatment groups and receive either the investigational drug (Orogin®) or the active control (Protazil®). The study for Orogin® is limited to 70 horses. This is a study that will be part of our Freedom of Information Summary and, because it is an FDA study, it has a few strings attached.

This is not a placebo controlled study, the horse will get a drug to treat EPM. The signed Owner Consent form, is required. The owner consent form informs the horse owner that this is an investigational drug. Your horse will not be considered for entry into the study without this form in place. If you haven't signed this form, you are not in this study. Horses can receive an alternate treatment at day 10, if there is no improvement. Any horse that is removed from the study drug before the end of the study will be called a treatment failure. The horses are examined again at 48 days, the expected time for Protazil® to exert an effect.

What does it take for a horse to qualify for enrollment? The documentation for qualification is called the Qualification Record. There are three conditions that must be met to qualify 1) the horse must have a provisional diagnosis of EPM due to Sarcocystis neurona. 2) The animal must exhibit a minimum of a Grade 2 deficit, and the easy one 3) the animal is 9.6 months to 30 years. A Grade 2 deficit is defined as “neurological deficit obvious at normal gaits or posture; signs are exacerbated with manipulative procedures.”

There is a second part for the qualification into the study and that is disqualification because there are situations that will disqualify a horse. The horse can't be in another study or be unsuitable for this study. The horse can't enroll if it has another disease. If the horse can't get up, grade 5, or the owner can't get medication into the horse (or multiple folks will treat the horse) it won't meet the study qualification standards.

There are some other forms (all one page with check boxes) for the veterinarian to fill out, the Physical Exam form at day 0 and 48, the Data Collection forms at day 0, 10, 27, and 48 and a Supplemental History form (these document the criteria used to support the diagnosis of EPM). There is a blood draw before treatment and day 10, the blood is collected in a couple of red top and lavender top tubes, accompanied by a Sample Collection form. Owners participate in the observations by completing check boxes on the Client Observation form that note behavior, appetite, respiration, and occurrence of diarrhea for 28 days, the end of the Protazil® therapy. Additional space is provided for owner comments.

So far, so good. But the first inclusion criteria, a provisional diagnosis of EPM, may be the most difficult requirement for this study. The diagnosis of EPM must be supported by testing, along with any other diagnostic testing used generally by the veterinarian for the horse. This ensures the diagnosis is correct. The FDA set the standard as CSF analysis by Western Blot to support the diagnosis.

We recognize that CSF taps are not generally obtained in the field and most veterinarians test serum, or don't test at all. We strongly hold that serum testing guides the correct treatment and that is so important. Hopefully data from this study will support that rationale, just like the Marquis® study, that CSF testing isn’t the best criteria to determine a horse that will respond to treatment. What we know now, that was not known a few years ago, is that diseases (like S. fayeri or autoimmune polyneuritis) look like EPM but will not respond to ReBalance®, Marquis®, or Protazil®. That is why treatinginflammation that is common to S. neurona, S. fayeri, or autoimmune polyneuritis is important. We want to use the serum analysis to make these points in our FOI.

However, until there is a paradigm shift at FDA the horse must have a CSF tap to participate. We provide a CE course (3 credits) and teach a technique to obtain a CSF tap, in the field, in a standing horse. Field sedation techniques, using drugs most veterinarians have on hand, allow a veterinarian to get a CSF sample from the side of the neck. With a bit of practice, the tap can be obtained in a couple of minutes. You can email us for the link or go to the Learn More tab and copy the link from the slide presentation that describes Pathogenes CE program. We will run the CSF testing at no cost.

We are far from finished in our quest to make EPM treatable and affordable and need your help with these studies. If you desire new equine treatments in the pipeline veterinarians and owners will need to be proactive. From idea, to models, to field testing and ultimate use, it's expensive and time consuming. It's also highly rewarding. This is our commitment to the horse community and any part you can play is appreciated. A veterinarian can call and discuss the protocol and time commitments.

 

We believe new treatments are transformational when talking about protozoal disease. What inspires transformational thoughts and ideas?  Some think it is a mysterious process, right out of the blue, a light comes on.  More likely, innovation springs from many years of questioning, hard work, vigorous testing, and careful analysis.

We have reached the proverbial 10,000 hours (ref: Malcolm Gladwell, Outliers) studying EPM, creating models, and we are ready for a final analysis.  Our research started using models that defined the disease process and indicated that we could develop a beneficial treatment.  Years later, it’s time to put the models aside and statistically test our model.  Our first placebo controlled study for NeuroQuel is available, limited to 60 horse owners.  This is a study that will be part of our Freedom of Information Summary and because it is an FDA study it has a few strings attached.

NeuroQuel is a drug that is designed to control the residual or recurrent clinical signs due to inflammation associated with Equine Protozoal Myeloencephalitis (EPM) following antiprotozoal therapy in horses.  The field study will enroll horses that have failed to improve, or have relapsed after licensed EPM treatment (Marquis, Protazil, or Rebalance). The horses will receive NeuroQuel and we will evaluate the response to the treatment.

This is a placebo controlled study.  The first form, the Owner Consent form, is required.  The owner consent form informs the horse owner that there is a chance (a statistically calculated chance, 1 out of 3) that the horse will receive a placebo treatment.  Your horse will not be considered for entry into the placebo controlled study without this form in place.  If you haven’t signed this form, you are not in this study.

Does the horse qualify for enrollment?  Not surprising, the documentation for qualification is called the Qualification Record. There are three conditions that must be met to qualify 1) the horse was previously diagnosed and treated for EPM.  2) The animal exhibits recurrent or residual clinical signs with a minimum of a Grade 2 deficit, and the easy one 3) the animal is 6 months or older weighing 600 pounds or more.  A Grade 2 deficit is defined as “a defect that is easily detected and exaggerated by backing, turning or neck extension.  The horse may sway at a walk.  There may be a wide based stance after tight circling.  The horse is weak on the tail pull-easily pulled off track and does not return to a normal walk for 3 steps.”

There is a second part for the qualification into the study.  There are situations that will un-qualify a horse.  The horse can’t be in another study or unsuitable for this study.  The horse can’t enroll if it has another disease.  If the horse can’t get up, or the owner can’t get medication into the horse (or multiple folks will treat the horse) it won’t meet the qualification criteria.

There are some other forms to fill out, the Physical Exam form, the Data Collection form at day 0 and day 14, and a Supplemental History form (these document the criteria used to support the original diagnosis of EPM).  There is a blood draw before and after treatment, the blood is collected in a couple of red top and lavender top tubes, accompanied by a Sample Collection form.  Owners participate in the observations by completing check boxes on the Client Observation form for behavior, appetite, respiration, and occurrence of diarrhea.  Additional space is provided for owner comments.

So far, so good.  But the first inclusion criteria is the most difficult requirement for this study.  The original diagnosis of EPM must be supported by testing and other diagnostic testing as needed. The FDA set the standard as CSF analysis to support the original diagnosis.  After bioassay for antibodies in the CSF the horse must have received a full course of an FDA approved treatment, per label instructions: dose, duration, and frequency, no loading doses or multiple treatments.  Treatment must be what the sponsor intended when they licensed their treatment.  Treatment must be within 90 days of enrollment.  At the end of the FDA approved treatment, if signs (minimum Grade 2) are present, then the horse will qualify for this study.  Also, if within 90 days the horse “relapses” with a Grade 2 deficit, then the horse will qualify for the study.

We recognize that CSF taps are not generally obtained in the field and most veterinarians  test serum, or don’t test at all.  We strongly hold that testing allows the selection of the correct treatment and can be accomplished by serum testing.  Hopefully data from this study will support that rationale, CSF taps aren’t the best criteria to determine a horse that will respond to treatment.

However, until there is a paradigm shift at FDA, we provide a CE course (3 credits) and teach a technique to obtain a CSF tap, in the field, in a standing horse.  Field sedation techniques, using drugs most veterinarians have on hand, allow a veterinarian to get a CSF sample from the neck.  With a bit of practice, the tap can be obtained in a couple of minutes. You can email us for the link or go to the Learn More tab and copy the link from the slide presentation that describes Pathogenes CE program.

We will continue to use models that advance our ideas because idea-modeling will result in more treatment options for horses and their caretakers.  For example we learned about the effect Sarcocystis fayeri, a common protozoal infection in horses, can have on neuromuscular disease.  Another example is the presence of autoimmune disease in horses, when treated early the horse can have several more years of useful life. A  limited enrollment, placebo controlled study gathers statistical data.  After the “reasonable expectation” of clinical benefit study a conditional license is issued and a larger field effectiveness study is conducted.

We are far from finished and need your to help in these studies if new equine treatments are desired in the pipeline.  From idea to models to field use, it’s expensive and time consuming.  It’s highly rewarding.  This is our commitment to the horse community and any part you can play is appreciated.

 

Some horses with equine protozoal myeloencephalitis (EPM) resolve their issues with treatment, these cases are simple.  Some horses continue to have gait problems that span years, they “relapse” year after year.  These are complex cases.  One common strategy is to treat the EPM-suspect horse and see what happens.  If the horse fails on treatment or “relapses” some suggest that the relapses “must reflect either new infections or reactivation of latent  infections” (MacKay 2008).

The comments about treating relapses states: “there is no obvious rationale for using a different drug to treat recrudescent EPM than was used to treat the primary presentation.  In my (MacKay) experience, regardless of the drug used, the response to treatment of each successive relapse is incrementally less complete.”  This approach leads veterinarians to extend the course of therapy, use higher doses of the same therapy, combine antiprotozoal, switch from one approved drug to another, institute “maintenance” antiprotozoal drug therapy…

Complex neurologic cases may be difficult to treat, not due to a lack of treatment effectiveness, but rather a lack of understanding of the pathogenesis of disease and the the drugs that should be used for the disease process.  Treatment response is a part of elucidating the disease in horses.Testing is critical to understanding what the disease process is in the horse.

Diagnosis of EPM is difficult and it is common knowledge that there are no “EPM” tests.   EPM is a syndrome that involves protozoal infection and signs that result from Sarcocystis-associated  infection.  An antibody test that detects protozoa won’t diagnose the inflammatory component of disease.  The presence or absence of antibodies can rule in or rule out the involvement of Sarcocystis. The inflammatory processes can linger long after the horses immune system resolved the active infection.  Inflammation makes EPM complicated to detect and treat.  The inflammatory process is not detected by circulating or CSF antibodies.

We are piecing together puzzling aspects of neuromuscular disease in horses and can help you understand some of those difficult cases.  Important aspects to consider are the presence/absence of Sarcocystis neurona antibodies, the serotype of the S. neurona infection, the host’s inflammatory response to protozoal infections, the presence of Sarcocystis fayeri toxins in horses, autoimmune antibodies, and the pharmacodynamics of treatment on both the horse and the parasites.

Published data clearly showed the clinical difference between the S. neurona challenged immune-competent horse and the S. neurona challenged immune-deficient horse.  Immune-competent horses cleared parasites from the blood and suffered clinical signs of disease.  The immune-deficient horses were unable to clear the parasitemia and surprisingly, did not show any signs of disease (Sellon 2004)! One take home message was that immune cells were involved in parasite clearance as well and clinical signs.  Interestingly, the immuno-competent horses did not have parasites that could be recovered indicating the parasites were eliminated.  Parasites were recovered from the immune-deficient horses. Understanding disease involves animal models, these horse experiments show the immune system impacts the results of infection. We have a disease model that makes a normal horse act like an immune-deficient horse and that helps us understand the disease processes and the effects of some treatments.

Another telling experiment showed that anti-protozoal drugs can delay the production antibodies--but not prevent disease (Furr).  The message is that antibodies are not a measure of disease, but they are a method to associate disease to a particular organism.  Horses on “maintenance” doses of antiprotozoal drugs may test negative for antibodies yet have treatable disease.  One reason that antibodies are decreased (or delayed) is that some anti-protozoal drugs alter the protozoa’s expression of antigens that are used as markers in antibody tests. Using antiprotozoals to prevent antibody production is not a rational approach to preventing “EPM”.

A recent topic is the contribution of Sarcocystis fayeri to neuromuscular disease in horses.  Previously S. fayeri was considered a common and benign muscle infection in horses.  Researchers at UC Davis and Pathogenes believe that Fayeri may not be so benign. The UC Davis group associated a slight increase in muscle cysts in horses with neuromuscular disease (over the number of cysts found in clinically normal horses) while we associated neuromuscular disease with inflammation and S. fayeri antitoxin.

We have determined that autoimmune antibodies, not detected by S. neurona antibody assays, are present in some “relapse” horses.  These horses are not effectively treated with antiprotozoal drugs.  They respond to judicious use of steroids.  It is this disease process that showed us the obvious rationale for using a different drug to treat recrudescent EPM than was used to treat the primary presentation.  Of course, testing and defining the presence of autoimmune antibodies is as critical as the proper treatment.

No doubt these additional conditions found in the “EPM” horse are rare.  In our experience these diseases account for 50% of the horses that are diagnosed with the rare condition EPM.  We assayed sera from 7601 horses with neuromuscular disease and found only 3745 were “EPM” suspect.  The other 3856 were clinically ill due to undetermined causes that included autoimmune disease and S. fayeri toxins.

In the past two years we have identified 195 horses with autoimmune disease that have not, and would not, respond to any antiprotozoal treatment protocol.  We also identified 121 horses presumably suffering from S. fayeri toxins.  These horses become worse with some antiprotozoal treatment protocols.

It is important to understand the components of equine neuromuscular disease and focus less on branding these cases as EPM.  When EPM is the correct diagnosis understanding the inflammatory component of the disease process is important because pathological inflammation goes hand-in-hand with infection.

Call us with questions, we are happy to share our approach with you.

 

S. neurona, electron micrograph

Apicomplexan parasites are intracellular protozoa that are responsible for a great range of diseases in man and animals. The family Sarcocystidae contain the cyst-forming coccidia, cysts form in muscle tissues of the prey-host. Carnivores (prey) eat the infected muscle tissue and that completes the parasites lifecycle. It is generally thought that muscle cysts (sarcocysts) cause no pathology in the prey-host, except perhaps in debilitated animals. A compromised host may lack a robust immune response that can limit the infection. Exposure to protozoa, and the resulting immunity, builds resistance to infections. It is well known that overuse of antimicrobials and antiparasitic agents was an unwise strategy, it is no different in the fight against Sarcocystis. An unintended consequence of chemical prophylaxis is an animal with no natural immunity.

Sarcocystis fayeri produces sarcocysts in horse muscles and like most Sarcocystis infections, this finding is considered incidental. Early research dismissed S. fayeri as a factor in equine protozoal myeloencephalitis (EPM). Experimentally infecting ponies with S. fayeri and evaluating the immune responses convinced the researchers that infections by two species neurona and fayeri, were indistinguishable using the IFAT test. Important molecular tests identifying S. neurona resulted in 22,076 nucleotide sequences. In contrast, S. fayeri has 15 reported sequences-all are the small subunit ribosomal RNA gene. Variability in the small subunit ribosomal gene is useful to identify Sarcocystis species. Incorrectly, S. neurona and S. falcatula were reported as the same organism based on synonymous regions of this gene.

Our investigations reveal the need for a reassessment of the pathogenesis of S. neurona infections in horses and a need evaluating the role of the immune responses in equine disease. There are reasons that S. fayeri should get a more serious look.

Sarcocystis neurona causes muscle weakness in horses but the parasite isn’t thought to develop cysts in horses. There are four horse-related species that develop cysts in horses: S. asinus, S. bertrami, S. equicanis, and S. fayeri. Canids, including dogs, are the reported definitive hosts for these organisms.

Generally sarcocysts are not associated with inflammation in horse muscles (examined by histopathology), although in some debilitated horses, muscle degeneration is reported. Equine sarcocystosis, considered a mild disease, but can cause fever, apathy, anorexia, myositis, difficulty chewing, muscle weakness, autoimmune disease and sometimes hair loss. Surprisingly the profound muscle weakness exhibited clinically doesn’t correlate with the mild lesions observed by histopathology. And this leads some to hypothesize that there is a toxin associated with muscle infections.

The toxin idea isn’t new. There were reports of Sarcocystis-cyst toxins, called sarcocystine, in 1899. A toxin was isolated from cattle muscle cysts and characterized one hundred years later. Yet the possibility of muscle toxins causing disease in horses hasn’t been evaluated. Sarcocystine causes disease in people. A toxin found in raw horsemeat was associated with human food poisoning. The toxin was isolated from S. fayeri sarcocysts and toxic effects were evaluated in rabbits. The protein toxin, histopathological lesions, animal feeding experiments, rabbit enterotoxin assays, enzymatic digestion experiments, and heat/acid lability assays are similar between S. cruzi and S. fayeri-cyst toxins.

So far, initial research on the enterotoxin from S. fayeri sarcocysts is in humans. It would be interesting to explore a relationship between a S. fayeri sarcocystine and myositis in horses.

There is enough molecular information to investigate sarcocystine as a cause of muscle weakness in horses. The proteomics suggest the S. fayeri sarcocystine is homologous to proteins of Eimeria tenella and Toxoplasma gondii. Protein similarity, if high enough, would indicate conservation of the protein and a role in parasite survival. Protein similarity between organisms would also sink the protein as a good diagnostic to implicate an organism. The best it could be is a diagnostic for protozoal myositis.

It is possible that detecting the sarcocystine would benefit the treatment of sarcocystosis in horses.

Coccidiosis in horses is complicated. It is important to examine many factors before initiating practices that have unintended consequences. It took many animal infection studies to correct the false claim that S. neurona and S. falcatula were synonymous. False assumptions have plagued EPM research for 25 years. This has cost many horses their lives. Studies that examine the effect of equine coccidial infections and the immune response to infection should dominate the conversation. Initially clarifying the definition of EPM and the pathogenesis of disease are important.

Recent attention to daily prophylaxis to reduce antibodies against S. neurona may have unintended consequences in the disease EPM. One must weigh the need for antibody prevention (the consequence of prophylaxis) against the risk of neurologic disease and the consequences to the reduction of natural protective immunity against coccidiosis in the horse. Natural immunity holds S. fayeri in check and probably minimizes the effect of cyst toxins on the infected horse.

Overuse of antimicrobials led to superbugs. The human pharmaceutical industry will spend the many millions of dollars to develop new antimicrobials—if they can. The animal pharmaceutical industry will not spend any dollars on developing new anti-protozoals for the treatment of EPM, especially if there is a resistant superbug. Unintended consequences of prophylaxis may be the release of toxins from S. fayeri cysts. A veterinarian may misdiagnose a toxic event for an active infection—how can one distinguish these cases?

Reference paper: Ultrasound-guided cervical centesis to obtain cerebrospinal fluid in the standing horse  Anthony Pease, Ashley Behan, George Bohart Vet Radiol Ultrasound 2012 53(1) 92-5

In some cases a cerebrospinal fluid analysis is used to support a presumptive diagnosis of equine protozoal myeloencephalitis.  The presence of antibodies against S. neurona is considered, by some, as evidence that parasites are in the central nervous system.  Research done so far on on oocyst challenge infections fails to support this view.  We believe that the inflammatory component of the EPM syndrome should not be ignored.

There is a need to define cases that are associated with S. neurona and those that are unassociated with protozoa; for some clinicians a CSF tap may be the defining test.  Testing CSF fluid will help us obtain the data to make an assessment of the utility of CSF analysis in field selection of treatable cases of EPM in a statistically meaningful study.  We don’t charge for antibody determination on CSF fluid when it is submitted with a serum sample for SAG 1, 5, 6 testing.  The veterinarian-signed paperwork must accompany the submissions for the no charge CSF testing.

Resistance to field collection of CSF are justified.  The lumbosacral space is used for CSF collection in the standing horse.  The limits of this procedure are technical expertise, blood contamination, and the distance from the cranium, the proposed site of the lesion.  In the field the risk of trauma to the veterinarian is great due to the close proximity of the clinician to the rear limbs.

Alternatively, a sample can be obtained from the atlanto-occipital space under general anesthesia.  Experience with obtaining an AO tap is a plus with this technique.  The risk of injury during recovery from general anesthesia in an ataxic horse is high.  And transporting an ataxic animal is not advised.

 

The ultrasound-guided cervical centesis was used in normal horses in the above referenced paper and may be a viable alternative for field veterinarians that want a sample from an ataxic horse.  A brief survey of veterinarians indicates that this was not a procedure that is in common use for acquiring a CSF sample from an ataxic horse, therefore we are reviewing the procedure here. The hypothesis is  that obtaining a tap at C1-2, with assistance from ultrasound, may provide an alternative to collection of CSF in a standing horse.  The horses used in the Pease study were in stocks.  The horses were sedated with detomidine hydrochloride followed by morphine.  The area is aseptically prepared prior to the sample collection.  A nose twitch is used when the needle is placed.

A 10-4 MHz, microconvex curvilinear transducer (oriented dorsoventrally) is placed at the level of C1-2.  An 18g, 3.5 inch needle with stylet is advanced to the dura mater, in a dorsomedial direction approximately 4 cm through the dura mater (with the stylet) and into the subarachnoid space.  The CSF sample is then collected with gentle suction (changing syringes after the initial 5 ml decreases blood contamination).  Post collection observations are conducted for 48 hours.  We have a demonstration video of this procedure available on line. Call for information.