Serum Antibodies Against a Reactive Siteof Equine Myelin Protein 2 Linked to
Polyneuritis Equi Found in Horses
Diagnosed with EPM
Siobhan P. Ellison, Pathogenes Inc.
Tom Kennedy, Eleven Bravo Inc.
Austin Li, Senior Scientist, Pathogenes Inc.
Lark Schweiss, Pathogenes Inc.
Corresponding Author: Siobhan P. Ellison DVM PhD
15471 NW 112 Avenue,
Reddick, FL 32686
Phone: 352-591-3221
Fax: 352-591-4318
e-mail: sellison@pathogenes.com

KEY WORDS: Equine protozoal
myeloencephalitis, Polyneuritis equi,neurodegenerative disorder in horses,
Sarcocystis neurona
ABSTRACT
Equine protozoal myeloencephalitis (EPM)
is the most commonly diagnosed infectious
neuro-degenerative condition in horses andneuroinflammation is present in all definitively diagnosed cases. Historically EPM
is difficult to treat. Polyneuritis equi (cauda
equina neuritis) is a rarely recognized noninfectious, chronic relapsing, peripheral
neurodegenerative disorder in horses. We
propose that polyneuritis equi may be associated with Sarcocystis neurona infections
because we demonstrate the presence ofantibodies against equine myelin protein andS neurona surface antigens in 78% (n=55)
of serum from ataxic horses with a presumptive diagnosis of EPM. Serum antibodies
against S neurona were detected in a horsewith histopathologically confirmed immune
mediated polymyositis and intramyofiber
sarcocysts.

164

INTRODUCTION
Equine protozoal myeloencephalitis (EPM)
is the most commonly diagnosed infectious neuro-degenerative in horses. Little
attention has been paid to the inflammatorycomponent of disease despite the presence of neuroinflammation all definitively
diagnosed cases of EPM. Histopathologic
examination of a horse with EPM overwhelmingly reveals central nervous system(CNS) inflammation without the associated CNS presence of protozoa. It remains
dogma that the clinical signs of EPM are
due to presence of parasites in the CNS, andthat the area of parasitization determines thesigns observed.1 It is more likely that clinicalsigns of EPM are due to a combination ofparasitization and inflammation. Peripheral
polyneuritis (polyneuritis equi) causes signs
similar to EPM and is a rarely recognized,
auto-immune, chronic relapsing, disorder inhorses. We associate polyneuritis equi with
Sarcocystis neurona infections by demonstrating the presence of serum antibodies
against myelin proteins and S neurona surVol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

face antigens in ataxic horses.
Figure 1. Amino acid sequence of MPP with putative
Serum antibodies against S
epitope homology with S neurona and IL6 by colored lines
neurona were detected in a
beneath the sequence. The broken gray line above thehorse with histopathologicallysequence indicates non-disease inducing peptide and theconfirmed immune-mediatedsolid black line indicates the putative T cell epitope for thepolymyositis and intramyofiber
clinical and histological disease.
sarcocysts.
A large number of clinically normal horses have circulating antibodies to S neurona.1
Sarcocystis neurona seropositive horses can have neurologic
disease that is unrelated to S
neurona. An unusual neurodegenerative disease with clinicalsigns similar to sarcocystosis ispolyneuritis equi. Detection ofanti-equine myelin P2 antibodies in horses with polyneuritis
equi was first reported incause of polyneuritis equi is that it is im1981.5 6 The pathology and clinical finding
mune mediated. Likely causes are an allergicof polyneuritis equi were initially describedneuritis in response to antigen released byin 1897 in Germany, and has since beentrauma, infection, or a post-infectious allerreported in Europe and the United States.7
gic neuritis. Polyneuritis equi is comparable
Polyneuritis equi is a progressive, chronin its pathogeneses and lesions to the humanic, and often relapsing-remitting disease
Guillain-Barre syndrome and its laborathat affects the sacral and coccygeal nerves
tory model, experimental allergic (autoimleading to paralysis of the tail, rectum, andmune) neuritis (EAN).7 Similar associations
bladder, with loss of sensation in the sacral
between anti-myelin proteins and diseasedermatomes with surrounding hyperesare made in humans with multiple sclerosis
thesia. This disease is often accompanied(MS).
with cranial nerve paralysis, particularly the
Sarcocystis neurona infections can result
facial and trigeminal nerves. The cranialin signs of involvement of cauda equinae,
deficits may precede clinical signs associthus the diseases look similar. A causal relaated with involvement of the sacral and coctionship between S neurona infections andcygeal nerves. Clinical signs of polyneuritis
polyneuritis equi has not been established.
equi can include behavior changes, ataxia,
We hypothesize that immune reactions to S
proprioceptive deficits, and possibly a sideneurona infections may initiate inflamma879
winding gait. Antemortem diagnosis oftion that can develop into polyneuritis equi.
polyneuritis equi by histopathology and
Untreated neurologically abnormal horses
circulating myelin protein serum antibodmay have serum antibodies against equine68
ies have been described. Rostami andneurogenic myelin protein P2, (MP2), and S
Gregorian indicated that anti-myelin serum
neurona.5 6 7 8 9
antibodies may differentiate this T cell mediEvidence suggests that levamisole canated peripheral neuritis from clinical signs
alter
immune mediated disease and theassociated with conditions that induce CNS
clinicalcourse of MS in humans.10 Levamilesions in horses.
sole HCl inhibits the production of IL6, a
The etiology of polyneuritis equi ispleiotropic cytokine that can be
unknown. The prevailing theory as to thepro-inflammatory, in mice, dogs, and huIntern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

165

mans.11 12 13 14 A clinical response to levamisole HCl in horses with chronic, relapsing
signs of EPM has been reported.2 In thisstudy, a clinical response to levamisole HCl
was investigated.
MATERIALS AND METHODS
Sarcocystis neurona SnSAG 1, 5, 6 ELISA
was performed as previously reported.15
A 22 amino acid peptide (myelin protein
peptide, MPP) that corresponds to theneuritogenic site on equine myelin protein
associated with peripheral demyelinating
disease, Figure 1, was synthesized (United
BioSystems, Herndon VA) and used as
antigen (MPP). Antigen coating dilution
was optimized using a checkerboard titration
with equine sera positive for anti-myelin
antibodies. Sera was assayed by indirect
ELISA using a modification of the Fordyce
procedure using MPP coated and blocked
plates.6
The plates were incubated for 2 hours
at 37oC, washed, and alkaline phosphataseconjugated anti-horse whole molecule(Sigma, St. Louis MO) at 1:3000 was added
and incubated for 30 minutes at 37oC. The
plates were washed and reacted with PNPP
for 15 and 30 minutes. Plates were read ona Molecular Devices ELISA plate reader, thereciprocal of the last dilution positive (OD
0.518 or greater OD 405) was recorded as
the titer. A positive titer was a value >8 indicating we accepted a value that was equal
to 8 and greater.
Positive Control Sera for MPP
One neurologically abnormal, 12-year-old,
Warm Blood gelding (#10523) with serum
antibodies against S neurona SAG 1, 5,
6 also had serum antibodies against MPP(titer 64) before any treatment. The horse
was diagnosed with immune-mediatedpolymyositis and intramyofiber sarcocysts
by histopathology (Oregon State University
Veterinary Diagnostic Laboratory). Sera
from this horse was used as a positive control in the assays.

166

Screen Sera for MPP Antibodies by
ELISA
Sera from horses (172) with clinical signs of
EPM and 10 neurologically normal horses
were tested by MPP ELISA.
Pooled sera from positive horses andsera from horse 10523 were used as a positive controls for each MPP ELISA.
Fifty-five sera from ataxic horses, preand post-EPM treatment, were tested for
SnSAG 1, 5, 6 and MPP antibodies.
Sera from horses that met study entrance
criteria were selected for evaluation. Study
entrance criteria included horses that were
ataxic (grade 1-4), under a veterinarians
care, treated with levamisole HCl (1mg/kg once daily for 10-24 days), an adjunctto anti-protozoal therapy, received a posttreatment exam, and had sera available forpre/post analysis. Horses were not included
if there was no veterinarian exam, only one
serum available, and horses had multiple
therapies within 90 days. Fifty five neurologically abnormal horses were tested for serum antibodies against S neurona (SAG 1, 5,
6), C-reactive protein, and MPP by ELISA.
Gait assessment scores (GAS) were assigned
using the following criteria:• GAS 0 no abnormalities;• GAS 1 gait deficit just detected
at a walk, drags limbs, circumducts
limbs(s) on tight circling, mild weakness noted on tail pull-easily pulled off
track, but regains normal stride in 1-2
steps;• GAS 2 gait deficit easily detected
and exaggerated by backing, turningor neck extension, swaying at walk,displays a wide based stance after tightcircling, weakness on tail pull-easily
pulled off track, and does not return tonormal stride for more than 3 steps.
• A GAS 3 was a gait deficit prominent on walking, turning or neck extension, unable to perform tight circles
without falling backwards, weakness
on tail pull easily pulled off track andalmost falls, and doesn’t regain normal
Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

Table 1. Sera from 55 ataxic horses were examined for antibodies
against S neurona and myelin protein peptide (MPP). The majority ofhorses were positive for MPP.

tion >10 µgrams/
ml was detected bycapture ELISA in
66% (n=34) of the
MPP + (78%) MPP - (22%)
samples before treatS. neurona SAG + (n=31, 56%)
24 (56%)
7 (58%)
ment and a serum
CRP concentration
S. neurona SAG – (n=24, 44%)
19 (44%)5 (41%)<10 µgrams/ml was
detected in 33%
stride.
(n=17) of the samples (data not shown).
• A GAS 4 was a horse that stumbles
Successful response to treatment meaoften, tripping spontaneously during
sured
by a change in gait score was evident
exam, falls spontaneously and will fall
in
23
(86%)
of horses that were positive by
upon backing or tight circles, weakMPP
ELISA
and 25 (89%) of horses thatness on tail pull pulled off track, andwerenegativeby MPP ELISA (Table 2).
could fall.

• A GAS 5 was a recumbent horse,
unable to rise without assistance, maylean on a wall for support, spins toregain balance, and may have seizures.
The GAS assigned by a veterinarian were
evaluated for success of treatment by evaluating a change in gait by the difference inscore before and after treatment. Successful
response to treatment was a change in GAS
that was >0 and a treatment failure was achange in GAS that was < 0.
RESULTS
Screen Sera for MPP Antibodies by
ELISA
One hundred seventy two sera were tested
for serum MPP antibodies by MPP ELISA.
Seventy seven of 172 (45%) sera obtained
from neurologically abnormal horses werepositive for antibodies (MPP ELISA titer >8) establishing that MPP reactive antibodies
are detected in some horses.
Fifty five sera from ataxic horses, preand post-EPM treatment, were tested for
SnSAG 1, 5, 6 and MPP Antibodies
Thirty one (56%) of sera from 55 ataxic
horses were seropositive for S neurona and24 (44%) were seronegative (Table 1). The
majority of the horses (78%) were seropositive for MPP antibodies (Table 1). Twelve
horses (22%) were MPP seronegative.
Fifty-one sera were evaluated for serum
C-reactive protein concentration (CRP) by
capture ELISA. A serum CRP concentraIntern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

DISCUSSION
Serum antibodies against S neurona and
MPP were detected in a horse with histologically confirmed polymyositis andintramyofiber sarcocysts. We detected
antibodies against MPP in 45% of the serum
samples obtained from ataxic horses in thisstudy. We did not detect MPP antibodies innormal horse serum (data not shown). A
slight majority of horses with antibodies
against S neurona were MPP positive inthis study suggests that some of the clinicalsigns of EPM may be due to an autoimmune peripheral neuritis. Equine protozoal
myeloencephalitis was suspected in horses
with circulating S neurona antibodies and anabnormal gait.
Table 2. A change in gait assessment score(GAS) was evaluated post treatment. A
successful response to treatment was notedin the majority of horses. There was nodifference between treatment successes inhorses that were MPP seropositive versus
MPP seronegative (MPP measured prior
to treatment). This indicates that treatment
was unassociated with location of neurological disease because MPP is detected inhorses with peripheral neuropathy and notneuropathy in the central nervous system.
(GAS) =0

(GAS) >0MPP +4 (14%)

23 (86%)MPP -3 (11%)

25 (89%)167

It is recognized, from molecular analysis
and population genetics, that SnSAG1,SnSAG5, and SnSAG6 surface antigens will
predominate in the S neurona strains thatcirculate in nature and infect horses.1 Highly
specific bioassay using surface antigens
SAG 1, 5, and 6 were useful to detect serum
antibodies in horses with suspected EPM
and were used in this study.12 13Generally, antibodies against S neurona
and clinical neurological abnormalities
are used to presumptively identify horses
with EPM. Inflammatory lesions are often
found in neural tissues postmortem fromhorses with EPM. Criteria for a histological diagnosis of EPM includes the presenceof multifocal, asymmetric myelitis, with orwithout encephalitis, consisting of infiltrates
of lymphocytes, macrophages, and occasional eosinophils forming wide cuffs of cells
around blood vessels.14 The predominantmicroscopic lesion is reported as multifocal
to coalescing areas of hemorrhage, nonsuppurative inflammation, and small foci of
necrosis.1 Perivascular cuffing by mononuclear cells can be evident in some areas.
It is rare to find protozoa in neural tissues on
post-mortem exam using immunohistochemistry, PCR, or culture. Polyneuritis equi isassociated with the presence of equine myelin protein 2 auto-antibodies.5 6 Histologicallesions associated with chronic polyneuritis
equi are nonsuppurative inflammation of extradural nerve roots with perineural fibrosis
and Wallerian degeneration. The inflammatory infiltrate can consist of lymphocytes,
macrophages, and foci of neutrophils. Also,
perineural fibrosis and perivascular lymphocytic cuffs with hemorrhage are foundin affected areas.4 6 Demyelination is likely
the primary pathophysiogical event with degeneration and fibrous tissue replacement ofaffected nerves. No viral, bacterial, fungal,
or protozoal agents are associated with thelesions in polyneuritis equi.
Several studies indicate that MPP is areasonable assay antigen to detect peripheral
neuropathy in the horse. Myelin proteins arelargely conserved across species allowing

168

comparison of studies from lab animals.
Peripheral and CNS derived myelin P2 in
the horse is practically identical to othermyelin P2 binding structures.16 Induction ofexperimental autoimmune neuritis (EAN) by
whole peripheral nerve myelin P2 protein,or partial peptides derived from the wholeprotein, results in an inflammatory demyelinating disease in several species.17 The
induction of EAN with myelin protein is
dose-dependent.17 The neuritogenic site
within the myelin protein was determined byinducing clinical and pathological signs of
EAN in rats. A shorter synthetic peptide ofmyelin protein was ineffective at inducing
pathological disease. The neuritogeniccontaining myelin protein peptide is a logical choice for detecting anti-myelin antibodies in equine serum because this peptide
contains the neuritogenic site that experimentally induces clinical signs of autoimmune neuritis.14
Anti-MMP antibodies are expected topreferentially detect peripheral neuropathies.
Rats immunized with neuritogenic peptidesof myelin protein develop extensive inflammatory demyelinating changes in ventral
roots and sciatic nerves, but no central nervous system changes. Further, there was apositive correlation between the clinical severity of experimental disease, and the doseof the neuritogenic peptide in immunized
rats. Lymph node cells from neuritogenic
peptide-immunized rats generated reactive T cells specific for the MP2 peptide.17
These data demonstrated a cellular immune
response by T helper cells to the peptide.
Rostami and co-workers concluded
that the antigenic site for EAN induction inrats is located within the 53-78 amino acidresidues of the myelin protein peptide andthat the neuritogenic peptide may contain an
important T cell epitope (8-11 amino acids)
for the induction of experimental disease
as shown by adoptive transfer of disease
to naïve rats.18 They assert that at least one
T cell epitope for the induction of disease
resides within the 53-78 residues of myelin
P2 protein, which may be the antigenic siteVol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

for the disease process.18 Interestingly, they
showed that the induced cellular immune
response to the MP2 protein was milder
than induced responses to the neuritogenic
peptide. The T cell line lost its proliferative
activity to myelin protein activity, whereas
the neuritogenic peptides activity was notaffected on multiple challenges.
Based on the foregoing, it is logical touse the putative disease-inducing peptide
to detect anti-myelin antibodies in horses
with suspected peripheral neuropathy. The
absence of MPP antibodies in some ataxic
horses in this study may indicate that clinical signs are due to central neuroinflammation and not peripheral neuroinflammation.
Detection of serum antibodies to MMP may
differentiate a T cell mediated peripheral
neuritis from clinical signs associated withconditions that induce central nervous system lesions in horses. However, it would bepossible to have both central nervous systemand peripheral neurodegenerative diseaseassociated with EPM in the same animal.
Protozoa rarely remain in the CNS ofhorses that continue to exhibit progressive
clinical signs of neurodegeneration, leading to speculation that the pathogenesis of
disease is initiated by parasites, but disease progression is not parasite dependent.
One may speculate as to the relationshipof protozoa-mediated inflammation andcytokine mediated encephalomyelitis bymolecular analysis. A molecular (BLAST,
Basic Logical Alignment Tool NCBI) analysis of the amino acids 57-78 (MMP) of theneuritogenic peptide against the Sarcocystis
neurona NCBI data bank reveals homology
with amino acids from surface antigens (SnSAG) 4, 5, 6, 1 of S neurona and equine IL6.
Surface antigens 6, 1, and equine IL6 show
sequence identity to amino acids 69-77, 7174, and 74-77, respectively. The sequencehomology is within the neuritogenic regionof the protein.
Based on our study results, homology
with the reactive antigenic sites for myelin
protein-associated autoimmune polyneuritis
supports the association of the pathogenesis

of neuritis in EPM horses with S neurona
antigens. Further, our results support that
IL6 may be the reactive T cell epitope inhorses with autoimmune polyneuritis. Surface antigen SAG 4, a conserved Sarcocystis
surface antigen, is identical to amino acidsof MPP at residues 60-68. This homology
is within the non-disease inducing 61-72
residues of myelin protein. The homologous
amino acids of surface antigen SnSAG 5
also is inside the non-reactive part of thepeptide at residues 64-68. It remains to be
determined if the three dimensional structureof myelin P2 protein grants significance for
SAG 4 and SAG 5 antigens, or a combination of these antigens, in the inflammatorycomponent of EPM. Sarcocystis neurona
display major surface antigens during infection, and it is possible that these S neurona
antigens or antibodies against them reactwith T cell epitopes that induce neurodegenerative disease in horses. A possible mechanism of the neuroinflammatory componentof EPM syndrome can be speculated if
clinically ill horses have serum antibodies
against SAG 1, 5, 6 and MPP as we demonstrated.
Supporting the association of themechanism of disease with equine cytokine
IL6 is the clinical response to levamisole
HCl because one known action of levamisole HCl is the inhibition of the productionof IL6. 9 A clinical response was apparent
in 48 of 55 treated horses in this study. It
would be beneficial to differentiate horses
with IL6 mediated peripheral neuropathies
versus central neurodegenerative conditions
because treatment may differ.
Our data supports that some, but not all
horses may have peripheral neuritis and at
least one etiology of polyneuritis equi may
be Sarcocystis neurona infections (Table1). It was not determined how long MPP
antibodies remain in the successfully treatedhorses. Our data indicates that levamisole
HCl may treat both peripheral and central
neuroinflammation associated with protozoal infections.12 There was little difference between horses with and without serum MPP

Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

169

antibodies that show a positive responseto treatment (Table 2) indicating anothercondition, central neuroinflammation, may
be present in some horses. Levamisole HCl
is potentially beneficial to alleviate clinicalsigns of peripheral neuritis equi associatedwith S. neurona infections, alleviating clinical signs in more than 85% of the treatedhorses (Table 2).
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