SAG 6 associates host specific cytokine IL6 signaling pathways to the pathogenesis of disease. The focus

of EPM research should be inflammation.

Thank you: Ellis Greiner, Mike Grigg, Jared Wendte, Tom Kennedy, David Lindsay

Siobhan P. Ellison DVM PhD

• SnSAG6 was lethal for sea otters in California. Shellfish, contaminated with S. neurona are responsible for the infections .

Cutler showed that S. falcatula does not infect horses. Immunoconversion was not detected using a 17 kDa protein on SN immunoblots.

Marsh shows that horses with EPM produce antibodies against S. falcatula. Marsh indicates that SN and SF are likely multiple related species (that may infect different IH).

Howe/Zang showed that a 17 protein is expressed by virulent SN and does not differentiate between SN and SF.

37R experiments, Trojan horse model, and Levamisole HCl implicate clinical signs to the functional WBC

A BLAST analysis of equine IL6 signaling proteins show low level homology with SAG 1/5 (host specific) and SAG 3, microneme, and enolase (non-host specific) proteins.

LEVAMISOLE HCL down regulates IL6

Experiments that relate IL6 to S. neurona

Clinical signs of EPM abate with levamisole treatment. Parasite proteins may function in equine inflammatory IL6 signaling pathways to cause disease.

Day 1

Day 10

IL 6 classic signaling is anti-inflammatory and trans-signaling is proinflammatory. IL6 is species specific gp 130 is not.

• IL6 binds IL-6R to activate gp130• IL-6R

• Cognate receptor subunit found on liver and some leukocytes (neutrophils)

• IL-6R/IL 6• Formed on cognate cells to activate gp130 locally• anti-inflammatory, regenerative

• Trans-signaling: sIL-6R/IL-6 activates gp130• Expression of gp130 is ubiquitous• Binds gp130 via trans-signaling on many cells• sIL-6R/IL-6 crosses BBB• pro-inflammatory

gp130

IL6-R

IL6

IL6

SIL6-R

ANTI-INFLAMMATORY

PRO-INFLAMMATORY

SnSAG 6 and SfSAG 6 are almost identical, only the horse and high level sequencing of 33 markers differentiates the organisms.

SF SAG 6 strain does not express SAG 4

Score494 bits(1271) Expect5e-174 MethodCompositional matrix adjust. Identities259/283(92%) Positives267/283(94%) Gaps2/283(0%)

SnSAG6 1

SfSAG6 1

MTRAVLLTILLTLCSARVSLVKAANPRQATCANGQKTATKVENPGALQLVCPQQYQLNPAMTRAVLLT+LLTLCSARVSLVKAA+PRQATC NGQKTATKVENPGALQ+VCPQQYQLNP MTRAVLLTLLLTLCSARVSLVKAAHPRQATCVNGQKTATKVENPGALQVVCPQQYQLNPP

60

60

SnSAG6 61

SfSAG6 61

PANDAAGDMQVFGTEAADNAVALRGVLPAATYINANGATTLTVPQLPPKPVSVFIQCRQA ANDAAG+MQVFGTEAADN VAL+GVLPAATYINA+ A TLTVPQLPPKPVSVFIQCRQAAANDAAGNMQVFGTEAADNPVALQGVLPAATYINADDAVTLTVPQLPPKPVSVFIQCRQA

120

120

SnSAG6 121

SfSAG6 121

AQGAQQAGQCIIEVQVAGSPRLGLGPNTCAAQQSRIDFEIKAANEAAVFSCGAGLALLQQ QGAQQAGQCIIEVQVAGSPRLG PNTCAAQQSRIDFEI A NEAAVFSCGAGLAL+QQRQGAQQAGQCIIEVQVAGSPRLG-- PNTCAAQQSRIDFEITAGNEAAVFSCGAGLALVQQ

180

178

SnSAG6 181

SfSAG6 179

ASDDTCSKDQALPSGVALAAKEAGAVQLAFPQLPQNPLKICYICTPNGQRAEAAQRCEIHA DDTCSK+ QALPSGVA A KE GAVQL FPQLPQNPLKICYICTPNGQRAE AAQRCEIHALDDTCSKE QALPSGVASAQKEGGAVQLGFPQLPQNPLKICYICTPNGQRAEAAQRCEIH

240

238

SnSAG6 241

SfSAG6 239

VTVAGSGDGGNPGPTGAAPVGPAARSASALVLAVVAAGFFHFWVTVAGSGDGGNPGPTGAAPVGPAARSASALVLAVVAAGFFHFWVTVAGSGDGGNPGPTGAAPVGPAARSASALVLAVVAAGFFHFW

283

281

SAG 6

Genetic analysisGrigg showed that the S. molothrusdidelphis (falcatula) isolated from a bird was the Sf SAG 6 strain.

The Cutler experiment used bird intermediate host selectivity to show SF SAG 6 does not infect horses

A classic example of IH host selectivity: An immune deficient mouse selects SN while the budgie select SF. Biological assays are an important adjunct to sequence analysis, this experiment clarified that SN was not SF as suggested by some

sequence data.

*

*Mansfield may have shown cowbird hosts SN and SF

S. molothrusdidelphis (falcatula) did not cause clinical signs or induce antibodies against a 17 kDa protein of S. neurona in horses.

• One horse developed clinical signs due to sarcocystiasis (that was attributed to pre-experiment exposure based on antibody in serum and CSF against S. neurona).

• On WB: The ‘diagnostic’ protein was a 17kDa (low molecular weight) S. neurona antigen. Results suggest SF won’t cross react on SN immunoblots.

S. molothrusdidelphis S. neurona

SAG 1

SAG 5

SAG 4/3

SAG 2 WB

IFAT CSF SAG 4

AG3

Marsh showed that horses with EPM produce antibodies to SF (1), SF strains differ in protein expression (2), and antibody against SF detects

SN 17 kDa antigens by immunoblot (3).

*

*SAG 1 sera

SF Mu1

SAG minus sera

*Mu1 UCD1 Fl

α SN

α SF *

S. falcatula

αSN

α SF

SN

1.

2.

3.

Zhang (2008) determined that S. neurona SnSPR1, a 17 kDa protein can not distinguish SN and SF. Horses aren’t infected with falcatula therefore

anti-SAG 6 antibody is detected in horses are from neurona infections

The asexual stage of Sarcocystis found in the CNS of horses was named S. neurona with no knowledge of the hosts that transmitted the parasite.

Cutler, Marsh, Howe, Zhang demonstrate that it is necessary to distinguish between SF and SN to interpret the role of SAG 6.

SN SAG 6 can be detected in horse sera therefore SnSAG 6 infects horses

Is S. dasydidelphis the true S. neurona?

S. falcatula and S. neurona maybe more accurately understood as a group of closely related species

• Lost to antiquity is the origin of the name S. falcatula and the type specimen that could be used for genotyping.

“Although S. falcatula Stiles, 1893 was named as a parasite maintained through transmission among birds and opossums over 100 years ago, recent molecular evidence indicates that isolates designated as ‘S. falcatula may be more accurately understood as an assemblage of related species” (Dubey 2006).

• Based on the biology of Sarcocystis we can anticipate that S. falcatula strains will • express different, possibly mutually exclusive, surface antigens • relate to intermediate host specificity• S. falcatula avian parasite and won’t infect horses

Howe (2008) showed all SN tested express a 17 kDa, SnSAG2, antigen (1), the 37R strain is a mixed infection (2), and Gautam (2011) showed that SN SAGs show stage related

expression (3).

Howe 2008, Figure 3 left, and Table 1 footnote below, indicated that Sn-37R is a mixed infection

o 37R SN138 is a SAG 1 minus strain (sporocyst)o 37R SN744 is a SAG 1 strain (merozoite from a horse)

1. 2.

3.Strain Stage

A functional WBC is needed for clinical signs of EPM, shown by SN 37R strain infections in SCID (1) vs immunocompetent (2) foals.

Only the SAG 1 merozoites invaded the CNS (2) indicating the horse may show a parasite bias

Sporocyst SN 37-R 138* (SAG minus) Parasitemia and no clinical signs

Merozoite SN 37-R 744 (SAG 1) Parasitemia and no clinical signs

Sporocyst SN 37-R 138* (SAG minus) No parasites in CNS, Clinical signs

Merozoite SN 37-R 744 (SAG 1) Short lived parasitemia, Neuroinvasion, clinical signs

1.

2.

Strain Stage WBC

37 R strain infections, the Trojan Horse model, and levamisole HCl implicate the functional leukocyte in EPM

• 37R experiments show that WBC needs to be functional for clinical signs and neuro-invasion by the SAG 1 strain. The SAG 1-minus strain didn’t invade the CNS.

• Trojan horse model depends on functional leukocytes• S. neurona (SAG 1) infected leukocytes can deliver parasites into CNS• S. neurona (SAG 1) infected leukocytes produce clinical signs (neuroinflammation)

• Levamisole alleviates inflammation associated with clinical signs • Levamisole can block signs due to challenge (Ellison, unpublished)• Levamisole receptors are present on WBC and on Sarcocystis neurona• In humans and mice levamisole affects cytokine pathways: IL6 is down regulated

• Is there a relationship between virulent S. neurona strains and IL 6?• Equine virulent strains are SAG 1 and SAG 5

Strain Stage WBC

EPM is a biphasic disease. Acutely, strain specific down regulation of IFNγ may allow neuroinvasion. Antibody mediated,

proinflammatory pathways may characterize chronic disease

SN SAG 6 SF SAG 6 SN SAG 1, 5Immunoconversion in horses No immunoconversion in horses Immunoconversion in horses

No neuroinvasion No neuroinvasion NeuroinvasionClinical signs No clinical signs Clinical signs

0 DPC

3 DPC

4 DPC 12 DPC

17 DPC

0

50

100

150

Titer

3

4

5

6

9

10

PMA/I suppression

A mechanism for S. neurona strain virulence was identified by low homology between IL6-IL6r-gp130 and S. neurona SAG’s

SnSAG’s suggest neurovirulence in horses by SAG 1 and 5 strains but not SAG 6 via species specific IL6

SAG 1 and SAG 5, proteins or antibodies that bind them, may interact with equine IL6 pathways. S. neurona SAG 1 and 5 do not have cytokine structure.

* IL6 is species specific therefore this proposed mechanism would confer host cell selectivity by Sarcocystis

Levamisole and its effects on IL6 are well studied. Levamisole receptors on protozoa have been identified.

SnSAG1 SnSAG5 SnSAG2 SnSAG3 SnSAG4 SnSAG6 Microneme enolase

NCBI # ABW82530.1 ABD47681.1 AAO38736.1 AAO38737.1 AAO38738.1 ADG26773.1 AAM95973 AAV80211

Equine IL6 AAB62246.1 44% 35% -- -- -- -- 38%Opossum protein -- -- 100% -- 100% 100%gp130 AEB61458.1 -- -- -- 25% -- --

Eq IL6R XP_005610133 -- 63%

Clinically and experimentally parasites are not always found in the CNS. Dose is related to signs, not parasites.

Intermediate host specificity suggested in CNS disease

IL6 is species specific while gp 130 is not.

Acute (neuroinvasion) disease may require homologs that bind host IL6 receptors. Chronic (neuroinflammation) disease may involve antibody binding of non-species specific gp 130, via IL6 trans-signaling pathways at the blood/CSF barrier.

gp130

IL6-R

IL6

IL6

SIL6-R

ANTI-INFLAMMATORY

PRO-INFLAMMATORY

SAG 6The SAG 6 phenotype may be used implicate virulence factor’s for an IH:• SAG 1 and 5 show homology with equine specific IL6• SAG 1/5 merozoites may down regulate protective IFNɣ

SAG 6 may be the exception that proves the rule:• CNS invasion may require IL6 similarity • Clinical signs of EPM involves anti-SAG 1,5,6 to gp 130

Propose that pathogenesis of disease in a horse • Acute EPM involves stage related SAG’s 1 and 5• Chronic disease involves anti-SAG 1,5,6 • Other stimuli of neurotropic cytokines also show single protein IL6

homology: Borellia and EHV• Look for genetic predisposition to EPM by heredity of IL6r proteins

Possibly, host cytokines are managed by stage related, expressed proteins (SAG’s) that favor infection. Inflammation is induced by antibodies produced against immunodominant SAG’s that, in turn, react with host proteins.

Loop Trail 20 years

Pathogenes PathInflammation

QUESTIONS?

For further discussions meet Siobhan tonight at the clam bar!

SAG 6 has homology with human IL6

First 72 hours Antibody production day 17

gp130

IL6-R

IL6

sIL6-R

C-reactive protein

gp130

IL6

sIL6-R

IL6

Acute Disease Chronic DiseaseSN microneme

SN SAG 1, 5SN enolase

IFNγ

SN SAG 3

IL6

Parasite invasion α SAG 1, 5, 6

Parasite clearance

IL6