Siobhan Ellison• B.S. Microbiology• M.S. Agriculturual Microbiology• D.V.M.• Ph. D. Molecular Biology
About Pathogenes• Developing patent protected technologies for thediagnosis and treatment of disease since 1999
When Pathogenes Started• EPM was cloaked in mystery.• The source of EPM wasn’t known. The natural host wasn’t known.• There were no definitive tests, no effective treatments.• EPM was a late finding in animals with severe neurologic signs.• EPM was largely a diagnosis of exclusion, made after all other causes ofataxia had been ruled out.
When Pathogenes started• Other Sarcocystis species confounded research. Papers were publishedwith what are now known to be mixed cultures. These old papers canstill cause confusion.• Research was hampered by researcher’s inability to reliably infect studyanimals. Even massive doses of parasites only resulted in about 3%infection rate.
Pathogenes has• Devoted 17 years of research focused on EPM in horses• Developed techniques to infect 93% of study animals• Developed methods to grow S. Neurona in tissue culture• Recovered S. Neurona from the CNS in experimentally infected animals• Fulfilled Koch’s Postulates using S. Neurona as the cause of EPM in horses• Owns the sole patent for the “model of disease” for EPM in horses• This model is necessary for proving effectiveness of treatment
PATHOGENES• Developed laboratory assays for identification of phenotypes, and assaysto evaluate effectiveness in treatment of affected horses• Defined effective drugs for treatment of EPM in horses• Actively supports clinicians in the field with guidelines, educationprograms, and live telephone support• Developed tools for identification of S. Neurona infection in marinemammals• U.S. Navy service animals are used naval operations• Pathogen identification in unexplained marine animal deaths
EPM and Sarcocystis neurona• Sarcocystis Neurona is the proven cause of Equine ProtozoalMyeloencephalitis (EPM) in horses.• S. Neurona is a pathogenic parasitic protozoa. The natural host is theopossum. Horses are exposed to opossum feces in the field while eating.• In general, parasites cause mild disease in their native hosts• Horses are not part of the normal life cycle of the parasite makingthe horse an aberrant or “dead end” host• S. Neurona causes devastating neurologic illness in horses
Genotype vs Phenotype• Genotypes (strains) refer to DNA sequences, defined by 35 molecularmarkers• Genotype identification requires molecular biological tools (DNAsequencing) available for research, but not useful for medical testing• Phenotype refers to parasite expressed antigens• Phenotype patterns identify specific species of Sarcocystis.• Identification of phenotype is clinically very useful• “SAG” stands for Surface Antigen• Phenotypes are defined by 6 surface antigens, numbered 1-6• SAG 1, 5, 6 are unique to S. neurona
S. Neurona in horses• There are three unique phenotypes:• SAG 1• SAG 5• SAG 6• SAG 1 & SAG 5 are responsible for the majority of animal disease(Wendte 2011)
Antigen vs Antibody• An ANTIGEN is produced by the parasite.• An ANTIBODY is produced by the horse immune system in response tothe antigen.• Our tests look at the amount of antibody the horse immune system isproducing
Antibody is made to phenotypes• The infected horse makes an antibody to the surface antigen.• S. Neurona antibody is detected by serum antibody tests• Pathogenes has developed antibody tests. They’re patented. All S. Neurona antibody tests usePathogenes testing products.• The antibody test is a highly sensitive and specific ELISA test• SAG 1, 5, 6 “ELISA” is reported as a titer which shows how muchantibody is in the animal’s serum.• The higher the titer, the more antibody there is in the serum• We measure at intervals. A change in titer over time• can identify active disease• can be used to evaluate response to treatment
Does a positive antibody test mean a horse has epm?• No, it only means a horse has been exposed to S. Neurona• Most horses exposed to S. Neurona will not develop EPM• 80% of horses with EPM will have a positive antibody test• It takes 17 days from infection to positive antibody tests. The 20%either are tested before the 17 days, or exposure to antiprotozoal drugshas delayed antibody production.• The titer will rise with active infection and it will fall after the parasitesare killed• We now know that EPM = parasites plus INFLAMMATION
Inflammation• Recognition of the role inflammation is a breakthrough• For many years all research was dedicated to the parasite…• and none to inflammation
S. Neurona Induces Inflammation• Clinical signs of EPM are due to inflammation• Established in 1985, proven in 2001-2007• Inflammation in CNS may or may not be accompanied by parasitesin the CNS
Why Static Drugs Don’t Work“These results confirm that treatment with ponazuril at 5.0 mg/kgminimizes, but does not eliminate, infection and clinical signs of EPM inhorses.”Furr M, McKenzie H, Saville WJ, Dubey JP, Reed SM, Davis W. J Parasitol. 2006Jun;92(3):637-43Static drugs such as Marquis® and Protazil® do not kill the parasite, they slow or stopgrowth of the parasite, relying on the horse immune system to kill the parasite.Unfortunately, the parasite down-regulates the immune response in the horse, so static drugsdo not clear the infection. Decreasing the parasite load may decrease symptoms but relapseis very common.Static drugs do not treat inflamation.What is needed is a drug that kills parasites and another drug that treats inflammation. Thatis the Pathogenes approach.
Static Drugs Won’t Prevent EPM, But They Will Delay It…Prophylactic administration of ponazuril reduces clinical signs and delaysseroconversion in horses challenged with Sarcocystis neurona.Furr M, McKenzie H, Saville WJ, Dubey JP, Reed SM, Davis W. J Parasitol. 2006 Jun;92(3):637-43
S. Neurona CAUSES NEUROInflammation• Remember that inflammation can be reversed quickly• Remember that “inflammation” is a very general term• Inflammation is mediated through several pathways• Prostogandin is only one of the commonly known pathways,• Prostaglandin mediated inflammation responds to NSAID’s and steroidsbut• IL6 is a different pathway, lesser known, but very important in EPM• S. neurona neuroinflammation is not prostaglandin mediated• S. neurona induced neuroinflammation does not respond to NSAIDS
S. Neurona Neuroinflammation Responds toLevamisole• To treat EPM acutely:• Remove parasites with a protocidal drug• Stop inflammation with levamisole• Then you must recognize and treat chronic inflammation• Chronic is inflammation present longer than 30 days• Chronic inflammation also responds to levamisole
To Treat EPM• IDENTIFY the cause• ELIMINATE the cause• TREAT INFLAMMATION• REPEAT TITERS• REPORT RESULTS BACK TO PATHOGENES• Test to identify parasite antibody SAG 1, 5, 6• Eliminate the cause of disease with a protocidal drug• Test to determine initial level of inflammation• C-reactive protein ELISA• Modulate the immune response with levamisole• Follow up test to follow antibody levels and inflammation• Report your results. You are part of the research.
Is S. neurona neuroinflammationmediated through the cytokine IL6 ?or could we be putting the cart before the horse?or could we be putting the cart before the horse?• We determined that levamisoletreatment is effective even in chronic,relapsing cases of EPM.• Levamisole is thought to be effective intreatment of IL6 mediated inflammationin other models• We infer that IL6 is probably themediator of EPM neuroinflammationOnly circumstantial evidence:
We feel that S. neurona induces IL6 mediatedneuroinflammation that is sensitive to levamisole
We’re still up in the air on a fewdetailsBut we know, if it isn’t the rightdrug...more drug is not better
C Reactive Protein• A non specific indicator ofinflammation
C-reactive protein measuresinflammation• CRP is an acute phase protein• Production is stimulated by IL6 cytokine• Normal 7.4 µ/ml; High is over 10 µ/ml• Not specific to S. neuronaNormal Pneumonia Enteritis Arthritis EPMN= 10 10 10 10 139Av CRP 7.4 19 16 11 17
Neuroinflammation isInflammatory Encephalitis• IL6 is a pro-inflammatory cytokine• IL6 works in the periphery• IL6 crosses into the brain (receptor mediated)• IL6 is inversely related to the neurotransmitter serotoninpathogenIL6Serotonin
• Serotonin is released from the pre-synaptic neuron where it crosses the synaptic cleft and attaches tothe post-synaptic receptors.Serotonin is recycled in the cleft by SERT, serotonin transporter protein
Parasites+inflammation=EPMWe looked at the other half ofthe equation, the inflammationhalf. Ellison 2010Establishment 1985
Levamisole metabolism in the horse• Levamisole Aminorex90 minutesserotoninIL6Levamisole is metabolized to Aminorex so the studies of Aminorexshould be considered when studying LevamisoleAminorex increases serotoninand decreases IL6
SERT is the transporterprotein that is responsible forserotonin reuptake.Aminorex binds SERT, thetransporter protein.When reuptake is inhibited,there is more serotoninavailable in the cleft tostimulate the post synapticreceptorsAn increase in serotonin turns
But there’s more to the storySerotonin acts as a negative feedback molecule on autoreceptorslocated on the pre-synaptic neuron. This decreases the productionof serotonin when serotonin is high.So we increase serotonin but the feedback mechanism responds byadjusting the level back downwards.Levamisole is an indirect serotonerginicagonist in the horse, its effects areimmediate, within hours.
Our tools that help with the diagnosis and treatment of EPM• SAG 1, 5, 6 ELISA testing• Multiple testing to follow antibody levels• CRP ELISA for following inflammation levels• Measures C-reactive protein that is increased in response to IL6inflammation• Multiple testing to follow inflammation• Decoquinate/levamisole for acute control of EPM• Decoquinate is a protocidal drug that does clear infection• Levamisole for treatment of neuroinflammation
Seeking FDA Approval• INAD 012092• decoquinate/levamisole tablets• Patent protected• INAD 012219• levamisole tablets for horses• Field efficacy trials are ongoing• Contact us to enroll an animal into our field trials
Main Points• As you know, previously recommended drugs don’t work• Bigger doses don’t help
Main Points• EPM is now treatable and curable• EPM is caused by parasites but clinical signs come from inflammation• You must treat both infection and inflammation• We have information and tools to guide your treatment