CE Course 0007692 FL 3 hours
The understanding of diagnosis and
treatment of Equine Protozoal
Pathogenes Inc. Siobhan P. Ellison DVM PhD
Introduction and Basics
• Sarcocystis are protozoa that cause disease in animals.
• THIS DISCUSSION IS LIMITED TO SARCOCYSTIS
• Infection is called sarcocystiasis
• Infections with protozoa present in the diseased
animal is called sarcocystosis
Incongruity between S. neurona infection (antibody in serum)
and the occurrence of clinical disease (EPM) has frustrated
researchers and clinicians.
• Merozoites, the asexual stage of S. neurona, can
be found in the CNS of horses with
sarcocystosis. This is rare.
• Sarcocystiasis results in horses with serum
antibodies against S. neurona.
• 60-80% of horses have detectable antibody to strains that may or
may not cause EPM
Sarcocystiasis vs. Sarcocystosis
• The terms distinguish infection, past or present,
determined by antibody [sarcocystiasis] and the active
parasite in a diseased animal [sarcocystosis].
• Both conditions are identified by clinical signs
• Treat for parasite or inflammation based on testing.
• Each commercial diagnostic test measures different
Treating for infection or inflammation…
• Serial testing can determine if the protozoa is present by
observing an increase in titer after treatment
• Targeting specific inflammatory paths is beneficial both
sarcocystiasis and sarcocystosis
• If no protozoa are present treat the inflammation…
• Anti-protozoal won’t treat inflammation
• Inflammation can present as relapse cases!
A Brief History
Our understanding is based on some incorrect notions that are important
• Sarcocystis are generally named from the stages of the
lifecycle of the parasites within the hosts they infect:
• The intermediate host that harbors the cyst stage or
• the definitive host that sheds sporocysts.
• A combination of the two hosts indicates the life
cycle of the parasite.
Exception to the Rule:
• Sarcocystis neurona was named for the asexual stage of
an undefined protozoa found in a horse with
sarcocystosis in 1991 (Dubey):
• “The name S. neurona is suggested, however the final
identity of the cultured organism will have to be
confirmed with further study.”
• Concentrating on a parasite in the CNS changes diagnosis and
expectations for treatment.
• Naming S. neurona before the hosts were known complicated
the ensuing science.
• Molecular and biological differences (that were beyond our
understanding) provided confounding information that
impact the diagnosis of EPM.
• The name S. neurona presumes the organism has a
predilection for the CNS that it does not show. S. neurona has
a predilection for the leukocyte!
Parasites are not always present in the CNS of EPM horses.
• The expectation that parasites are in the CNS
decreased the importance of inflammation to the
clinical signs of EPM.
• Inflammatory responses induced by Sarcocystiasis are
responsible for the clinical signs of EPM.
• The leukocyte and its inflammatory responses are
required for clinical signs of EPM.
Several species of Sarcocystis cause clinical signs of EPM.
• The opossum (Didelphis virginiana) is the definitive host for
• Several intermediate hosts support the life cycle of S. neurona
• The armadillo (Dasypus novemcinctus) and the raccoon (Procyon
• S. falcatula does not cause EPM (Cutler).
• S. dasydidelphis and S. procyondedelphis* cause inflammation but
not organisms in the CNS. (Dubey 2001).
• * proven by experimental infections
An abnormal neuro exam is the hallmark
• Idiopathic encephalomyelitis clinical signs due
unrecognized etiology-often identified as EPM.
• is a sequel to sarcocystiasis or sarcocystosis…and other
infections such as Lyme
• EPM is diagnosed by physical and neurological
examination, supported by ancillary diagnostic tests
to determine etiology.
Overview of sarcocystiasis
• Horses ingest sporocysts, visceral infections stimulate
immune responses (antibody and inflammatory
• Most horses control and eliminate infection, antibody
is produced, inflammation can persist after infection.
• S. procyondidelphis infections can cause inflammation
but not always disease.
Overview of sarcocystosis
• Protozoa are transported to the CNS from the gut or
the viscera to the CNS by leukocytes.
• The strain of the organism may be crucial to
invasion into the CNS.
ACVIM Consensus 2002
• The consensus opinion was intended to serve as an
aid to equine clinicians attempting to establish a
diagnosis of EPM in horses presented for evaluation
of neurological disease.
• The ACVIM 2002 consensus opinion is outdated!
ACVIM consensus 2002
• “Thorough physical and neurological examinations
are the primary and most important diagnostic
procedures for evaluation of horses suspected of
• “Finally, a favorable response to treatment, especially
when subsequently followed by a relapse of similar
clinical signs, is also supportive of a diagnosis of
EPM in the living horse.”
• “EPM” tests detect antibodies to S. neurona.
• Tests detect IgG or IgG & IgM.
• “EPM” tests do not detect inflammation.
• Tests are “validated” on characterized sera based on
the laboratory criteria that run the tests.
• Antibody detection is based on antibody to S. neurona
• Serum or CSF can be used in tests
• Some tests strongly suggest CSF to strengthen interpretation
• All antibody tests use S. neuronaSAG1 strains!
• C-reactive protein detects inflammation due to active
infections (protozoa, virus, bacteria)
• “The reluctance to perform a spinal tap due to risk,
cost or inexperience is understandable and although
not the preferred approach, a positive serum IgG test
in the presence of neurologic signs and history
compatible with EPM, supports a diagnosis of
EPM.” ACVIM 2002, Cornell 2011
• Some tests: IFAT, WB, and SAG 2, 4/3 strongly suggest CSF
testing to strengthen “probability” that antibody relates to EPM
Two Inclusive tests
• Inclusive tests use antigens common to all Sarcocystis
as diagnostic antigens.
• Down side: cross-reactivity of antibody against
common antigens is an issue.
• IFAT, Western Blot, SAG 2, 4/3 ELISA
• Strongly advise using CSF to increase diagnosis
• Exclusive tests use antigens only displayed on S.
• Down side: the possibility that a rare, undiscovered
strain exists that displays an alternate SAG antigen.
• SAG 1, 5, 6 ELISA’s do not require CSF
Western blot (WB) Parasite proteins are separated by molecular weight into reactive
IFAT Parasites are immobilized on slides
ELISA Plates are coated with immunodominant, purified recombinant
This is an example of a western blot taken from Sarcocystis of
Animals and Man. The blot shows the difference in protein
bands from several stages of parasite growth. The M 36 lane
represents the proteins seen at 36 days of culture while the M
60 lane shows the proteins after 24 more days of culture.
This exemplifies the difficulty of homogenous antigens for
Antigen selection for diagnostic tests
WB Inclusive Semi-quantative; subjective interpretation of various “bands”
that vary with the testing laboratory.
IFAT Inclusive Subjective interpretation of indirect detection of surface
immunofluorescence. Antigen detection varies with fixation
Inclusive Quantative, detects SAG’s 2, 4/3 shared by Sarcocystis, is not
1, 5, 6
Exclusive Quantative, detects SAG’s 1, 5, 6 unique to pathogenic
Sarcocystis neurona, phenotype specific
Surface antigens used in tests
IFAT SnSAG1 S. neurona (UCD1)
EBI WB SnSAG1 S. neurona (Sn2)
Neogen WB SnSAG1 S. neurona (Sn2)
The rSAG1 ELISA SnSAG1 SnPath1 (equivalent to UCD 1 based on nuclear and aa
sequences, mAb binding
rSAG 5 ELISA Synthetic DNA based on published sequences for SnSAG5 (horse)
rSAG 6ELSA Synthetic DNA based on published sequences for SnSAG6
SAG2, 4/3 ELISA SnSAG1 (SN3)
All tests use SAG 1 phenotype S. neurona! To detect other phenotypes the
phenotype antigens must be used (SAG 5 and SAG 6 ELISA) or antigens
common to all Sarcocystis must be used, this decreases the specificity of the
tests (IFAT, SAG 2, 4/3 ELISA).
Test Validation for S. neurona
• Validation methods vary widely.
• True positives (TP) are from horses with parasites
confirmed in the CNS at post-mortem exam.
• “EPM” often means that inflammatory lesions, but
no parasites were confirmed, in the CNS of horses.
Validation of tests
Test Validation samples obtained from:
WB (Granstrom 1993) Several hundred necropsies, field cases
WB (Rossano 2000) Six (6) true positive EPM horses
IFAT Eight (8) true positive EPM horses
SAG 1 (Ellison 2001) Six (6) true positive EPM horses
SAG 5, 6 (Ellison 2010) Several thousand field cases
SAG 2, 4/3 (Howe 2013) 44 “EPM” by inflammatory lesions
Researchers validate tests using the most pure “true negative” and
“positive” samples that represent the antigen used in the detection method.
However, using disease free animals from an endemic area for the “true
negatives” lowers the specificity of tests.
Recommended diagnostic sample
Test Recommended for best interpretation
WB (Granstrom 1993) Serum and uncontaminated (RBC) CSF
WB (Rossano 2000) Serum and uncontaminated (RBC) CSF
IFAT Serum and uncontaminated (RBC) CSF
SAG 1 (Ellison 2001) Serum
SAG 5, 6 (Ellison 2010) Serum
SAG 2, 4/3 (Howe 2013) Serum and uncontaminated (RBC) CSF, AI or C-value to
determine contamination of CSF. “Serum titer alone is
poor indicator of disease.”
Interpretation of results
• Presence of antibody indicates sarcocystiasis.
• In most cases, antibody and clinical signs indicate
• A negative result, except in acute onset or a treated
horse, indicates no infection.
The significance of the test results
WB Negative, suspect, weak positive, positive.*
WB(Rossano) Negative, suspect, weak positive, positive.*
IFAT Based on mathematical modeling/simulation. Cross reactivity with non-pathogenic S.
ELISA SAG 1, 5, 6 Titer >8 on a phenotype is a positive test. Coupled with CRP indicates active
ELISA 2, 4/3 Serum:CSF <100 indicates antibody in CNS, serum titer > 4000 “correlates better
with EPM” Cross-reacts with SFCornell SAG 4 but not SFFla1 SAG 4
*ACIVM comment specific to WB: “In situations in which test sensitivity
and specificity are determined from samples that had neurological disease
(suspect EPM) incidence of disease is high leading to skewed
results…diagnostic efficiency depends on positive predictive value.
TEST RESULTS ARE MORE MEANINGFUL WHEN DISEASE PROBABILITY IS HIGH. TESTS VALIDATED
WITH TRUE NEGATIVE SERA INSTEAD OF NEGATIVE SAMPLES FROM AREAS OF ENDEMIC DISEASE
WILL OVERESTIMATE SPECIFICITY.
Surface AntiGens of S. neurona
• Six immunodominant SAG’s: 1-6
• Mutually exclusive expression SAG 1, 5, 6 allows
• Stage related expression SAG’s 2-5
• Genotype II
• Genotype VI
• Genotype I
• Genotype III
• Genotype V
• Genotype IV
SAG 1 and 5 strains dominate animal
disease caused by S. neurona
The molecular genetics is very complicated!
• SAG 1 strains used for antigen blots may not detect
SAG 5 and 6 strains.
• There are two SAG 1 genotypes. They differ based
on 30 molecular markers, but display identical SAG 1
• Strain SN3, SAG1 genotype, displays a SAG 4
protein that is common to S. falcatula.
Tests that detect SAG 4 of falcatula
• Can be false positive tests for sarcocystosis!
• This is why so many horses tested by immunoblot
are positive but don’t have clinical signs of EPM!
• The presence or absence of SAG genes and selective
display of proteins are clinically relevant issues!
An academic understanding of SAG
• S. neurona has stage related expression of SAG’s 2, 3, 4, 5.
• Antigens must be displayed to elicit an immune response.
• Brain stages don’t always express S. neurona SAG’s
confounding the reliance on CSF antibody.
• Antiprotozoal treatment can affect the display of certain
SAG’s by inhibiting the growth of some stages!
SAG expression is stage related
• Stage related expression (Gautam and Grigg) in SAG
2, 3, 4, and 5.
• SN6 and SN7 (Dubey) showed protein differences
with long term culture.
• The journeyman’s understanding of SAG expression:
Changes in antigen expression can affect
interpretation of test results.
SAG 1, 5, 6 ELISA’s
• SAG 1, 5, and 6 are specific to S. neurona and
represent all the known genotypes.
• Statistically shown that CSF didn’t increase diagnostic
value with SAG 1 ELISA in a challenge study.
• Antibody levels increase with duration of infections.
• A four-fold increase in titer indicates active infection.
S. Falcatula (one strain) did not cause disease in
horses--but some strains can induce antibodies
• S. falcatula does not cause disease in horses when S.
falcatula (FLA1) was used in challenge studies.
• But Marsh showed that horses with EPM have
antibodies against S. falcatula tested by immunoblot!
• S. falcatula SAG 6 and S. neurona SAG 6 are cross
react on non-specific tests
One instance in which antibody can
confound the detection of disease
• SAG 4 proteins vary with strains that are biologically
different between at least 2 S. falcatula strains (Marsh,
Grigg) SnSAG 6 SFCornellSAG4 SFFla1SAG4 Horse infection
S. neurona + + - +
S. Falcatula Cornell - + - -
S. Falcatula FLA1 + - + -
CSF testing can help differentiate infections when this cross reactive antigen
is used. This is not an issue when specific S. neurona antigens are used.
Sarcocystosis or Sarcocystiasis and
• Inflammation is detected by clinical exam
• Parasite mediated neuroinflammation
• Cause and effect: parasites may not be present in CNS but
inflammation is demonstrated during or after infection.
• Non-parasite mediated neuroinflammation
• After treatment or after resolution of infections
inflammation can persist.
The role of C -reactive protein (CRP) in
• CRP is an acute phase inflammatory protein induced by
the cytokine IL6 (appears with infections, non-specific).
• CRP is an enzyme that releases IL6 into the circulation
• The soluble IL6/IL6 receptor can cross the blood brain
• IL6/IL6 receptor is pro-inflammatory in the CNS.
• IL6 inflammation does not require parasites in the CNS!
Serum CRP Test
• Format: ELISA CRP levels are reported in µg/ml
• Validation: CRP levels reported in infections with protozoa, bacteria, virus.
• Related to EPM (n=2720):
Total Sarcocystiasis IE idiopathic encephalomyelitis
Clinical signs 1532 582 950
CRP elevated 652 226 (39%) 426 (45%)
Clinical signs were observed in 90% of the animals tested for serum CRP levels. Horses with clinical signs
showed an elevated CRP (43%). Horses with evidence of EPM (antibody and clinical signs) had an elevated CRP
in 39% in the tested sera. Horses without antibodies to S. neurona had an elevated CRP in 45% of the sera.
Interpretation of CRP
• A value of >9 µg/ml indicates active infection with
protozoa, virus, or bacteria-i.e. no etiology identified
• Treating the infection will result in a rapid drop in
• CRP can monitor effective therapy.
What does CRP mean?
• The evidence suggests 45% of horses with clinical
signs don’t have active protozoal infections!
• Anti-protozoal therapy is expensive and ineffective in
• Treating the inflammation can be effective.
• Stage related expression of SAG’s won’t alter CRP
Effects of host on antibody tests
• Anti-protozoal treatment affects antibody production
and detection. (Furr)
• Duration of infection: infections less than 17 days,
affect antibody detection.
• Erroneous conclusions are drawn when samples that
have treatment-induced alteration of antibodies are
used to evaluate tests.
Johnson incorrectly states “strains that lack SAG 1
predominate in the mid-Atlantic region”
• Validation sera/CSF in the Johnson study:
• Confirmed positive = non-surviving horses (post-
treatment) with inflammation in CNS tissues.
• Suspect positive = surviving horses with neurologic signs
w/o other cause and response to treatment.
• Confirmed negative = non-surviving horses with other
CNS disease or no CNS lesions.
Conclusions about testing
• Testing serum for antibodies to S. neurona can be
valuable to manage the EPM case.
• Different diagnostic tests can not be directly
• An understanding of the test, its validation, and what
the results mean are important to clinical application.
Treatment of EPM
• First generation
• Sulfonamides and pyrimethamine
• Second generation
• Trizine antiprotozoal agents
• Third generation
• Decoqunate (antiprotozoal) and levamisole (immune
Clinical Field Study 1
Clinical Field Study 2
141-240 141-188 141-188 141-268
Design Demonstrate the safety and effectiveness for the treatment of Equine Protozoal Myeloencephalitis (EPM).
Controls No N/A N/A N/A
97 Horses 113 Horses 12 Horses 214 Horses
26 Horses 47 Horses 7 Horses 42 Horses
Breed Any Any Any Any
Age 9 months – 32 years 2 – 30 years 2 – 19 years 9.6 months – 30 years
Sex F/MC F/MC F/MC F/MC
NA Greater than 3 months Any treatment accepted NA
Yes Yes optional Yes
>1 >2 >2 ≥ 2
≥ 1 @ 90 days
≥ 1 end treatment (28
NA Yes NA NA
Video Review yes Video: 18 of 24 (75%) NA Video: 10 of 24 (42%)
1X 47 to 5 mg/kg 7 to 5 mg/kg 68 to 1 mg/kg
1X 1X 1X 1X
1X-16 of 26 (61.5%)
1X-14 of 26 (53.8%)Gait and WB
success duration >180
5 mg/kg–28 of 47 (60%) 5 mg/kg-7 of 7 (100%) 1 mg/kg–28 of 42 (67%)
Orogin and NeuroQuel
• Orogin (INAD 012092)
• For the treatment of EPM due to S. neurona in horses
• Anti-protozoal (decoquinate) immune modulator (decreases IL6) (levamisole)
• Undergoing safety and effectiveness studies
• NeuroQuel (INAD 012219)
• For the treatment of residual or recurrent clinical signs associated with S. neurona
• Immune modulator (levamisole)
Therapeutic agents for EPM
Drug FDA approval Action Failure due to:
ReBalance INAD 141-240 pyrimidine synthesis Ineffective dose in CNS
Marquis INAD 141-188 pyrimidine synthesis regrowth of parasites after
Protazil INAD 141-268 pyrimidiine synthesis regrowth of parasites after
Orogin INAD 012-092* parasite
* Currently undergoing license process under MUMS
Therapeutic agents for IE
• Unique therapy for the treatment of inflammation due to
S. neurona infections and post-infection inflammation
• Safety and effectiveness studies underway
• Synergism between toltrazuril and trimethoprim or
• It is not advised to use trimethoprim with
• Orogin is the only treatment designed to treat
inflammation associated with disease.
• “..is most likely caused by the failure of
maintenance of coccidiocidal concentrations of the
standard treatment drugs in the CSF as a result of
either lack of ability of these agents to pass through
the blood-brain barrier or the short elimination
half-lives of these agents in horses.”
• 2 out of 3 horses relapse with standard treatment.
Adverse Reactions with
• Anemia, neutropeinia, thromboctopenia, leukopenia,
• Teratogenic, neonatal disorders, abortion.
• Affects breeding performance of stallions.
• JAVMA VOL 242 FEB 15 2013
Effectiveness and relapse with
• 67% effectiveness, 5-17% relapse
• Prevent relapse with Protazil (diclazuril) by dosing 7
mg/kg (7X). (MacKay 2008)
• Re-growth of parasites when drug is removed. (Lindsay
• Side effects rare
• JAVMA VOL 242 FEB 15 2013
Effectiveness and relapse with
• Effectiveness 62%, 10% relapse^
• Rob MacKay (2008) recommends 35 mg/kg/day
for four days (7X) and treatment duration should
be extended to 2 months.
• FDA approved dose is 5 mg/kg/day for 28.
• *JAVMA VOL 242 FEB 15 2013 ^MacKay 2008
Post treatment with triazines
• CSF Western blots were positive in 90% (Diclazuril)
treated horses after 6 to 12 months.
• CSF Western blots were positive in 75% of
(Toltrazuril) treated horses after 6 to 12 months.
• Does CSF presence of antibody detected by
immunoblot indicate active infection?
• MUMS INAD 012092
• Licensing process 2/5 years
• Intern J App Res Vet Med 2012
• 93% treatment response based on clinical
examination (Ellison Lindsay 2012).
• MUMS INAD 012219
• Licensing process 2/5 years
• For the treatment of residual or recurring signs of
EPM after anti-protozoal therapy.
• Levamisole HCl
Levamisole in humans
• Works through IL6 inflammatory pathway
• Decreases serotonin transport protein to increase the
level of serotonin at synapse.
• Immune modulation of proinflammatory response.
• pH and temperature affect breakdown products in
• Hanson 1991
• Stable in dry form
• Studies underway to determine effects of breakdown
products (paste, liquid) on leukocytes.
• Effects on S. neurona
• Pathogenic protozoa have levamisole receptors
• Effects on leukocytes
• Leukocytes have cholinergic levamisole receptors
• Studies are underway to determine the mechanism of
levamisole suppression of leukocyte invasion by S. neurona
and modulation of the cytokine responses to infection.
Conflicts of Interest SAG 2, 4/3
• S. Reed is a consultant for EDS Solutions
• J. Morrow and A. Graves licensed SAG 2, 4/3 assays
• D. Howe is the inventor on patents covering SAG
EPM is a manageable disease!
• EPM can be managed effectively.
• Management requires an understanding of the
underlying pathology and what tests and treatments
• Providers of tests and treatments should provide the
necessary information to manage cases successfully.
Conflicts of Interest SAG 1, 5, 6
• S. Ellison
• Is an inventor on the SAG 1 patent
• Is the inventor of Merozoite Challenge Model
• Is an inventor on the SAG 6 assay
• Is the inventor on decoquinate/levamisole patent for the treatment of
EPM in horses
• Is the inventor on levamisole for the treatment of IE for horses
• Is the holder of INAD 012092, INAD 012219
• Is the inventor of SAG 1 vaccine
3 CE hours
• Please call 352-591-3221 for clarification of any topic
in this presentation.
• Complete the quiz and email your comments to
• Qualified veterinarians will receive a certificate of
completion upon review of the email.
• 1. True or False?
• Clinical signs of EPM can be due to infection, inflammation, or
both. Residual or recurrent signs after antiprotozoal therapy
may be due to untreated inflammation.
• 2. True or False?
• The incongruity between S. neurona infection (sarcocystiasis)
and the occurrence of clinical signs may be due to tests which
detect cross-reactive antibodies.
• 3. True or False?
• Inflammation can be induced by S. neurona and
remain after protozoa are eliminated. These clinical
signs can give the impression the horse has relapsed.
• 4. True or False?
• Not all strains of S. neurona can enter the central
nervous system and infect CNS tissues.
• Extra credit: What phenotype infections account for
the majority of animal disease by S. neurona.
• 5. True or False?
• The IFAT and 2, 4/3 ELISA strongly suggest testing
CSF for an accurate prediction of infection while the
SAG 1, 5, 6 ELISA does not require CSF testing.
• 6. True or False?
• C-reactive protein is present when inflammation due
to infection is present. This test won’t distinguish the
etiology of infection but can be used to monitor
success of treatment.