epm 2012 updated
TRANSCRIPT
EPM 2012EQUINE PROTOZOAL MYELOENCEPHALITIS
Understanding EPM CE coordinator Siobhan Ellison DVM PhD
Identify, treat, and prevent EPM in horsesRecognize Idiopathic Encephalomyelitis
Updated!
Understand the pathogenesis of the disease EPM
Email any questions to [email protected]
Topics Strategy Semantics The role of inflammation in EPM Treatment of parasites and inflammation Rehabilitation Cure, re-infection, relapse
CE Course Goals
Siobhan P. Ellison graduated from UF
BS 1972 Agricultural microbiology MS 1974 Equine Leptospirosis and Recurrent uveitis
Periodic ophthalmia –chronic relapsing inflammation DVM 1983-current -practice equine medicine Ocala, Florida PhD 1998-2000 Clone and Express S. neurona SAG 1
Founded Pathogenes, Inc. in 1999—EPM Strategy Developed S. neurona antibody tests using SAG 1, 5, 6 genes Developed Equine Model to produce EPM in horses Tested efficacy of licensed antiprotozoal drugs Re-discovered Decoquinate as a therapy for equine coccidia Developing therapies for treatment of neuromuscular disease in horses Recognized the role of inflammation in EPM
Identify infections Effective treatment
Immune modulation Prevent relapses
Solve the EPM problem
Strategy for EPM Case Management Involves Immune Modulation1-Antibody detection test that identifies phenotypes 1, 5, 6 of S. neurona 2-Identify the right condition/conditions3-Treat the condition4-Monitor and prevent relapse
Peer Reviewed PublicationsEllison, Siobhan and Lindsay, David. Decoquinate Combined with Levamisole Reduce the Clinical Signs and Serum SAG 1, 5, 6 Antibodies in Horses with Suspected Equine Protozoal Myeloencephalitis. 1, 2012, Int J App Res Vet Med, Vol. 10.
Ellison, SP., Witonsky, S. Evidence that antibodies against recombinant SnSAG1 of Sarcocystis neurona merozoites are involved in infection and immunity in Equine Protozoal Myeloencephalitis. Ca J Vet Res 2009 July:73(3): 176-183
Ellison, SP, Kennedy T, Li A, Schweiss L. Serum Antibodies Against a Reactive Site of Equine Myelin Protein 2 Linked to Polyneuritis equi found in horses Diagnosed with EPM. 2015. Intern J Appl Res Vet Med. 164-170.
Ellison SP, Kennedy TJ, Li A. Neuritogenic Peptides Derived from Equine Myelin P2 Basic Protein Detect Circulating Antibodies in Ataxic Horses. 2015. Intern J Appl Res Vet Med. 175-181.
Ellison SP et al. Development of an ELISA to detect antibodies to rSAG1 in the horse. J App Res Vet Med 1:4. 318-327. 2003
Ellison SP et al. Experimental infection of horses with S. neurona merozoites as a model for Equine Protozoal Myeloencephalitis. J App Res Vet Med 2:2. 79-89. 2004.
Ellison SP et al. Early Signs of Equine Protozoal Myeloencephalitis J App Res Vet Med 1:4. 272-278. 2003
Ellison SP et al. In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells. Vet Parasitol 95: 251-61. 2001.
Ellison SP et al. Molecular characterization of a major 29 kDa surface antigen of Sarcocystis neurona. Int J Parasitol 32: 217-225. 2002.
Yang J., Ellison S., Gogal R., Norton H., Lindsay D D., Andrews R., Ward R., Ward D., Witonsky. Immune response to Sarcocystis neurona infection in naturally infected horses with equine protozoal myeloencephalitis. 2006 Jun 15;138(3-4):200-10. Epub 2006 Mar 23.
Witonsky S., Ellison S., Yang J., Gogal R., Norton H., Yasuhiro S., Sriranganathan N., Andrews F., Ward D., Lindsay D. In vitro suppressed immune response in Horses experimentally infected with Sarcocystis neurona J Parasitol. 2008 Mar 12:1.
Updated!
We're basically agreed; let's not quibble over semantics.
EPM: Horse with parasites in CNS (could be obtained by culture).
Presumptive EPM: Horse with neurological signs Response to treatment
Sarcocystosis has signs of disease and Sarcocystiasis does not.
Sarcocystiasis: Sarcocystis (neurona, falcatula, fayeri) in gut or organs infection and induces immune response; subclinical disease
IE: Idiopathic encephalitis due unknown etiology
Species specific: Mutually exclusive display of SAG 1, SAG 5, or SAG 6 Horse only “sees” serotype
Genus markers: Variable SAG 2, 3, 4, and microsatellite markers
S. neurona: One of 3 serotypes, or 6 genotypes or 12 antigen types
Strains: Defined by genotype Horses can’t distinguish genotype
Virulence: Infects horses, neuro-virulent can invade CNS
Disease Overview EPM is a uncommon neurological disease of horses in the United
States and South America. Sarcocystiasis is a common infection in horses—horses commonly have antibodies.
Inflammation causes the signs associated with EPM and IE.
USDA has strict definitions for EPM, diagnostic tests, kits, and antibody tests. There are no USDA licensed EPM diagnostic tests.
Since 1970 EPM cases have been reported in 48 States.
Horses are infected by the parasite when ingesting hay, feed or grass contaminated by opossum feces.
Our strategy to identify, treat and prevent disease
Identify horses that have or are at risk for EPMIdentify presence of antibody in serum
Identify serotype of S. neurona causing signsELISA –quantative
Treat parasite and inflammation for responseSigns and antibody= institute treatmentInflammatory treatment response in horses in 5 days
Identify autoimmune diseaseMP2/MPP antibodiesTreat with levamisole and steroids
Stimulate protective immunityserotype independent
Updated!
The equine population is 7.2 million
Sarcocystosis-disease Morbidity of EPM is 0.88%
of the population (63,360 horses) with active disease that need treatment/year
Frank Andrews LSU
Sarcocystiasis-exposure 62% of the equine
population (4,464,000)have infections and are at risk to develop EPM. USDA 2001 Consensus statement
11%
38%
11%11%
30%
Antibodies against Sarcocystis in Normal Horses
Mixed Negative SnSAG1 SnSAG5 S. falcatula
Two antigen types are responsible for most of animal disease. Only SAG 1 and 5 phenotypes cause EPM in horses. Sarcocystis neurona is defined by 35 genotypes, 12 antigen types and three
(phenotypes).
SAG 1• Genotype II• Genotype VI
SAG 5• Genotype I• Genotype III• Genotype V
SAG 6• Genotype IV
Drug treatment is difficult unless the serotype is identified. Treatment fails in triazine resistant strains. Peptide 1, 5, 6 ELISA’s identify serotype and can assist therapy decisions.
PHENOTYPE
Genotyping involves sequence of SAG’s (1, 3, 4, 5, 6) 9 microsatellite markers, and 33 microsatellite types. SAG’s 2, 3, 4 are common to all Sarcocystis
Serotype testing yields more information
It takes 10x more triazine drugs to kill SN2 and SN138 strains than the SN6 genotype.
Severe clinical EPM cases are 92.8% SnSAG 1 phenotypes
SAG 1• Genotype II• Genotype VI
SAG 5• Genotype I• Genotype III• Genotype V
SAG 6• Genotype IV
SN2, SN3, SN5, SnUK-3, SN744, SnCAT2, Sn15 OP
SN6, SN4, SnMu1, SnVT-1, SN7, Sn138, SNOt1
SnOTg
SnSAG 1 phenotype Pathogenes model
used SnSAG 1 phenotype induced moderate to severe disease 93% of horses (n=75) S. neurona SnSAG 1 enters CNS recover by isolation All animals produced antibody, titer that increased over time
93% of the published isolates from horses are SnSAG 1 phenotypes.
Neurovirulent in the horseStudies confirm the resistance of some SAG 1 and SAG 5
strains to triazine drugs. Field trial, experimental trials, in vitro
Published study showed the laboratory effectiveness of vaccination against rSnSAG 1 challenge prevents the clinical signs of EPM
SnSAG 5 phenotype Ohio model
used SnSAG 5 phenotype (SN138) induced mild disease in the transport studies S. neurona SnSAG 5 didn’t enter CNS in their studies
Inflammatory encephalitis associated with S. neurona Some horses resolved infections by the end of the
trial
Only 7% of the published isolates from horses are SnSAG5 phenotypes.
Studies by Furr showed 40% of horses on Marquis therapy (5 mg/kg) prior to infection were not protected against disease.
SnSAG 6 Not yet found in horses…a hint that cross-protection
exists?
Responsible for epizootic in sea otters killing 1/3 of the California population in 2004
Horses have antibodies to SnSAG 6
Some horses with pure SnSAG 6 antibody evidence of infection are ataxic and these horses respond to treatment
Serotype SnSAG 6 is prevalent in horses. NOV. 2010
SAG124%
SAG59%
SAG631%
SAG 1&65%
SAG 1, 5 &617%
SAG 1&515%
Phenotype Distribution in EPM Horses
Frustrating FactsAntibody titer increase over time correlates with active disease.
No vaccine is on the market, not commercially viable
Effective therapy can induce protective immunity
Treatment with FDA licensed drugs fail in 61% of casesSuccessful treatment with FDA licensed drugs is less than 25% Rob
MacKay UF
Relapse is 50% in horses successfully treated with FDA approved drugs. Some say parasites may encyst…feeding horse tissue to opossums did not result in oocyst shedding—(Cutler UF)
Peptide ELISA detects the presence of antibodies, three serotypes of S. neurona, in serum
The Peptide ELISA test determines:
-presence of parasites by serotype-triazine resistant infections-pyrimethamine/sulfa resistant strains-endpoint to treatment-outcome-prevention strategy
1 2 3 4 5 6
Lege
nd 1
Lege
nd 2
Lege
nd 3
864 64 64
128
8
128
32 48
64128
25664
256 4
216
256
12864
64
64
32
32
8 16
ELISA Results for 8 horses with signs of EPM
SnSAG1 SnSAG5 SfSAG6
THERE IS NO STATISTICAL ADVANTAGE TO DETERMINE ACTIVE INFECTIONS BY CSF ANALYSIS SIOBHAN P. ELLISON
Clinical exam is the most important factor for identification of EPM and treatment efficacy for EPM suspect horses
Oroquin-10 field trial using compounded drug
Oroquin-10 Marquis Protazil ReBalance0
10
20
30
40
50
60
70
80
90
100
EPM drugs comparing efficacy, treatment duration
Efficacy Treatment duration
Decoquinate/levamisole in 150 horses (no adverse events reported).
Resolved--Tx duration
Legend is an 11 YO gelding that went from severe ataxia to good with 10 days treatment with Oroquin-10.
This horse had complicating idiopathic encephalitis
2015 UPDATE: AUTOIMMUNE DISEASE!!! SEE COURSE 3Updated!
Success No change 60% grp0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
94.2%
5.8% 12.7
IE
The IE Group IS INFLAMMATORY DISEASE
Chronic disease Unresponsive to Triazine or pyrimethamine/sulfa
Levamisole therapy alleviates signs in this
group
Trial results for 141 horses
RELAPSE: 12% ON OROGIN VERSUS 55% REPORTED WITH OTHER ANTI-
PROTOZOAL DRUGS
successful treatment of horses requires recognizing disease that is encephalomyelitis
due to S. neurona versus inflammatory etiologies:
IE = idiopathic encephalomyelitis
Some horses need longer duration levamisole and some have autoimmune disease.
2015 UPDATE: Levamisole in paste or liquid degrades by day 4 and is proinflammatory!!! Formulation matters.
Updated!
Enrollment criteria 012092:
Veterinarian trained on the protocol—complete EPM 2012 courseS. neurona antibody, clinical signs of EPM, not treated within 72 hours, 2-15 yo
Enrollment criteria 012219:
Veterinarian trained on the protocol—complete EPM 2012 courseTreated horse experiences “relapse” with conventional treatment within 90 daysPrior to EPM treatment horse was diagnosed with serum and CSF antibody
Levamisole has a place in the treatment of EPM if parasites are effectively killed with an antiprotozoal.
Ongoing trials: 012092 & 012219
Updated!Levamisole has an agonistic effect on PROTOZOAL
cholinergic receptors. This may cause sustained parasitemia
Identify, treat and prevent diseaseIdentify horses that have or are at risk for EPM
Identify presence of antibody in serum
Identify serotype of S. neurona causing EPMPeptide 1, 5, 6 ELISA –quantative
10 day therapySigns and antibody= institute treatmentTreatment response in horses in 5 days or lessIdentify idiopathic encephalomyelitis
Prevention therapy goal by stimulating protective immunityserotype independent
2014 UPDATE: SERUM CRP IDENTIFYS INFLAMMATION AND/OR AUTOIMMUNE DISEASE
Updated!
EPM suspect
ELISA Titer
Orogin
OK within 10 days Not 100% OK
ELISA titer 8-16 weeks 60% improve with plateau No clinical or Ab change
Re-exposure possible? Prevention Continue diagnostics
Antibody still elevated
Change duration Tx
Antibody decreases, slow imp. no progress, plateau, worse
LevamisoleDMSO IV, Steroids,
Levamisole
Idiopathic Encephalitis , determine etiology
94%
6%
13%
3%
Decision Tree
Updated!
Sarcocystis neurona Pathogenic protozoa Equine Protozoal Myeloencephalitis
EPM = parasites + INFLAMMATION Three unique serotypes: SAG 1, 5, 6
SAG 1 & SAG 5 are responsible for the majority of animal disease (Wendte 2011)
REVIEW
Genotype vs Phenotype Genotypes (strains)
defined by 35 molecular markers Can’t discern genotype clinically
Phenotypes are defined by 6 surface antigens SAG 1, 5, 6 are unique to S. neurona Discern serotypes by antibody ELISA, SAG 1,
5, 6 Establishment agrees it is unlikely there will
be any new serotypes of S. neurona discovered
REVIEW
Updated!
Antibody is made to serotypes
Antibody is detected by serum antibody test
SAG 1, 5, 6 ELISA is reported as a titer A change in titer can identify active
disease Antibody can indicate resistance to
treatment
REVIEW
S. Neurona induces inflammation
Signs of EPM are due to inflammation Established in 1985, proven in 2001-2007
Inflammation in CNS +/- parasites Inflammation can be reversed quickly
Inflammation is mediated through several pathways Prostaglandin mediated inflammation responds
to NSAID’s and steroids S. neurona induces IL6 inflammatory path that
is unresponsive to NSAID’s
REVIEW
S. Neurona neuroinflammation responds to levamisole
Remove parasites Requires a protocidal drug
Treat inflammation with levamisole Chronic inflammation
Inflammation longer than 30 days Also responds to levamisole May have autoimmune disease
Diagnose with CRP Update!New information starts at slide 45…
Is S. Neurona neuroinflammationmediated through the cytokine IL6?
We determined that levamisole treatment works in chronic/ relapsing cases of EPM.
Circumstantial evidence:
How does levamisole affect IL6?What is chronic/relapsing EPM?
Static drugs don’t prevent EPM…
Prophylactic administration of ponazuril reduces clinical signs and delays
seroconversion 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
…and don’t treat inflammation
“These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but
does not eliminate, infection and clinical signs of EPM in horses.” Furr M,
McKenzie H, Saville WJ, Dubey JP, Reed SM, Davis W. J Parasitol. 2006
Jun;92(3):637-43
Identify the cause and then treat inflammation
Test to identify parasite antibody SAG 1, 5, 6 Eliminate the cause of disease Remove parasites with a protocidal drug
Test to determine level of inflammation C-reactive protein ELISA Modulate the immune response with
levamisole
Update!Detect autoimmune disease with MPP/MP2 ELISA. Diagnosis alters treatment strategy
OUR THEORY IS THAT S. NEURONA INDUCES IL6 MEDIATED NEUROINFLAMMATION THAT IS
SENSITIVE TO LEVAMISOLE
We’re still up in the air on a few details…
Orogin Neuroquel Levamisole0
5
10
15
20
25
30
Provisional di-agnosis of EPM
Improved No change
A second population of horses with ataxia (no S. neurona antibody) have idiopathic encephalomyelitis…
These horses respond to different therapy. But before we discuss that…
…but more drug is not better…
because… (the next 8 slides explain our theory)
C-reactive protein measures inflammation
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. neurona Antibody against S. fayeri toxin is statistically
related to increased serum CRP Normal Pneumonia Enteritis Arthritis
EPMN= 10 10 10 10 139Av CRP 7.4 19 16 11 17
Updated!
NeuroinflammationIdiopathic Encephalitis
IL6 is a pro-inflammatory cytokine IL6 works in the centrally and peripherally IL6 crosses into the brain coupled to a soluble
IL6 receptor
pathogenIL6
Serotonin
IL6 is inversely related to the neurotransmitter serotonin PHARMACOLOGY AND
EXPERIMENTAL THERAPEUTICS VOL 327:316-323 2…
Serotonin is released from the pre-synaptic neuron where it crosses the synaptic cleft and attaches to the post-synaptic receptors.
Serotonin is recycled in the cleft by SERT, serotonin transporter protein
SERT
IL6
serotonin
The presence of serotonin is necessary for expression of IL6, low levels induce and higher levels decrease IL6.
Net effect is high IL6 is pro-inflammatory
PARASITES+INFLAMMATION=EPM
Hey! Let’s treat inflammation! Ellison 2010
Establishment 1985
SERT
serotonin
IL6Aminorex
Aminorex binds SERT, the transporter protein, releasing serotonin to the post synaptic neuron and an increase in serotonin which turns off IL6
LEVAMISOLE METABOLISM IN THE HORSE
Levamisole Aminorex
90 minutes
serotonin
IL6Levamisole inhibits the production of IL6-RNA for 48 hoursUpdated!
Not so fast, my friends…Serotonin acts as a negative feedback molecule on autoreceptors located on the pre-synaptic neuron Serotonin receptors decrease the production of serotonin when it is too high
Levamisole is an indirect serotonin agonist and a cholinergic agonist in the horse, its effects are immediate, within hours.
Summary Levamisole in low doses may
modulate neuroinflammation via serotonin or in higher doses, may turn off the production of serotonin! High doses are proinflammatory.
C-reactive protein is an enzyme, stimulated by IL6, also cleaves IL6 into a soluble receptor that—when bound to IL6 attaches to cells and promotes inflammatory disease.
Horses with IE, idiopathic encephalomyelitis, may need a longer DURATION of levamisole or steroids if autoimmune disease is present.
Orogin Neuroquel Levamisole0
2
4
6
8
10
12
Provisional diagnosis IE
Improved No change
Treatment responses may indicate that there are two populations of ataxic horses
Orogin Neuroquel Levamisole0
2
4
6
8
10
12
Provisional diagnosis IE
Improved No change
Orogin Neuroquel Levamisole0
5
10
15
20
25
30
Provisional diagnosis of EPM
Improved No change
The underlying condition must be treated to get a response to treatment.
Effectiveness Trials Learn more at
pathogenes.com
Orogin® Field Study
NeuroQuel® Field Study
Orogin® Field Study Enrollment:
Call to discuss the case with us Complete
Qualification record Owner Consent record Gait Assessment Score sheet
Costs for participation are case dependent and vary with the trial (placebo controlled, no placebo)
NeuroQuel® Field Study Enrollment:
Call to discuss the case with us Complete
Qualification record Owner Consent record Gait Assessment Score sheet
Costs for participation are case dependent and vary with the trial (placebo controlled, no placebo)
Seeking FDA ApprovalFDA license assures products provide a safe and
effective treatment. The licensing process is expensive and time consuming for us. The rewards for participation in our field trials outweighs the required paperwork.
Our 16 years of research redefined EPM and provided solutions for you and your clients. Your participation in these final steps to licensed products for horses supports our ongoing research. If you participated and completed our field work you are a member of our VIP program.
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