Effects of Experimental Sarcocystis neurona-Induced

Research ArticleEffects of Experimental Sarcocystis neurona-Induced Infectionon Immunity in an Equine Model

S Rochelle Lewis12 Siobhan P Ellison3 John J Dascanio14 David S Lindsay5

Robert M Gogal Jr56 Stephen R Werre5 Naveen Surendran17 Meghan E Breen18

Bettina M Heid1 Frank M Andrews910

Virginia A Buechner-Maxwell1 and Sharon G Witonsky1

1 Department of Large Animal Clinical Sciences Virginia-Maryland Regional College of Veterinary MedicineBlacksburg VA 24061 USA

2 Rangiora Veterinary Centre Rangiora 7400 New Zealand3 Pathogenes Inc PO Box 970 Fairfield FL 32634 USA4College of Veterinary Medicine Lincoln Memorial University Harrogate TN 37752 USA5Department of Biomedical Sciences and Pathobiology Virginia-Maryland Regional College of Veterinary MedicineBlacksburg VA 24061 USA

6Department of Biosciences and Diagnostic Imaging College of Veterinary Medicine University of Georgia Athens GA 30602 USA7 Rochester General Hospital Research Institute Rochester NY 14621 USA8Natural Vet Palm Beach Juno Beach FL 33408 USA9Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Tennessee Knoxville TN 37996 USA10School of Veterinary Medicine Veterinary Teaching Hospital and Clinics Louisiana State University Baton Rouge LA 70803 USA

Correspondence should be addressed to Sharon G Witonsky switonskvtedu

Received 17 June 2014 Revised 21 August 2014 Accepted 15 September 2014 Published 12 November 2014

Academic Editor William Van Bonn

Copyright copy 2014 S Rochelle Lewis et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM) affecting 05ndash1 horses in theUnited States during their lifetimes The objective of this study was to evaluate the equine immune responses in an experimentallyinduced Sarcocystis neurona infection model Neurologic parameters were recorded prior to and throughout the 70-day study byblinded investigators Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression Allexperimentally infected horses displayed neurologic signs after infection Neutrophils monocytes and lymphocytes from infectedhorses displayed significantly delayed apoptosis at some time points Cell proliferation was significantly increased in S neurona-infected horses when stimulated nonspecifically with PMAI but significantly decreasedwhen stimulatedwith S neurona comparedto controls Collectively our results suggest that horses experimentally infected with S neuronamanifest impaired antigen specificresponse to S neurona which could be a function of altered antigen presentation lack of antigen recognition or both

1 Introduction

Equine Protozoal Myeloencephalitis (EPM) represents themost commonly diagnosed neurologic disease of horseswithin the United States [1ndash3] Horses are aberrant hostsand are commonly infected by ingestion of the Sarcocystis

neurona sporocyst through contamination of feedstuffsThe majority of horses exposed to this parasite generatea protective immune response and do not develop clinicalsigns However for the small percentage of horses thatdevelop EPM the clinical signs are classically those asso-ciated with asymmetric neurologic deficits including gait

Hindawi Publishing CorporationJournal of Veterinary MedicineVolume 2014 Article ID 239495 16 pageshttpdxdoiorg1011552014239495

2 Journal of Veterinary Medicine

abnormalities ataxia weakness and focalmuscle atrophy [3ndash5] Annual losses in theUnited States are estimated to be $554to $1108 million [2]

In order to developmore efficacious treatments vaccinesand diagnostic assays it is important to first determine ifthere is an immune signature that dictates whether a horsewill develop a protective response or neurologic diseaseWhile protection has been linked to CD4 and CD8 cell-mediated response and interferon-gamma (IFN120574) produc-tion [6ndash9] the specific pathway(s) of immune cell responsesassociated with development of disease in the equine are stillpoorly characterized Interestingly a number of studies havereported an immune profile of decreased CD4 expressionsurface antigen 1- (SAG1-) induced cell proliferation PMAIstimulated cell proliferation and IFN120574 in EPM affectedhorses [10ndash13] It has been proposed that stress-inducedimmune dysregulation increases disease susceptibility ashorses subjected to stressful conditions (ie shipping show-ing training and pregnancy) tend to have an increasedincidence of disease [14ndash17] Therefore new studies designedto explore the specific immune consequences of EPM in thehorse would facilitate new drug discoveries and diagnostictests whichwould have great economic and emotional impactfor both the individual horse owner and the US equineindustry

Previously we determined that both naturally and exper-imentally infected horses have suppressed leukocyte prolif-eration responses when stimulated with combined mitogensPMAI [12 13] The purpose of the present study was todetermine whether this defect was due to an increase inapoptosis or decrease in cellular proliferation of a specificsubset of leukocytes as well as to identify altered immuneresponses during disease progression in horses experimen-tally challenged with S neurona

2 Materials and Methods

21 Horses Nine-quarter horse and quarter horse crossesranging in age from 18 months to 11 years consisting ofeight mares and one gelding were used in this study Allhorses were vaccinated prior to transport to Virginia Techagainst equine influenza tetanus West Nile Virus Easternand Western Equine Encephalitis and Equine Rhinopneu-monitis Upon arrival the horses were also vaccinated againstrabies Historic records indicated that none of the horses hadexhibited clinical signs of neurological disease Horses weremaintained on pasture and fed grass hay and pelleted ldquosweetfeedrdquo in accordance with normal energy requirements forweight gain for the study duration Horses were acclimatedfor two weeks prior to assessment of baseline immunefunction and serology

22 Physical and Neurologic Examinations Complete phys-ical examinations were performed on all the horses uponarrival at Virginia-Maryland Regional College of VeterinaryMedicine (VMRCVM) Baseline physical and neurologicexams were performed (Figure 1) All care and maintenance

of the horses was in accordance with the guidelines estab-lished by the Animal Use and Care IACUC committee atVirginia Tech and the study was approved by the IACUCcommittee All horses underwent a neurologicallamenessexamination two weeks prior to the start of the study Inves-tigators Lewis (RL) and Witonsky (SW) performed blindedneurologic exams separately for each horse including baselinescores A 97-point scoring system was used to evaluate thehorsesrsquo neurologicallameness status and is described in detaillater in Table 4 The scoring system was based on a protocolestablished by Ellison et al [18ndash20] which had been usedfor several previous United States Department of Agriculture(USDA) approved EPM studies Briefly the scores rangedfrom zero (normal) to 3 (severely affected) for 22 differentparameters with a maximum total score of 97 (Table 4)The parameters measured included but were not limitedto cranial nerve function ataxia weakness (paresis) andspasticity The 22 parameters that were evaluated includeddropping feed tongue tone apprehension of feed droolinglip facial nerve and eyelid paresis attitude muscle atrophycauda equine weakness lameness conscious proprioceptionbased on crossing over of limbs abduction of hind limbstripping paresis circling pivoting and toe dragging of limbshypermetria inconsistent placement and tail pull Whena horse displayed a neurologic or lameness deficit theappropriate score was assigned and the horse was observedclosely for two weeks prior to initiation of the study If thehorsersquos status was unchanged during this period the animalwas subjected to a more thorough neurologic evaluation(described in the following section) to minimize the possibil-ity of a preexisting EPM infection A few horses displayed gaitand conformation abnormalities that resulted in ameasurableelevated baseline neurologiclameness score however theseabnormalities were determined to not be attributable to anEPM infection We predicted that an S neurona infectionwould alter the baseline score thus horses with a preexistingminimal measureable baseline neurologiclameness scoreremained in the study

23 Serum and CSF Sample Collection (Day minus5) prior toInfection After a two-week acclimatization period (day minus5)peripheral blood samples were collected and horses wereanesthetized CSF was obtained from the atlanto-occipital(AO) joint space using a 3510158401015840 18 G spinal needle and sub-mitted along with the serum to a commercial laboratoryforSnSAG1 ELISA and to the laboratory at VMRCVM forroutine cytology [19 21] CSF collection was also performedon postinfection day 73

24 S neurona Culture Merozoites were obtained by atechnique described by Ellison et al [18 20] BrieflySnSAG1 merozoites were isolated from CNS tissue froma horse previously diagnosed with EPM These merozoiteswere then maintained in continuous culture Merozoiteswere cultured in complete media (RPMI with L-glutamine(Mediatech Herndon VA) hepes buffer 25mM (Mediat-ech Herndon VA) 2 heat inactivated fetal bovine serum(FBS) (Atlanta Biologics Lawrenceville GA) 50 IUmL of

Journal of Veterinary Medicine 3

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Acclimatization Infection

Study period

Treatment

minus21 minus5 0 2 7 10 14 21 28 35 42 56 70 73 120

Trea

tmen

t gro

ups a

lloca

ted

Figure 1 Experimental design for S neurona infection study Following a 2-week acclimation nine horses had baseline neurologicexaminations immune function analysis and CSF taps with samples submitted for SnSAG1 analysis Horses were randomly assigned toeither control or S neurona experimentally infected treatment groups Peripheral blood was collected on day minus5 day minus1 days 0ndash10 day 14day 21 day 28 day 35 day 42 day 56 and day 70 for infection or immune function assays CSF taps (AO = atlanto-occipital space) wereperformed on day 5 and postinfection day 73

penicillinstreptomycin solution (Sigma Chemical CompanySt Louis MO) 1 sodium pyruvate solution (MediatechHerndon VA))

25 Horse Treatment Selection and Infection Horses wererandomly assigned to two treatment groups As there is nosex bias with EPM there was no selection for or designationof sex with a treatment group Each group contained horsesthat had a baseline score ofgt0 after initial exam for unrelatedreasons as previously discussed On the day before infection(day minus1) blood was collected from the left jugular vein of allhorses into lithium heparinized blood collection tubes Fivehorses were designated to receive the parasite in infected hostleukocytes intravenously and constituted the experimentallyinfected group the remaining four served as the nonin-fected control horses and received uninfected autologouslymphocytes Briefly 10mls of peripheral blood collectedfrom each horse in the infected groupwas cultured with 6000live merozoites and incubated overnight at 37∘C 5 CO

2

Peripheral blood from control horses was also incubatedovernight without merozoites using identical conditionsEach day the same amount of peripheral blood was obtainedfrom each horse and the previous dayrsquos (infected or control)

autologous blood was administered Horses were infectedfor 10 days Blood was collected for specific assays on thefollowing days day 0 day 2 day 14 day 21 day 28 day 35day 42 day 56 and day 70 [20]

26 Peripheral Blood Smear and Mononuclear Cell (PBMC)Isolation Leukocyte differentials were obtained from directblood smears made from each horse at each time point priorto leukocyte enrichment Slides were stained with modifiedWright stain (Wrightrsquos Giemsa Sigma) PBMCs were thenisolated by density gradient centrifugation [22]Whole bloodsamples were diluted with phosphate buffered saline (PBS)(Mediatech Herndon VA) Samples were enriched usingLymphoprep (Lymphoprep Greiner NJ) Cells were washed2xrsquos with PBS and enumerated

27 Peripheral Blood Leukocyte Enumeration Blood leuko-cyte cellularity was determined with a Multisizer 3 CoulterCell Counter (Beckman Coulter Fullerton CA) using asample volume of 10 120583L diluted with 10mL of PBS Cells (5 to10 120583m diameter) were enumerated Samples were diluted toa final concentration of 5 times 106mL in complete RPMI 1640


with L-glutamine hepes buffer 25mM 10 heat inactivatedFBS penicillin (50 IUmL) and streptomycin (50 IUmL)[22]

28 Cytologic Analysis Cytospins were performed onenriched peripheral blood leukocyte fractions to assesscell morphology lymphocyte purity and neutrophilcontamination Briefly 50120583L aliquots of cells (5 times 106mL)were combined with 150120583L of PBS in a slide cytospinchamber and centrifuged for 5min 50timesg 23∘C (Cyto-tekcentrifuge Sakura Finnetechnical Co Tokyo Japan) Theslides were air dried stained with modified Wrightrsquos stainand enumerated by light microscopy One hundred cellswere counted across 10ndash15 fields [22]

29 Analysis of Peripheral Blood LeukocyteMarker ExpressionA 100120583L cell suspension (5 times 106mL) was stained with50120583L of the following diluted monoclonal antibodies (1 100in PBS was added to individual wells of each sample)CD4+ (mouse anti-equine CD4+ antibody 1mgmL cell lineHB61A IgG1 VMRD Pullman WA) CD8+ (mouse anti-equine CD8+ antibody 1mgmL cell line HT14A IgG1VMRD Pullman WA) B-cell (mouse anti-equine CD5+antibody 1mgmL cell line B29A IgG2a VMRD Pull-man WA) and DH59BCD172a antibody (mouse anti-equine IgG1 1mgmL DH59B VMRD Pullman WA) tostain monocytes and granulocytes (mouse anti-equine IgG11mgmL DH59B) Cells were incubated with the primaryantibody for 20min at 4∘C Cells were then washed withPBS and 50 120583L of the secondary antibody was added to thewells The secondary antibody added to the B-cell was PErat anti-mouse IgG2a (for conjugation to B-cell antibody)(Pharmingen San Diego CA) at a dilution of 1 100 FITCrat anti-mouse IgG1 (Pharmingen San Diego CA) was usedfor conjugation to CD4 CD8 and DH59b antibodies at a1 100 dilution Unstained control samples received PBS Theplate was washed and cells were resuspended in 200120583L ofcold PBS for flow cytometric analysis 7-AAD (MolecularProbes Eugene OR) if required in each step was addedat this time (see below) Cells were analyzed on an EPICSXL flow cytometer (Coulter Hileah FL) Samples were rununtil a minimum of 5000 (with an optimal count of 10000)leukocytes or lymphocytes were captured (dependent onsubset) as described below [12 13 22]

210 Analysis of Cell Viability and Apoptosis by 7-Amino Acti-nomycin D (7-AAD) Following cell surface staining above1 120583g of 7-AAD in 200120583L PBSwas added to each sample Plateswere incubated for a maximum of 30min on ice in the darkGreater than 5000 gated events per sample were collectedby the EPICS XL flow cytometer Based on the intensity ofstaining cells were classified by their subset as 7AAD dull(live cells) 7AAD moderate (early apoptosis) and 7AADbright (late apoptosis) [12 13 22]

In order to study the effect of PMAI on apoptosisadditional cells were plated in three separate 96 well roundbottom plates and PMAI was added (final concentration20 ngmL PMA 10 pgmL ionomycin) to each of one set of

samples Plates were incubated for 24 48 and 72 hrs in acontrolled environment (37∘C 5 CO

2) with nonstimulated

samples included as controls At designated time points (2448 and 72 hrs) cell surface and 7-AAD staining of the cellswere performed [12 13 22]

211 Carboxyfluorescein Succinimidyl Ester (CFSE) StainingAliquots of the cells (2 times 106) were resuspended in 5120583M ofCFSE (Molecular Probes Eugene OR) for 10min at 37∘C[23] Cells were then washed plated and stimulated with orwithout PMAI for 72 hrs in a controlled environment (37∘C5CO

2) Following incubation primary and secondary anti-

bodies were added as previously described Flow cytometrywas then performed 5000 or 10000 cells gated events persample The number of cellular divisions for each subsetwas determined [23] PMAI-treated merozoite-treated andmedia only wells were included for each stain

212 Live Merozoite Preparation For assessment of antigenspecific proliferation and apoptosis merozoites were used forstimulating cells in some assays (ie lymphocyte proliferationassays 7AAD CFSE) [24] Live S neurona merozoites ofthe SN-37R strain were grown and maintained in Africangreen monkey (Cercopithecus aethiops) kidney cells (CV-1 cells ATTC CCL-70 American Type Culture CollectionRockville MD USA) The S neurona merozoites were har-vested from CV-1 cells by removing the complete media(RPMI with L-glutamine hepes buffer 25mM 2 heat inac-tivated FBS 50 IUmL of penicillinstreptomycin solution1 sodium pyruvate solution) including the merozoitesThe suspension was filtered through a 3 120583M filter and spunat 1500 rpm (350timesg) for 10min at room temperature andresuspended in fresh complete media The merozoites wereenumerated with a hemocytometer and then resuspended to1 times 105mL with complete media containing 10 heat inac-tivated FBS Merozoites (100120583Lwell) were then incubatedwith the equine leukocytes [24]

213 Lymphocyte Proliferation Assays Plates containing100 120583L aliquots of each horsersquos enriched lymphocytes (2 times106mL) in triplicate wells were cultured together with 100120583Lof the appropriate mitogen or live merozoites (1 times 105mL)or media only [1 12 13 22] Final concentrations of mitogensin the wells were 5 120583gmL ConA (Sigma Chemical CompanySt Louis MO) 1 120583gmL PWM (Sigma Chemical CompanySt Louis MO) 20 ngmL PMA (Sigma Chemical CompanySt Louis MO) and 10 pgmL ionomycin (Sigma ChemicalCompany St Louis MO) Cell spontaneous proliferationwas assessed with cells cultured in triplicate wells containingcomplete media (200 120583Lwell) Cells were incubated for48 hrs at 37∘C in humidified 5 CO

2and then pulsed with

1 120583Ci3H-thymidine Plates were harvested 18ndash24 hrs laterusing a Filtermake Harvester (Packard Bioscience BillericaMA) Stimulation indices were calculated for each mitogenby dividing the mean counts per minute (CPM) of wells withmitogen by themeanCPMof the unstimulated cells inmediawhich represented spontaneous proliferation [12 13 22]


Table 1 SnSAG1 serum and CSF titers for all horses before and after experimental infection

Horse Infection status Initial serumSnSAG1 titer

Initial CSFSnSAG1 titer

Postexperimentserum SnSAG1

titer

PostexperimentCSF SnSAG1

titer1 Control lt2 lt2 32 lt12 Removed from study lt2 lt2 16 13 S neurona-infected lt2 lt2 80 24 Control lt2 lt2 lt2 25 S neurona-infected lt2 lt2 32 26 S neurona-infected 4 lt2 32 lt17 S neurona-infected lt2 lt2 80 18 Control lt2 lt2 32 lt19 S neurona-infected lt2 lt2 32 lt1Serum and CSF SnSAG-1 analysis was performed prior to and following the conclusion of the experiment (after Day 73) Infection status is marked as controlor S neurona-infected Horse 2 received a dose of the parasite in error and was excluded from statistical analysis

214 EPM Treatment Poststudy Horses in the merozoite-infected group were treated with trimethoprimsulphadi-azine (20mgkg sulphadiazine) and pyrimethamine (1mgkg) for a minimum of 12 weeks or until they returned totheir preinfected neurologic status and the SnSAG1 ELISAreturned to within normal reference range Upon completionof the study all the horses were placed in suitable homesthrough adoption

215 Statistics and Data Analysis Horses 3 5 6 7 and 9received ten doses of the protozoan in autologous lympho-cytes via intravenous injection and constituted the experi-mentally S neurona-infected group Horses 1 2 4 and 8served as controls Horse 2 received 1 dose of S neurona inerror andwas removed from the study Data from horse 2 wasnot included as either an infected or noninfected animal

Standard residual plots were used to assess model ade-quacy and logarithmic values were also analyzed for manydata sets in order to stabilize the variance between individualdata points Normal probability plots showed that logarith-mic values lay closer to a straight line

Since there were more than two treatments to be assessedin this study analysis of variance was conducted using theglimmix procedure of the SAS system (version 92) (SASInstitute Inc Cary NC)This procedure was utilized for mostof the individual analyses performed including results of flowcytometry neurologic scoring over time and lymphocyteproliferation assays As there were dramatically fewer timepoints available for CD8+ cells in late apoptosis (ie not alltime points contained sufficient cell numbers in late apoptosisto accurately analyze the data) an Exact Wilcoxon two-sample test was performed on CD8+ cells in late apoptosis

Results were expressed as an adjusted 119875 value and werereferenced against the mean value for each group plusmn standarddeviation The actual number of horses used to generate theresults was at times different for select tests due to limiteddata collection of some samples or reduced cellular survivalin these individuals Significance was set at 119875 lt 005

3 Results

31 Complete Blood Count and Differentials All CBC andblood cytologic profiles for the 9 horses were within accept-able reference intervals prior to initiation of the study (datanot shown)

32 Serum and CSF Analysis All horses had serum titersof lt2 except for horse number 6 which had a titer of 4at day 5 A titer ge32 is considered significant in cases withaccompanying neurologic signs including in experimentallyinduced cases with neurologic signs that received 6000organismsday for 10ndash14 days (Table 1) For this study horseswere only infected for 10 days By day 73 after inoculation allS neurona-infected horses had serum titers ge32 consistentwith active infection S neurona-infected horses 3 5 and 7had positive CSF titers which were ge1 Serum from horses 1and 8 had titers that were ge2

33 CSFCytology Several horses had increased red blood cell(RBC) levels which were attributed to iatrogenic contamina-tion (Table 2)We believe that this did not adversely affect theSnSAG1 ELISA results as all horses had a serum titer of lt2prior to the initiation of the experiment

34 Neurologic Examination Scores While all 5 S neurona-inoculated horses exhibited neurologic signs and had anincreased neurologic score over the course of the studyhorses varied as to when their neurologic deficits were mostsignificant (Figure 2) Initial neurologic signs were notedon day 5 of infection and on day 10 after infection AllS neurona challenged horses displayed progressive clinicaldisease with moderate to severe neurologic signs (Figure 3)Theneurologic signs included butwere not limited to cranialnerve deficits changes in attitude and ataxia The level ofneurologic deficits was higher than anticipated and as aresult the decision to stop the infection process on day 10was made based on genuine concern for the health of the


Table 2 Baseline CSF cytology data and SAG1 titer on Day minus5

Horse Infection status Color Transparency WBCuL RBCuL Glucose(mgdL)

Protein(mgdL)

SnSAG1ELISA titer

1 Control Colorless Clear 1 1575 491 624 lt22 Removed from study Colorless Clear 2 158 502 591 lt23 S neurona-infected Colorless Clear 0 1 493 451 lt24 Control Colorless Clear 1 52 509 282 lt25 S neurona-infected Colorless Clear 1 0 486 738 lt26 S neurona-infected Colorless Clear 2 11 513 420 lt27 S neurona-infected Colorless Clear 1 0 508 334 lt28 Control Colorless Clear 0 0 522 371 lt29 S neurona-infected Colorless Clear 0 0 565 557 lt2Results of cytologic analysis of the preexperiment CSF sample including SAG-1 titer are shown Results presented include infection status color transparencywhite blood cell (WBC) and red blood cell (RBC) count glucose protein and SnSAG titer Horse 2 received a dose of the parasite in error and was excludedfrom statistical analysis

horses According to Ellison (via phone communication)horses already displaying this level of neurologic deficitswould become progressively worse from this time point andit was therefore anticipated that these horses would progressto a Grade II status by day 60 after infection (the standardlevel of ataxia induced with this dose and model) [18ndash20]Neurologic scores for horse 3 peaked within the first 14 daysafter infection and horses 5 6 7 and 9 peaked betweendays 42ndash56 of the experiment All experimentally S neurona-infected except horse 7 and non-experimentally infectedcontrol horses except horse 8 had a higher score on day56 All S neurona-infected horses demonstrated a detectablelevel of ataxia compared to control horses at days 7 21 and 70(data not shown) Experimentally infected horses exhibited apeak in cranial nerve signs prior to the peak in ataxia thelatter of which was more pronounced towards the end of thestudy period Logarithmic values were used in the statisticalanalysis in an attempt to stabilize the variance and as thesewere closer to a straight line on the normal probability plotsA significant difference (119875 lt 005) between the controland infected populationrsquos mean overall neurologic score waspresent on day 70 A trend towards a difference (005 lt 119875 lt01) was seen at days 7 and 28

35 Enriched Peripheral Blood Leukocyte Cytology Cytologicexamination of the enriched peripheral blood leukocytes wasnot remarkable across treatment or time (data not shown)The average differential count obtained from these enrichedcell suspensions was 6 neutrophils 875 lymphocytes and65 monocytes

36 Lymphocyte Proliferation Enriched peripheral bloodlymphocytes from S neurona-infected horses cultured withPMAI demonstrated a significantly lower stimulation indexcompared to controls on day 35 (Figures 4 and 5) Noconsistent differenceswere observed in proliferation responseof S neurona-infected cells to ConA (T-cell) PWM (B-cell)or antigen specific- (merozoites-) induced proliferation as

detected by (3H)-thymidine incorporation throughout thestudy (data not shown)

37 Peripheral Blood Leukocyte Cell Surface Marker Expres-sion Cell surface marker expression of fresh and culturedleukocytes in general did not differ between the twotreatment groups with the exception of neutrophils The Sneurona-infected group had a significantly higher percentageof neutrophils at days 0 and 14 (data not shown)

38 Apoptotic Changes in Immune Subsets For cells cul-tured with media only PMAI and merozoite stimulatedsamples viable early and late apoptotic populations weredefined based on 7-AAD staining (Figure 6) All sampleswere analyzed using the same electronic gates Most data arepresented in the context of significant differences between theexperimentally infected versus non-experimentally infectedcontrol animals (Table 5)

In cells cultured with media only the percentage of CD8+cells undergoing apoptosis tended to be higher in S neurona-infected horses In PMAI cultured wells neutrophils (PMN)had decreased apoptosis (day 14) and viability (days 35 and42) (data not shown) Monocytes (mono) had decreasedapoptosis (days 21 28 and 35) and decreased viable cells at(days 42 and 56) CD4+ andCD8+ cells had increased viabilityat days 7 and 70 CD8+ cells had significantly increasedapoptosis present at day 42 In contrast B cells had increasedviability on days 21 and 28 In cells cultured with merozoitesthere were individual treatment groups with increased apop-tosis at select time points but no consistent changes acrosstime (data not shown) Apoptosis was increased inCD4+ cells(days 35 and 70) and CD8+ cells (days 42 and 70)

39 Alterations in Immune Populations Based on CFSE Pro-liferation Samples were gated on live versus dyingapoptoticgates and then cell divisions were marked (Figure 7 Table 3)[23] For each time point stimulation and immune cellsubset data were gated and statistically analyzed based on


Table 3 PMAI and merozoite stimulated proliferation as determined by CFSE

(a)

PMA Percent CD4 Percent CD40 divisions

Percent CD41 division

Percent CD42 divisions


Percent CD4gt3 divisions

Day 2 CD4Control 358 plusmn 468 52 plusmn 567 3923 plusmn 565lowast 1147 plusmn 086lowast 132 plusmn 032lowast 019 plusmn 009

Day 2 CD4Infected 4374 plusmn 437 3344 plusmn 708 498 plusmn 486 1886 plusmn 215 2892 plusmn 059 0442 plusmn 018

Day 28 CD4Control 347 plusmn 081 512 plusmn 1302 4663 plusmn 13 322 plusmn 090lowast 162 plusmn 029lowast 250 plusmn 008lowast


(b)

PMAI Percent CD8 Percent CD80 divisions





Day 2 CD8Control 1049 plusmn 13 5427 plusmn 239lowast 384 plusmn 37lowast 940 plusmn 101lowast 144 plusmn 156lowast 044 plusmn 008


MerozoitesDay 28 CD8Control 2655 plusmn 065 2926 plusmn 236lowast 6305 plusmn 226lowast 1011 plusmn 08lowast 1185 plusmn 026lowast 123 plusmn 001


Day 56 CD8Control 1763 plusmn 226 6557 plusmn 232lowast 2846 plusmn 1661lowast 728 plusmn 636lowast 012 plusmn 009 0 plusmn 0


Day 70 CD8Control 231 plusmn 204 6467 plusmn 145lowast 3753 plusmn 1416lowast 178 plusmn 109lowast 009 plusmn 009lowast 014 plusmn 007lowast


Lymphocytes from control and S neurona-infected horses were stained with CFSE and then cultured with no stimulation PMAI or live merozoites for 72 hrat 37∘C 5 CO2 Samples were then stained for CD4 (a) or CD8 (b) and the number of divisions per sample was determinedThe average number of cells perdivision plusmn SEM was determined for control and infected horses Statistical differences are marked as lowast119875 lt 005

quantitative differences in number of proliferations presentper sample The significant differences are presented inTable 3 to provide the pattern of response CD4+ cells (days2 7 21 and 56) and CD8+ cells (days 2 21 and 28) fromS neurona-infected horses had increased numbers of divi-sionsproliferation when stimulated with PMAI When cul-tured with live merozoites CD4+ cells from the S neurona-infected horses had decreased cell divisionsproliferationon day 28 but increased divisionsproliferation on day 42CD8+ cells had decreased divisionsproliferation in infectedhorses at days 28 56 and 70 suggesting a limited ability ofleukocytes to respond to merozoites Due to a consistentlylimited cell sample recovery after 72 hrs of culture the B cellsand monocytes were not evaluated

4 Discussion

Studying EPM in horses is difficult due to the challenges ofdeveloping a reliable infectionmodel In the present study wewere able to validate Ellisonrsquos model of infection by showing

that experimentally S neurona-infected horses developedneurologic clinical signs as well as generating positive serumand CSF titers Using this model we made several notewor-thy observations regarding the equine immune response toactive S neurona infection We observed mitogen-induceddifferences in leukocyte apoptosis and proliferation duringinfection and after infection Of interest our data suggest thatS neurona infection alters the ability of antigen presentingcells to stimulate CD4+ and CD8+ proliferation which willbe the subject of future studies

All S neurona infected horses had increased serum andCSF antibody levels after infection Horses with SnSAG1CSF titers ge1 indicated intrathecal production of antibodiesand thus active infection The lowest detection titer forSnSAG1 CSF as performed by Pathogenes was changed bythe company during our study period Prior to the exper-iment the first dilution performed was 1 2 Therefore thelowest negative titer for the test was lt2 During the timein which we performed our experiment (thus applicable topostexperiment CSF SnSAG1 results) Pathogenes lowered


Table 4 Neurologic scoring sheet for observations of clinical parameters (total possible 97)

Category Action Description Number Description 2

Eating

Drops feedlt14 lb 1

(max = 3)ge14 lb le 12 lb 2

gt12 lb 3

Tongue tone decreasedNormal mastication 1

(max = 3)Abnormal mastication 2

Paresis 3

Abnormal feedprehension

Normal 0(max = 2)Unable to masticate 1

Bites food 2

Head

DroolingDoes not drool 0

(max = 2)When eating 1

Continuously 2

Lip paresis

Normal 0

(max = 3)Perceptible when eating 1

Perceptible continuously 2

Lip hangs 3

Facial nerve paresis

Normal 0

(max = 3)Perceptible 1

Moderate 2

Severe 3

Eyelid paresis

Normal 0

(max = 3)Ventrally away from eye 1

Over 14 of eye 2

With corneal lesion 3

Attitude

Normal 0

(max = 3)Depressed 1

Aggressive 2

Somnolent 3

Muscle Muscle atrophy

Normal 0

(max = 3)Just perceptible 1

Immediately noticeable 2

Severe atrophy 3

Rear end Cauda equina

Normal 0

(max = 3)Holds tail rigid 1

Dribbling urine 2

Rectum paretic 3

Movement

Weakness

Normal 0 Left fore (LF)right fore (RF)left hind (LH)right hind (RH)

(max = 12)

While walking 1

Markedly reduced strength 2

Can pull horse over easilyrecumbent 3

Lameness

None seen 0

(max = 3)Just noticed at walk 1

Head bob or hip drop at walk 2

Reluctant to use limb 3


Table 4 Continued


Consciousproprioception

Crossing over limbs

Normal 0

LF RF LH RH(max = 12)

Slightly slow to place limb 1

back to normal positionNo resistance to abnormal placement oflimb

2

Horse unaware of limb position andunable to correctly place limb

3

Abduction of hind limbs

Normal 0

LH RH (max = 6)Slightly slow to place limb back to normalposition

1

No resistance to abnormal placement oflimb

2


3

Tripping

None seen 0

(max = 3)Sometimes 1

Often 2

Often and falls to knees 3

Limbs

Paresis

Normal 0

(max = 3)Detectable at normal gaits butexacerbated by manipulative actions

1

Obvious at normal gaits or postures 2

Very prominent at normal gait 3

Circling

Normal 0Counterclockwiseclockwise (max = 6)

Occasional abnormal limb placement 1

Does not move normally in circle 2

Refuses to circlefalls 3

Pivoting

No pivoting seen 0Counterclockwiseclockwise (max = 6)

Pivots occasionally on inside hind limb 1

Pivots frequently 2Will not pick up hind leg-pivotscontinuously

3

Toe dragging

No toe dragging seen 0

Left Right (max = 6)Occasionally drags a hind limb 1

Frequently drags hind limb 2

Constantly drags hind limbs 3

Backing up

Hypermetria

Normal movement 0

(max = 3)Slightly hypermetric in one limb 1Moderately hypermetric in onelimbslight hypermetria in both hindlimbs

2

Very obvious hypermetria 3

Inconsistent placement

Normal placement 0

(max = 3)Slightly wide based on placement of hindlimbs

1

Wide based on hind limb placement 2Places limbs very abnormally (touchingvery wide) almost falls 3


Table 4 Continued


Tail pull At the walk

Normal 0

Left Right (max = 6)Slight lack of resistance to tail pull 1Easily pull horse off track 2Horse almost falls when tail pulled 3

Maximum totalpossible 97

Horses were examined biweekly and scored based on a variety of different parameters listed above For most categories a score of zero represented a normalresponse to evaluation of this parameter and a score of three was the maximum neurologic score possible The scoring system was based on that used inprevious experiments by investigators using the intravenous method of infection

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 3Horse 5Horse 6

Horse 7Horse 9Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)

Figure 2 Neurologic scores by (a) individual total non-experimentally infected neurologic score control (top) and (b)S neurona experimentally infected (bottom) horses All horseswere scored biweekly via a comprehensive neurologic panelEach parameter was assessed a score from 0 (normal) to 3 (veryabnormal) with a maximum of 97 (a) Total neurologic score isreported Baseline = preacclimatization score Week minus1 = scorefollowing acclimatization but prior to infection Week 1 = firstweek of infection with S neurona Results are reported by baseline-individual horse response by day and the average response of thehorses by treatment by day

005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1

Logarithmic transformation of neurologic scores with time

Figure 3 Log-transformed neurologic scores (all parameters) overtime Horses were scored biweekly for a multitude of differentparameters in order to detect neurologic signs The average neuro-logic score was log-transformed and plotted against experimentaltime to equalize variance between samples Error bars represent onestandard deviation about the mean Baseline = preacclimatizationscore Week minus1 = score following acclimatization but prior toinfection Week 1 = first week of infection with S neurona Errorbars represent one standard deviation about the mean

the negative titer to lt1 This perceived reduced sensitivityin our prechallenge samples unlikely impacted our dataas all horses had negative serum titers at this time ofcollection Based on a postchallenge positive serum titerit appeared that control horses 1 and 8 may have had anatural exposure to S neurona Additionally based on controlhorse 4 having a positive CSF titer this suggests naturalexposure in which S neurona was sequestered from theimmune response until entering the CNS Throughout thestudy all the horses were kept together in a fenced pastureand while the hay was kept in a covered barn prior to feedingthe potential for natural exposure was possible When thestudy was performed ELISAs to determine the antibodiesto different SAGs were not available and unfortunately thesera from these horses are no longer available Thus wewere unable to determine if the ldquonaturally exposedrdquo horseswere infected by a non-SAG-1 expressing strain Howeverthese two control horses never developed positive CSF titers


Table 5 Tables of significant differences in viability for (a) PMAI and (b) merozoite stimulated cells

(a)

Immune cell Stimulation Gate Day Time (hrs) Change

CD4 PMAI Viable 7 24 Increase late apop

72 Increase viable

CD4 PMAI Apopdying 7 24 Decrease late apop

14 24 Decrease late apop

CD8 PMAI Viable

7 48 Increase viable

42 48 Increase early apop


72 Decrease early apop

Neutrophils PMAI Viable

14 72 Decrease early apop

35 24 Decrease viable

48 Decrease late apop


Monocytes PMAI

Viable




48 Increase early apop

72 Increase late apop


Viable56 24 Decrease viable

70 48 Increase late apop


B-cells PMAI

Viable









Apopdying21 24 Increase early apop



(b)


CD4 Merozoite

Viable 35 24 Increase late apop


Apopdying70 24 Increase viable

24Decrease early and

late apop

72Decrease viableincrease apop

CD8 Merozoite 42 24 Increase early apop



(b) Continued


Neutrophils Merozoite

28 24 Decrease early apop56 24 Increase viable

Viable 70 Increase viable earlyLater increase early

and late apop72 Increase early apop

Monocytes Merozoite 56 24 Increase viablecontrol


B-cells Merozoite

Live

0 24 Increase late apop2 48 Increase late apop14 48 Decrease viable21 24 Increase viable

72 Increase early apop28 24 Increase viable35 48 Increase late apop70 24 Increase early apop

Dying

35 72 Increase viable56 72 Decrease viable70 24 Decrease viable

72 Increase early apopThe immune cell subset treatment viabledying gate day time point and significant change are reported

005

115

225

335

4

Day

Log transformed stimulation index (PMAI)

ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD

Figure 4 Differences in log-transformed stimulation indices forincubation with PMAI between S neurona experimentally infectedand control horses Control horses had a significantly higherstimulation index for PMAI responses on Day 35 with a patterndemonstrating higher indices in the control group towards theend of the experiment Lymphocyte proliferation assays using the(3H)-thymidine assay were performed with a variety of mitogensincluding the pan-leukocyte stimulant PMAI Stimulation indiceswere calculated by dividing the average counts per minute forcells stimulated with each mitogen by the counts per minute forspontaneously proliferating cells Average stimulation index waslog-transformed and plotted against time for control (blue) andinfected (white) horses The error bars represent standard deviationabout the mean The blue star indicates a significant difference (119875 lt005) Day 0 = first day of experiment (Day 1 of infection)

It is possible that natural exposure to S neurona couldhave affected the immune responses of these control horsesand thus the relative differences between experimentallyinfected and non-experimentally infected horses requiredcareful review of the data However we do believe that thesignificant findings of the S neurona experimentally infectedhorses compared to the control horses even with possiblenatural exposure of some are still valid For most cases wepredict that the findingswould have been evenmore dramaticwithout possible natural exposure

With regard to the merozoite-infected leukocyte modeldeveloped by Ellison et al we were able to confirm suc-cessful infection and manifestation of clinical EPM [18ndash20]However we only infected the horses for 10 days based onthe severity of clinical neurologic signs According to DrEllison (personal communication) these signs were moresevere than typical of Day 10 If the infection process had beenallowed to continue for 14 days we predicted that the clinicalsigns and possibly the immune responses would have beenmore pronounced Our primary rationale for discontinuingthe experimental infection protocol at day 10 was for thesafety of the horses

The utilization of the different mitogens (ConA PWMPMAI) in culture allowed us to assess the T cell B celland pan-leukocyte response in these horses during activeS neurona-induced infection With PMAI-induced prolif-eration we observed a significant decrease in proliferativeresponse of infected horsesrsquo lymphocytes at day 35 This


00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)

Figure 5 Individual PMAI stimulation indices for non-experimentally infected (a) and S neurona experimentally infected (b) horsesLymphocyte proliferation assays using the (3H)-thymidine assay were performed with a variety of mitogens including the pan-leukocytestimulant PMAI Stimulation indices were calculated by dividing the average counts per minute for cells stimulated with each mitogen bythe counts per minute for spontaneously proliferating cells Results are reported by individual horse response and the average response of thehorses by treatment by day

was in contrast to previous studies where the decreasedproliferation response was present at multiple time points[12 13] However we did observe a pattern of decliningproliferation late in the infection stage Possible explanationsfor the lack of additional differences would be (a) differencesin the stageseverity of infection in the facts that these horseswere infected with a lower total dose compared to otherstudies with experimentally infected horses and that (b) someof the control horses were natural exposed to S neurona anddeveloped similar decreased PMAI responses thus maskingthe decrease in PMAI response of experimentally infectedhorses The lack of more consistent significant differencesin immune cell subsets and proliferation could be due toindividual variability among horses with different geneticbackgrounds possible natural exposure and dose of infectionused as well as individual differences in immune responsesAdditionally it is possible that the S neurona infectioncould have caused transient changes in the immune responsebased on the dynamic nature of the immune response (iedevelopment of protective response versus development ofdisease) Therefore we focused on differences when anoverall trendwas seen even if there were just a few significantdifferences

Analysis of the apoptosis and CFSE results revealedthat there were multiple time points in which unstimulatedCD8 cells (days 0 14 35 and 42) from experimentallyinfected horses had increased apoptosis With regard toPMAI stimulation neutrophils had decreased viability atdays 35 and 42 monocytes had decreased apoptosis at days21 28 42 and 56 B-cells had increased viable cells at days21 and 28 and decreased viable cells at day 35 which mayhave adversely impacted the observed decrease in PMAI-induced proliferation Based on theCFSE experiment PMAI

stimulation demonstrated increased proliferation in CD4(days 2 7 21 56 and 70) and CD8 cells (days 2 21 and 28)whereas cells stimulated with merozoites resulted in changesin CD4 (decreased day 28 increased day 42) and decreasedCD8 proliferation (days 28 56 and 70) With regard todetermining whether suppressed PMAI proliferation wasdue to increased apoptosis or decreased proliferation asonly day 35 was statistically significant our interpretationof the possible mechanisms is very limited In examiningthe day 35 data there is a spontaneous increase in CD8apoptosis and with PMAI stimulation there is a decrease inviable neutrophils and B-cells Based on these data plausiblemechanisms for the decreased PMAI induced responsewould be decreased PMN B-cell and CD8 viability withincreased apoptosis of the CD8 cells and will require furtherinvestigation

In regard to the changes in immune response duringactive S neurona infection the observations of increasedPMAI-induced proliferation with CFSE and decreased pro-liferation to merozoites in the S neurona-infected horsessuggest that both CD4+ and CD8+ cells were at a height-ened stage of activation to nonspecific stimuli (ie PMAI)With merozoite stimulation S neurona-infected horses haddecreased cellular proliferation This decreased responsecould possibly be associated with an impaired ability ofantigen presenting cells (APC) to process and present Sneurona antigen to CD4+ andCD8+ cellsMerozoitesmust beprocessed and presented by antigen presenting cells (APC)which is a time dependent process in order to stimulate T-cells It is also possible that S neurona impairs the abilityof antigen presenting cells to present novel antigen to theCD4+ and CD8+ cells We do not know if this is specificto S neurona or any antigen Another possibility is that the


1000

800

600

400

200

0

SS S

S

Dying lymphocytes128

Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)

Figure 6 Gating for apoptosis data 2a gate of live versus dying lymphocytes after (a) 24 hrs (b) 48 hrs and (c) 72 hrs (d) A sample of the7-AAD gating of viable early and late apoptotic cells

CD4+ and CD8+ cells had a decreased ability to respondto S neurona (T-cell specific response) due to a loss insecondary signals Alternatively CD4+ and CD8+ cells fromS neurona-infected horses could be more susceptible to themerozoites This would result in decreased CFSE-mediatedproliferation of merozoite-infected cells from infected versuscontrol horses

In this study we were not able to distinguish a unique andconsistent change in equine immune response to S neuronainfection and relate it to susceptibility Decreased viability ofneutrophil populations was noted and likely was due to activeinfection The observed decrease in monocyte apoptosiscould be an effect of active infection or a direct inhibitoryeffect of S neurona on monocyte function (as demonstratedwith other protozoan) [25] Likewise observed increased B

cell viability could also be associated with response to activeinfection and will be evaluated in future studies Additionalstudies are needed to further define these mechanisms

To summarize the results from this study demonstratethat horses experimentally infected with S neurona developclinical disease and generate antibodies to S neurona inthe serum and CSF These S neurona-infected horses hadaltered immune cell subset expression which changed dur-ing disease progression Furthermore the functionality ofthese immune cells as measured by proliferation as well asviability and degree of cell death was also intermittentlyaffected Most interestingly S neurona-infected horses haddecreased antigen specific proliferation responses comparedto non-experimentally infected horses however non-antigenspecific responses (ie mitogen-induced) were not decreased


1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103

3 divisions 2 divisions 1 divisionNo divisiongt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

ApopLive lymphocytes

Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103

3 divisions 2 divisions 1 division

No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)

Figure 7 Effects of PMAI and merozoite stimulation on peripheral leukocyte proliferation as detected using CFSE Lymphocytes fromcontrol and S neurona-infect horses were incubated with CFSE and cultured with (a) PMAI or (b) unstimulated for 72 hrs at 37∘C 5 CO

2

Cells were washed and stained for leukocyte markers and proliferation based on number of divisions was determined (c) PMAI stimulatedand (d) unstimulated

throughout most of the study These data suggest that theprocess between antigen presenting cell (monocytedendriticcell) andor T cell antigen recognition may be impaired inS neurona-infected horses The nature of this defect in thehorsersquos immune response could possibly be associated withthe particular stage of development of clinical EPM followingexposure to S neurona but needs to be investigated furtherunder conditions in which the infection duration is longerStill these preliminary experiments raise interesting newquestions regarding the precise immune response of cellularsubsets to infection with S neurona

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to acknowledge the following sources offunding for this project Patricia Bonsall Stuart Equine Foun-dation and the Virginia Horse Industry Board paramutualracing funds from Virginia Polytechnic and State University


and a graduate assistantship to Rochelle Lewis from theOfficeof Research and Graduate Studies at the Virginia-MarylandRegional College of Veterinary Medicine The authors wishto thank Dr Jennifer A Pierro (SAMS) and Dr Brina Lopez(LAMS) at the University of Georgia College of VeterinaryMedicine for critical review of this paper

References

[1] R J MacKay D E Granstrom W J Saville and S M ReedldquoEquine protozoal myeloencephalitisrdquoThe Veterinary Clinics ofNorthAmerica Equine practice vol 16 no 3 pp 405ndash425 2000

[2] J P Dubey ldquoMigration and development of Sarcocystis neuronain tissues of interferon gamma knockout mice fed sporocystsfromanaturally infected opossumrdquoVeterinary Parasitology vol95 no 2ndash4 pp 341ndash351 2001

[3] NAHMS Equine Protozoal Myeloencephalitis (EPM) in the USCEAHmdashUSDA APHIS Fort Collins Colo USA 2001

[4] C K Fenger D E Granstrom A A Gajadhar et al ldquoExper-imental induction of equine protozoal myeloencephalitis inhorses using Sarcocystis sp sporocysts from the opossum(Didelphis virginiana)rdquo Veterinary Parasitology vol 68 no 3pp 199ndash213 1997

[5] C K Fenger D E Granstrom J L Langemeier and S StamperldquoEpizootic of equine protozoal myeloencephalitis on a farmrdquoJournal of the American VeterinaryMedical Association vol 210no 7 pp 923ndash927 1997

[6] S G Witonsky R M Gogal Jr R B Duncan Jr and D SLindsay ldquoImmunopathologic effects associatedwith Sarcocystisneurona-infected interferon-gamma knockoutmicerdquo Journal ofParasitology vol 89 no 5 pp 932ndash940 2003

[7] S GWitonsky RM Gogal Jr R B Duncan andD S LindsayldquoProtective immune response to experimental infection withSarcocystis neurona in C57BL6 micerdquoThe Journal of Parasitol-ogy vol 89 no 5 pp 924ndash931 2003

[8] D C Sellon D P Knowles E C Greiner et al ldquoDepletionof natural killer cells does not result in neurologic diseasedue to Sarcocystis neurona in mice with severe combinedimmunodeficiencyrdquo Journal of Parasitology vol 90 no 4 pp782ndash788 2004

[9] S G Witonsky R M Gogal Jr R B Duncan Jr H Nor-ton D Ward and D S Lindsay ldquoPrevention of meningoencephalomyelitis due to Sarcocystis neurona infection in miceis mediated by CD8 cellsrdquo International Journal for Parasitologyvol 35 no 1 pp 113ndash123 2005

[10] S J Tornquist L J Boeder D E Mattson C K CebraR J Bildfell and A N Hamir ldquoLymphocyte responses andimmunophenotypes in horses with Sarcocystis neurona infec-tionrdquo Equine Veterinary Journal vol 33 no 7 pp 726ndash729 2001

[11] J A Spencer S E Ellison A J Guarino and B L BlagburnldquoCell-mediated immune responses in horses with equine pro-tozoal myeloencephalitisrdquo Journal of Parasitology vol 90 no 2pp 428ndash430 2004

[12] J Yang S Ellison R Gogal et al ldquoImmune response toSarcocystis neurona infection in naturally infected horses withequine protozoal myeloencephalitisrdquo Veterinary Parasitologyvol 138 no 3-4 pp 200ndash210 2006

[13] S G Witonsky S Ellison J Yang et al ldquoHorses experimentallyinfected with Sarcocystis neurona develop altered immuneresponses in vitrordquo Journal of Parasitology vol 94 no 5 pp1047ndash1054 2008

[14] W J Saville S M Reed D E Granstrom et al ldquoSeroprevalenceof antibodies to Sarcocystis neurona in horses residing in OhiordquoJournal of the American VeterinaryMedical Association vol 210no 4 pp 519ndash524 1997

[15] W J Saville S M Reed P S Morley et al ldquoAnalysis ofrisk factors for the development of equine protozoal myeloen-cephalitis in horsesrdquo Journal of the American VeterinaryMedicalAssociation vol 217 no 8 pp 1174ndash1180 2000

[16] W J Saville R W Stich S M Reed et al ldquoUtilization of stressin the development of an equine model for equine protozoalmyeloencephalitisrdquoVeterinary Parasitology vol 95 no 2ndash4 pp211ndash222 2001

[17] P S Morley J L Traub-Dargatz K M Benedict W J ASaville L D Voelker and B AWagner ldquoRisk factors for owner-reported occurrence of equine protozoal myeloencephalitisin the US equine populationrdquo Journal of Veterinary InternalMedicine vol 22 no 3 pp 616ndash629 2008

[18] S Ellison T Kennedy and K Brown ldquoEarly signs of equineprotozoal myeloencephalitisrdquo International Journal of AppliedResearch in Veterinary Medicine vol 1 pp 272ndash278 2003

[19] S Ellison T Kennedy and K Brown ldquoDevelopment of anELISA to detect antibodies to rSAG1 in the horserdquo Journal ofApplied Research in Veterinary Medicine vol 1 no 4 pp 318ndash327 2003

[20] S Ellison E Greiner K Brown and T Kennedy ldquoExperimentalinfection of horses with culture-derived Sarcocystis neuronamerozoites as a model for equine protozoal myeloencephalitisrdquoInternational Journal of AppliedResearch inVeterinaryMedicinevol 2 pp 79ndash89 2004

[21] I G Mayhew A deLahunta R H Whitlock L Krook and JB Tasker ldquoSpinal cord disease in the horserdquo Cornell Veterinaryvol 68 supplement 6 pp 1ndash207 1978

[22] S Witonsky R M Gogal Jr V Buechner-Maxwell and SA Ahmed ldquoImmunologic analysis of blood samples obtainedfrom horses and stored for twenty-four hoursrdquo AmericanJournal of Veterinary Research vol 64 no 8 pp 1003ndash10092003

[23] A B Lyons ldquoAnalysing cell division in vivo and in vitro usingflow cytometric measurement of CFSE dye dilutionrdquo Journal ofImmunological Methods vol 243 no 1-2 pp 147ndash154 2000

[24] D S Lindsay and J P Dubey ldquoDirect agglutination test for thedetection of antibodies to Sarcocystis neurona in experimen-tally infected animalsrdquo Veterinary Parasitology vol 95 no 2ndash4pp 179ndash186 2001

[25] P Keller F Schaumburg S F Fischer G Hacker U Groszlig andC G K Luder ldquoDirect inhibition of cytochrome c-inducedcaspase activation in vitro by Toxoplasma gondii reveals novelmechanisms of interference with host cell apoptosisrdquo FEMSMicrobiology Letters vol 258 no 2 pp 312ndash319 2006


abnormalities ataxia weakness and focalmuscle atrophy [3ndash5] Annual losses in theUnited States are estimated to be $554to $1108 million [2]

In order to developmore efficacious treatments vaccinesand diagnostic assays it is important to first determine ifthere is an immune signature that dictates whether a horsewill develop a protective response or neurologic diseaseWhile protection has been linked to CD4 and CD8 cell-mediated response and interferon-gamma (IFN120574) produc-tion [6ndash9] the specific pathway(s) of immune cell responsesassociated with development of disease in the equine are stillpoorly characterized Interestingly a number of studies havereported an immune profile of decreased CD4 expressionsurface antigen 1- (SAG1-) induced cell proliferation PMAIstimulated cell proliferation and IFN120574 in EPM affectedhorses [10ndash13] It has been proposed that stress-inducedimmune dysregulation increases disease susceptibility ashorses subjected to stressful conditions (ie shipping show-ing training and pregnancy) tend to have an increasedincidence of disease [14ndash17] Therefore new studies designedto explore the specific immune consequences of EPM in thehorse would facilitate new drug discoveries and diagnostictests whichwould have great economic and emotional impactfor both the individual horse owner and the US equineindustry

Previously we determined that both naturally and exper-imentally infected horses have suppressed leukocyte prolif-eration responses when stimulated with combined mitogensPMAI [12 13] The purpose of the present study was todetermine whether this defect was due to an increase inapoptosis or decrease in cellular proliferation of a specificsubset of leukocytes as well as to identify altered immuneresponses during disease progression in horses experimen-tally challenged with S neurona

2 Materials and Methods

21 Horses Nine-quarter horse and quarter horse crossesranging in age from 18 months to 11 years consisting ofeight mares and one gelding were used in this study Allhorses were vaccinated prior to transport to Virginia Techagainst equine influenza tetanus West Nile Virus Easternand Western Equine Encephalitis and Equine Rhinopneu-monitis Upon arrival the horses were also vaccinated againstrabies Historic records indicated that none of the horses hadexhibited clinical signs of neurological disease Horses weremaintained on pasture and fed grass hay and pelleted ldquosweetfeedrdquo in accordance with normal energy requirements forweight gain for the study duration Horses were acclimatedfor two weeks prior to assessment of baseline immunefunction and serology

22 Physical and Neurologic Examinations Complete phys-ical examinations were performed on all the horses uponarrival at Virginia-Maryland Regional College of VeterinaryMedicine (VMRCVM) Baseline physical and neurologicexams were performed (Figure 1) All care and maintenance

of the horses was in accordance with the guidelines estab-lished by the Animal Use and Care IACUC committee atVirginia Tech and the study was approved by the IACUCcommittee All horses underwent a neurologicallamenessexamination two weeks prior to the start of the study Inves-tigators Lewis (RL) and Witonsky (SW) performed blindedneurologic exams separately for each horse including baselinescores A 97-point scoring system was used to evaluate thehorsesrsquo neurologicallameness status and is described in detaillater in Table 4 The scoring system was based on a protocolestablished by Ellison et al [18ndash20] which had been usedfor several previous United States Department of Agriculture(USDA) approved EPM studies Briefly the scores rangedfrom zero (normal) to 3 (severely affected) for 22 differentparameters with a maximum total score of 97 (Table 4)The parameters measured included but were not limitedto cranial nerve function ataxia weakness (paresis) andspasticity The 22 parameters that were evaluated includeddropping feed tongue tone apprehension of feed droolinglip facial nerve and eyelid paresis attitude muscle atrophycauda equine weakness lameness conscious proprioceptionbased on crossing over of limbs abduction of hind limbstripping paresis circling pivoting and toe dragging of limbshypermetria inconsistent placement and tail pull Whena horse displayed a neurologic or lameness deficit theappropriate score was assigned and the horse was observedclosely for two weeks prior to initiation of the study If thehorsersquos status was unchanged during this period the animalwas subjected to a more thorough neurologic evaluation(described in the following section) to minimize the possibil-ity of a preexisting EPM infection A few horses displayed gaitand conformation abnormalities that resulted in ameasurableelevated baseline neurologiclameness score however theseabnormalities were determined to not be attributable to anEPM infection We predicted that an S neurona infectionwould alter the baseline score thus horses with a preexistingminimal measureable baseline neurologiclameness scoreremained in the study

23 Serum and CSF Sample Collection (Day minus5) prior toInfection After a two-week acclimatization period (day minus5)peripheral blood samples were collected and horses wereanesthetized CSF was obtained from the atlanto-occipital(AO) joint space using a 3510158401015840 18 G spinal needle and sub-mitted along with the serum to a commercial laboratoryforSnSAG1 ELISA and to the laboratory at VMRCVM forroutine cytology [19 21] CSF collection was also performedon postinfection day 73

24 S neurona Culture Merozoites were obtained by atechnique described by Ellison et al [18 20] BrieflySnSAG1 merozoites were isolated from CNS tissue froma horse previously diagnosed with EPM These merozoiteswere then maintained in continuous culture Merozoiteswere cultured in complete media (RPMI with L-glutamine(Mediatech Herndon VA) hepes buffer 25mM (Mediat-ech Herndon VA) 2 heat inactivated fetal bovine serum(FBS) (Atlanta Biologics Lawrenceville GA) 50 IUmL of


Neu

rolo

gic e

xam

s an

d ba

selin

e im

mun

e fun

ctio

n

9 ho

rses

obt

aine

d-ba

selin

e ne

urol

ogic

exam

s

AO

taps

AO

taps

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ysLy

mph

ocyt

e pro

lifer

atio

n as

says

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys


Study period

Treatment

minus21 minus5 0 2 7 10 14 21 28 35 42 56 70 73 120

Trea

tmen

t gro

ups a

lloca

ted




2


























titer








3 Results








Protein(mgdL)

SnSAG1ELISA titer












(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























Neu

rolo

gic e

xam

s an

d ba

selin

e im

mun

e fun

ctio

n

9 ho

rses

obt

aine

d-ba

selin

e ne

urol

ogic

exam

s

AO

taps

AO

taps

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ysLy

mph

ocyt

e pro

lifer

atio

n as

says

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys

Lym

phoc

yte p

rolif

erat

ion

assa

ys


Study period

Treatment

minus21 minus5 0 2 7 10 14 21 28 35 42 56 70 73 120

Trea

tmen

t gro

ups a

lloca

ted




2


























titer








3 Results








Protein(mgdL)

SnSAG1ELISA titer












(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References















































titer








3 Results








Protein(mgdL)

SnSAG1ELISA titer












(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References































titer








3 Results








Protein(mgdL)

SnSAG1ELISA titer












(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References





























Protein(mgdL)

SnSAG1ELISA titer












(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References




























(a)










(b)
















4 Discussion







Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References





























Eating

Drops feedlt14 lb 1


gt12 lb 3



Paresis 3



Bites food 2

Head



Continuously 2

Lip paresis

Normal 0



Lip hangs 3


Normal 0


Moderate 2

Severe 3

Eyelid paresis

Normal 0


Over 14 of eye 2


Attitude

Normal 0


Aggressive 2

Somnolent 3


Normal 0



Severe atrophy 3


Normal 0


Dribbling urine 2

Rectum paretic 3

Movement

Weakness


(max = 12)

While walking 1



Lameness

None seen 0





Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























Table 4 Continued



Crossing over limbs

Normal 0




2


3


Normal 0


1


2


3

Tripping

None seen 0


Often 2


Limbs

Paresis

Normal 0


1



Circling





Pivoting




3

Toe dragging





Backing up

Hypermetria

Normal movement 0


2



Normal placement 0


1



Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























Table 4 Continued



Normal 0




0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days

Horse 1Horse 4

Horse 8Average

minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(a)

0

2

4

6

8

10

12

14

7 14 21 28 35 42 49 56 63 70Days



minus4

minus2

Neu

rolo

gic e

xam

(max

=97

)

(b)


005

115

225

335

4

Base

line

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 9

Wee

k 10

Ln(n

euro

scor

e)

Week

ControlInfected

lowast

Wee

kminus1






(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References




























(a)



72 Increase viable



CD8 PMAI Viable










Monocytes PMAI

Viable










B-cells PMAI

Viable












(b)


CD4 Merozoite





late apop





(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























(b) Continued








B-cells Merozoite

Live



Dying



005

115

225

335

4

Day


ControlInfected

Base

line

Day

0

Day

2

Day

7

Day

14

Day

21

Day

28

Day

35

Day

42

Day

56

Day

70

Log(

stim

ulat

ion

inde

x)timesplusmn

SD






00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70

Horse 1Horse 4

Horse 8Average

Stim

ulat

ion

inde

x

Days

(a)

00

50

100

150

200

250

300

350

400

0 2 7 14 21 28 35 42 56 70



Stim

ulat

ion

inde

x

Days

(b)







1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























1000

800

600

400

200

0

SS S

S


Live638

FS FS10008006004002000

(a)

1000

800

600

400

200

0

SS S

S


Live452

FS FS10008006004002000

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Live344

(c)

12

9

6

3

0

Num

ber o

f cel

ls

10minus1

100

101

102

103

Viable871

Early apoptosis122

Late apoptosis068

FL4 LOG 7-AAD

(d)







1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References



























1000

800

600

400

200

0

SS S

S

FS FS10008006004002000

Apop

Live lymphocytes

Monocytes-granuloc

(a)

10minus1

100

101

102

103

10minus1

100

101

102

103


FL1 LOG CFSE

FL2

LOG

CD8

(b)

1000

800

600

400

200

0

SS S

S

FS FS10008006004002000


Monocytes-granuloc

(c)

10minus1

100

101

102

103

10minus1

100

101

102

103


No division

gt3 divisions

FL1 LOG CFSE

FL2

LOG

CD8

(d)


2





Acknowledgments




References




























References


























Documents

Effects of Experimental Sarcocystis neurona-Induced