Neuritogenic Peptides Derived from
Equine Myelin P2 Basic Protein Detect
Circulating Antibodies in Ataxic Horses
Siobhan P Ellison
Tom J Kennedy
Austin Li
Corresponding Author: Siobhan P. Ellison, DVM PhD
15471 NW 112th Ave
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, EPM,
C reactive protein, CRP(65%) in these horses that had a presumptive diagnosis of EPM. No cross-reactivity
between the antigens was observed. An
evaluation of the agreement between theassays (McNemar’s test) suggests as CRP
values increase, the likelihood of a positive
MPP ELISA also increases. Clinical signsof EPM may be due to an immune-mediatedpolyneuropathy that involves complex invivo interactions with the IL6 pathway
because MPP antibodies and elevated CRP
concentrations were detected in some horses
with S. neurona sarcocystosis.

ABSTRACTPolyneuritis equi is an immune-mediatedneurodegenerative condition in horses that is
related to circulating demyelinating antibodies against equine myelin basic protein
2 (MP2). The present study examined
the presence of circulating demyelinating
antibodies against neuritogenic peptidesof MP2 in sera from horses suspected ofequine protozoal encephalomyelitis (EPM),
a neurodegenerative condition in horses that
may be immune-mediated. The goals of thisstudy were to develop serum ELISA tests
that may identify neuroinflammatory conditions in horses with EPM and indirectly
relate the pathogenesis of inflammation
to IL6 by serum C-reactive protein (CRP)
concentration. Circulating antibodies against
MPP, recombinant equine myelin P2 protein(rMP2), and CRP were assayed in 2 neurologically abnormal horses by ELISA in thisretrospective study. We detected antibodies
against MPP (40%), rMP2 (33%), and CRP

INTRODUCTION
Polyneuritis equi is a neurodegenerative
condition in horses that is related to circulating demyelinating antibodies against equine
myelin basic protein 2 (MP2). The clinicalsigns of polyneuritis equi (PE) are similar to equine protozoal myeloencephalitis(EPM).1,2,3,4 Equine protozoal encephalomyelitis (EPM) is also a neurodegenerative
condition in horses that may be due to an
immune-mediated pathogenesis of disease as
indicated by infection studies.5 Muscle wasting, an abnormal gait, and paralysis/paresisof cranial nerves are signs associated with

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

175both neurodegenerative diseases.
Table 1. Antigen sequence for MPP, SAG 6, SAG 1,
The etiology of PE is unknown but
and IL6 that are homologous to residues on equineis generally considered an immunemyelin basic protein 2.
mediated peripheral polyneuritis.
Sequence
Name
The clinical signs associated withGFKNTEISFKLGQEFEETTADN
MPP
EPM are due to inflammation associated with infections by pathogenic
EFEETTADSAG 6protozoa, in most cases Sarcocystis
FEET
SAG 1
neurona. The histopathologicalTTAD
IL6
lesions of both diseases have been
1,4
described. It was suggested thatis not pathogen specific, but may indicate
alterations in cytokine levels may
the mechanism of disease is attributable tocontribute to neuropathology seen in horses
cytokine IL6 pro-inflammatory pathways. A
with EPM, particularly T cell-mediated imBLAST (NCBI) analysis reveals that there
munity.5 It is possible that both diseases areare amino acid homologies between thelinked by a common disease mechanism thatneuritogenic peptide MPP contained within
stimulates proinflammatory pathways via the
PE and some S neurona surface antigens.
IL6 cytokine.
Sarcocystis neurona SAG 4 and SAG 5
Clinical signs and histopathologicalshare homology with MPP residues that arelesions that are similar to PE can be exin the non-disease producing regions of theperimentally induced in rats.5 Induction of
peptide. However, the S neurona SAG’s 6experimental autoimmune neuritis (EAN)
and 1 have sequence homology in the neuriin susceptible animals is accomplished bytogenic region of the MPP peptide.
injection of MP2 or neuritogenic peptides
Cytokine IL6-dependent stimulation of(MPP) derived from MP2, or by passive
the acute phase C-reactive protein (CRP)
transfer of lymphocytes from syngeneic aniresults in detectable serum CRP levels inmals sensitized to MP2. The neuritogenic
mice and may be similar in horses.7 The
site for experimental induction of EAN is loIL6 plasma buffering system in horses may
cated on 26 amino acids of MP2. Induction
preclude sensitive measure of IL6 in plasma,of disease in rats by adoptive transfer into
however, the serum CRP concentration may
naive rats suggested that the antigenic site
provide an indirect measure of IL6 activfor the disease process may due to at least
ity in equine disease as it does in dogs.8
one T cell epitope identified on MPP.6 The
The purpose of this study was to develop
neuritogenic peptide, MPP, contains a putaa serum ELISA to evaluate the presence oftive T cell epitope that corresponds to equine
MPP antibodies in horses and implicate thecytokine IL6. Equine IL6 shares amino acidpathogenesis of disease to IL6 pro-inflamsequence homology with residues within themation. A serum MPP ELISA may be usefulEAN-inducing site of MPP.6 Cytokine IL6
to elucidate the complex in vivo interactions
is a proinflammatory cytokine related to theassociated with neuroinflammation present
pathology associated with adoptive transfer
in PE and EPM.
of EAN that can be inhibited by anti-IL6 invivo. Levamisole HCl decreases the syntheMATERIALS AND METHODS
sis of IL6 in dogs and rats.9,10 Levamisole
Antigens
HCl was shown to alleviate clinical signs
A peptide (MPP) that corresponds to amino
of ataxia in horses with suspected EPM
acid residues 57-78 of equine myelin proand suggests the pathogenesis of diseasetein was synthesized (United BioSystems,
may be IL6 related if the mechanism of
Herndon, VA). Recombinant myelin
action of levamisole HCl in horses also
P2 protein (MP2) was produced in E coli
blocks IL6 production.6,7 Antibody reactiv(MyBiosource, San Diego, CA). Sarcocystis
ity in an ELISA that uses MPP as antigen

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

neurona 1, 5, 6 antiTable 2. Results showing the reactivity of sera against antigens
gens were produced as used in the ELISA. Rabbit α PMP2 was positive against MPP andpreviously described.6
MP2 and rSAG 1 and rSAG 6, but not rSAG 5. Control positive
The sequence of thesera CP bound antigens MPP, MP2, rSAG’s 1, 5, and 6.
common amino acids
MPP MP2 rSAG 1 rSAG 5 rSAG 6
are shown in Table 1.
Rabbit α PMP2
pos
pos
pos
neg
pos
Sera
CP
pos
pos
pos
pos
pos
A positive control S
neurona SAG 1, 5, 6
serum sample (CP) wastion plates coated with approximately 0.1µobtained from a 12-year-old thoroughbred
grams of protein in carbonate buffer. Plates
mare with a 5-week history of severe ataxia
were read on a Molecular Devices automatic(grade 4). Negative control sera was pooled
plate reader. Optimum concentrations forfrom 10 clinically normal yearling thorantigen coating and the detecting antibodies
oughbred horses that did not have antibodies
were determined by standard checker-board
against S neurona.
titration.
Sera from untreated horses (n=52) that
The equine P2 myelin protein ELISA
had clinical signs of ataxia with or without
described by Fordyce was modified bymuscle wasting determined by veterinarusing MPP or MP2 coated and blocked
ians that suspected EPM were analyzed forplates.6 Briefly, the plates were incubated
S neurona antibodies and retrospectively
for 40 minutes (MP2) or 2 hours (MPP) atassayed for antibodies against MP2, MPP,
37oC with diluted serum. The plates were
and CRP. Anti-MPP antibodies and serum
washed and alkaline phosphatase conjuconcentration of C-reactive protein wasgated anti-horse whole molecule (Sigma, St.
determined in 49 sera. Rabbits were immuLouis MO) was added and incubated for 30
nized with a synthetic 14 amino acid peptide
minutes at 37oC. Diluted control sera bindwithin amino acids 17-77 of human PMP2
ing to antigens was detected with alkaline
protein, affinity purified, and lyophilized
phosphatase conjugated anti-rabbit whole(Antibody Verify; Las Vegas, NV). Rabmolecule (Sigma, St. Louis MO). The plates
bit anti-PMP2 antibodies were diluted to awere washed and reacted with 4-nitrophenyl
concentration of 1 mg/ml as a stock reagent.
phosphate in carbonate buffer (pH 9) for 15
Alkaline phosphatase anti-rabbit was oband 30 minutes. A positive result was retained from Sigma Aldrich (St. Louis, MO).
corded if the OD was > 0.518. The reciprocal of the last dilution positive (OD 0.518 or
Assaygreater at 405nm) was recorded as the titer.
The ELISA was performed in 96 well
The serum concentration of C-reactive(HyBind, Greiner, Monroe, NC) microtitraTable 3. Raw OD 405 showing the binding of rSAG 1, 5, 6, MPP, and MP2 by S. neurona
positive sera (CP) in serial 2 fold dilutions 1:8 through 1:64, wells A-E. The raw OD 405
after reacting rSAG 1 and rSAG 6 wells with rabbit anti-PMP2 followed by ELISA using
S. neurona positive control sera, CP, are shown, wells F, G, and H.
rSAG 1rSAG 5rSAG 6

MPP

rMP2

rSAG1

rSAG5

rSAG6

1:8

1.937

1.944

1.935

1.935

1.921

1.509

1.944

0.841

1:32

1.897

1.896

1.540

1.824

1.865

1.296

1.896

0.753

1:16

1:64

A

1.922

1.894

B

1.929

1.893

C

1.916

1.505

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

D

1.898

1.632

E

1.890

1.640

F

1.500

1.473

G

1.929

1.893

H

0.680

0.619

177

Table 4. Results of sera tested by antigen showing the number of sera
that were positive and the percent of total sera tested.

Rabbit antiPMP2 slightly
blocked binding of
Number of sera%
S neurona positive
MP2 positive
18
33%
sera to rSAG 1 and
MPP positive
21
40%
significantly blockedbinding to rSAG 6
S. neurona positive
31
60%
antigens (Table 3).
S. neurona SAG 1 positive
29
56%
This antibody did
S. neurona SAG 6 positive
25
48%
not block binding torSAG 5 antigens.
CRP positive32
65%
The results of an
MPP and MP2 positive
7
13%
indirect ELISA using
MPP positive and MP2 negative
14
27%
rMP2, MPP, and S
MPP negative and MP2 positive
11
21%
neurona SAG’s asantigen are shown in
MPP negative and MP2 negative
20
38%
Table 4. The serum
CRP and MPP positive
12
24%
concentration of
CRP positive and MPP negative
20
41%
C-reactive protein
CRP negative and MPP positive
7
14%
was determined, apositive result was a
CRP and MPP negative
10
20%
value >10 µ grams
MP2 and S. neurona positive
17
33%
per ml (data not
S. neurona negative
21
40%
shown). The distribution of seroposiSAG 6 and MPP positive
12
23%
tive ataxic horses by
SAG 6 positive and MPP negative
13
25%
antigen is shown in
SAG 6 negative and MPP positive
9
17%
Chart 1.
SAG 6 and MPP negative
18
35%
Assuming MPPor MP2 indicate
SAG 1 and MPP positive
13
25%
a neuropathy, a
SAG 1 positive and MPP negative
15
29%
McNemar’s test was
SAG 1 negative and MPP positive
8
15%
used to evaluate
SAG 1 and MPP negative
16
31%
agreement between
the MPP and SAG 1,
SAG 5, and SAG 6
protein was measured by capture ELISA as
results.
For
each
of
these
comparisons, theper manufacturer recommendations (GenP
value
is
not
significant,indicatinga lack ofway, San Diego CA).
agreement/lack of cross-reactivity between
RESULTSthese antigens. There was no statisticallyRabbit anti-PMP2 antibody was positive
significant relationship between MPP andagainst MPP and MP2, and rSAG 1 and
CRP concentration, however the comparirSAG 6, but not rSAG 5 (Table 2). Control
son suggests as CRP values increase, thepositive horse sera (CP) was obtained fromlikelihood of a positive MPP ELISA also
an ataxic horse with a presumptive diagnoincreases, Chart 2.
sis of EPM, and was positive against MPP,DISCUSSION
MP2, rSAG’s 1, 5, and 6. Negative control
The neuritogenic peptide MPP may besera from horses without disease did not
useful to detect IL 6-mediated neurologicalbind any of the antigens MPP, MP2, rSAG 1,
dysfunction in horses with suspected EPM.
5, or 6 (data not shown).

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

Circulating antibodies
Chart 1. Antibody distribution by antigen binding in horses
against MP2 and the neuri- with suspected EPM.
togenic peptide MPP of
MP2 were detected in 32
of 52 horses with suspected EPM in this study. Not
all horses were positive
for antibodies against bothmyelin proteins (Table
4). Continued stimulation with whole myelin
protein induces disease
tolerance in rats without
loss of activity against theneuritogenic peptide MPP.
Therefore, chronicallydiseased animals may
Sarcocystis neurona challenged horses also
be MP2 negative/MPP positive on antibody
produce antibodies when challenged, howtests.5 It was not possible to correlate duraever, antibody levels are not directly relatedtion of disease in these horses because onset
to clinical signs or prognosis. It was reportedof disease was not clearly identified.
that as clinical disease progressed, the serum
titer increased in S neurona induced infecA diagnostic test that detects MP2
tions.12 Individual responses to infections
antibodies in horses with polyneuritis was
are possible with S neurona infections andsuggested by Fordyce.2 Not surprisingly, anan absence of circulating antibodies can beti-MP2 antibody titer was not a useful progdue to the variable expression of S neurona
nosticator of disease outcome in the Fordyce
surface antigens.12,13,14
study. Individual responses to immune
stimulation were observed in experimenIndividual immune responses are postally induced autoimmune neuritis in rats.5
sible with S neurona infections in horses aswell as an absence of circulating
antibodies due to variable expresChart 2. The relationship between MPP and CRP sesion of S neurona surface antigens.
rum concentration is charted. The line predicting theprobablity of an MPP seropositive result indicates that Homology with equine IL6 amino
as the serum concentration of CRP increases there is a acids at MPP residues 74-77 may
highr probablity of a positive MPP test indicating that implicate IL6 as a T cell marker
for the disease inducing cells inneuroinflammation (MPP positive) may be related tohorses. The cytokine IL6 induces(IL6-induced) CRP.
the production of CRP by the liver.
We found no statistically significant correlation (95% confidence)
between MPP seropositive samples
and serum CRP concentration inthis group of horses. However, theanalysis suggested a linear relationship.
In this study, as CRP value
increased, the probability thatthe sample is MPP seropositive
increased, Chart 2. Antibodies
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179

(against MPP) that cross-react or bind IL6
may be expected to reduce the cytokine
dependent production of CRP. That was
not seen. Cytokine IL6 is a multifunctional
cytokine that regulates pleiotropic roles in
inflammation.16 The soluble IL6 receptor(sIL6r) is an IL6 agonist, and the controlof many IL6 mediated events are regulated
via sIL6r. Possibly CRP affects IL6 mediated inflammatory events by releasing sIL6r
into the plasma.7 If this is true, one would
expect to measure IL6 in the plasma in acutedisease. The elevated CRP values in the
MPP seropositive horses may indicate thatthe neuritis is acute, and is mediated throughthe IL6 cytokine pathways. However, we did
not measure IL6 in this study. A sustained
elevation in anti-MPP in a horse that is CRP
negative may indicate a chronic neuritis as
seen in the hyper-immunized EAN rats.
The EPM-suspect horses were seropositive against antibodies to S neurona SAG
1, 5, or 6. There is amino acid sequencehomology between S neurona SAG’s 1, 4, 5,
and 6 and MPP.6 In order to investigate theproteomics of SAG proteins in the neuroinflammatory disease process, we identified
sequence homology between S neurona
SAG’s and the neuritogenic peptide MPP
and MP2. We determined the binding ofequine sera to the MP2 and MPP proteins
and the relationship to SAG 1, 5, and 6.
Control horse sera was seropositive for all
the proteins used in this study. A monospecific polyclonal antibody against aminoacid residues was specific to MP2 bound
MPP, rSAG 1, and rSAG 6, but not rSAG 5
antigens. Based on structural and antibody
binding data, anti-PMP2 antibodies presumably reacted with the neuritogenic peptide
MPP and not the non-disease producing residues of the equine myelin basic protein 2.
We showed that anti-PMP2 could
partially block binding of CP sera to rSAG
1 and rSAG 6, but not rSAG 5. Blocking
the binding of CP sera to antigens that had
homology in the disease-producing regionsof MPP, but not the antigens with homology to the non-disease producing regions of

180

MPP, may support that anti-PMP2 binds theactive disease producing residues of the antigen. The anti-PMP2 sera did not completely
block binding of CP sera to MPP, suggestinganti-PMP2 does not bind the same regions
on MPP as the sera from a diseased horse
but may react with multiple epitopes of MPP
that include the non-disease producing residues of MPP or additional antigens of MP2.
Anti-PMP2 antibody did not completely
block CP binding to rSAG 6, indicatingthat the antibodies anti-PMP2 and CP binddifferent epitopes of rSAG 6 that may
include the neuritogenic region of MPP at
residues EFEETTAD in this assay. The test
results using sera from these clinically ill
horses were evaluated by McNemar’s test
and showed insignificance between MPP or
MP2 ELISA and SAG 1, 5, or 6 ELISA test
results. The lack of agreement between The
ELISA tests indicate the antigens S neurona
SAG 1, 5, or 6 and MPP are not crossreactive.
IL6-mediated disease can be assayed using MPP and MP2. Assays that use MPP and
MP2 may be useful to elucidate the pathogenesis of disease associated with EPM, but
not the etiology. Possibly, early infections
with S neurona induces peripheral polyneuritis in horses that confounds a diagnosis ofEPM. It is possible that the proteomics ofparasites antigens facilitate inflammatory
disease. However, the SAG 1, 5, 6 ELISA
can indicate the etiology is due to S neurona because these assays measure different
antigens. If the inflammatory component of
EPM is IL6-mediated, blocking IL6 is a viable therapeutic strategy for diseased horses.
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