SEROPREVALENCE OF SARCOCYSTIS NEURONA IGG IN UNITED STATES SUBMISSIONS TO EQUINE BIODIAGNOSTICS (EBI): A RETROSPECTIVE ANALYSIS OF 2000-2005.

JK Morrow*, SM Reed** and DE Granstrom*** * Equine Biodiagnostics/IDEXX, Lexington KY, ** The Ohio State University College of Veterinary Medicine, Columbus OH *** USDA Animal and Natural Resources Institute, Beltsville MD

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QUALIFYING STATEMENTS

The results from non-US submissions were excluded from analysis. The results of all known infection studies were excluded from analysis. The results were counted in the state of the submitting veterinarian. Geographic regions within states were not distinguished. States in which 100 sera or less were submitted during the 6 year review period were Alaska, Hawaii, Idaho, North Dakota, Nevada, Rhode Island and West Virginia. States in which 200 sera or less were submitted during the 6 year review period were Maine, New Hampshire, New Mexico, South Dakota, Utah, Vermont and Wyoming.

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CENTRAL REGION RESULTS

24% N = 17 47% N = 471 70% N= 975 31% N = 180 73% 63% N= 1252 N = 338 72% 80% N = 1113 N = 759 68% N = 2687 72% N = 434 61% N = 2632

Serum POSITIVE Results for Central Region

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NORTHEAST REGION RESULTS

30% VT 32% N = 169 VT NH 56% N = 3808 MA CT N = 107

57% N = 2172 MD 81% N = 100 62% N = 1850

CT 49% N = 1065

NH 32% N = 186 MA 47% N = 742

NJ DE

NJ 48% N = 2100 DE 65% N = 221

MD 46% N = 1404

Serum POSITIVE Results for Northeast Region

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WEST REGION RESULTS

38% N = 1014 33% N = 168 55% N =291 36% N = 73 36% N = 112

21% N = 42

35% N =5518

31% N = 105

33% N = 745

31% N=483

33% N =137

Serum POSITIVE Results for West Region

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SOUTHWEST REGION RESULTS

75% N = 558 80% N = 718

80% N = 913

85% N = 318 80% N = 731

68% N = 4961

75% N= 548

Serum POSITIVE Results for Southwest Region

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SOUTHEAST REGION RESULTS

73% N=758 75% N = 1083 65% N=540 76% N = 270 72% N = 500

57% N=2066

Serum POSITIVE Results for Southeast Region

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Seroprevalence 21-40% 41-60% 61-85%

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Seroprevalence 21-40% 51-60% 41-50% 61-70%

71-85%

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Seroprevalence 41-40% 56-65% 76-85% 41-55% 66-75%

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States with the highest # serum submissions and seroprevalence:

State California Connecticut Florida Illinois Kentucky Maryland Nebraska New Jersey New York Ohio Pennsylvania Tennessee Texas Virginia Washington

#submitted %positive 5518 35% 1065 49% 2066 57% 1113 72% 2687 68% 1404 46% 1252 63% 2100 48% 3808 56% 2632 61% 2172 57% 1083 75% 4961 68% 1850 62% 1014 38%

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PUBLISHED SEROPREVALENCE STUDIES

EBI State California Colorado Florida Michigan Reference Vet Parasit (2001) 95:273-282 J Equine Vet Sci (1999) 19:122-126 Vet Parasit (2001) 95:273-282 JAVMA (2001) 48:113-128 #Horses 93 608 40 1121 %Positive 27% 34% 28% 60% 5518 745 2066 434

EBI 35% 33% 57% 72%

#Tests %Positive

Missouri
Montana Ohio Oklahoma Oregon Wyoming

Vet Parasit (2001) 95:273-282

Vet Parasit (2001) 95:273-282 JAVMA (1997) 210:519-524 J Vet Diag Invest (2003) 15:597-600 JAVMA (1997) 210:525-527 J Parasit (2003) 89:716-720

39
15 1056 798 334 117 276

54%
0% 54% 89% 45% 45% 7%

718
168 2632 913 291 2172 112

80%
33% 61% 80% 55% 57% 36%

Pennsylvania JAVMA (1997) 210:517-518

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SUMMARY

The overall seroprevalence from US submissions was 58%. The seroprevalence ranged from a low of 31% in Arizona to a high of 85% in Arkansas. The seroprevalence tended to be lower in the western states and the far northeast while higher in the southeastern central states. In states with moderate seroprevalence rates, 22-42% test negative and even in those with higher seroprevalence, up to 20% test negative. The western blot continues to be a useful diagnostic tool with a negative result essentially ruling out S. neurona caused EPM.

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Commercially Available Sarcocystis neurona IgG Tests

Source EBI/IDEXX Since June 1995 Reference (1) J Vet Diagn Invest (1993) 5:88-90 (2) Vet Parasit (1997) 68:199-213 (3) Proceedings International Equine Neurol Conf (1997) p.4 not published Validation Samples (1) 7 necropsy positive horses, serum (2) 5 infected and 2 uninfected foals, serum and csf (3) 295 necropsy cases, serum and csf ~40% were positive for EPM not published Test Format Antigen Used western blot Variation Interpretation 14.5 kDa

merozoite lysate none

Neogen

November 1996

western blot

merozoite lysate quantification of csf IgG based on serum levels merozoite lysate pre-blocking of blot with anti-S. cruzi IgG

17 kDa

Michigan State ~fall 1998 University

J Vet Diagn Invest (2000) 12:28-32

Serum only: 6 necropsy positive 45 from India 12 from Germany (1) 48 sera: 7 necropsy positive, neurologic 2 necropsy positive, non-neurologic (2) 20 OSU infected horses, serum and csf (4 non-infected horses not tested) 8 UFL infected horses, serum and csf 2 non-infected control horses 20 vaccinated (several pre-exposed) and 6 non-vaccinated horses, serum and csf 110 Necropsied horses, serum and csf 8 EPM positive 8 EPM suspect 94 EPM negative 6 horses infected with Ellison model, serum and csf

western blot

16 and 30 kDa

UC Davis

~ early 2004?

(1) J Vet Diagn Invest (2003) 15:8-13 (2) J Parasit (2004) 90:379-386

IFA

whole merozoites indirect fluorescent antibody endpoint tiiter= last dilution showing whole parasite fluorescence titer gives "probability" of EPM

Ellison/Antech ~late 2004

J App Res Vet Med (2003) 1:318-327

ELISA

recombinant

SAG1 ELISA

correlates a given titer

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Equine Biodiagnostics/IDEXX Sarcocystis neurona IgG western blot

ACVIM EPM Society SIG panel, Louisville KY Jennifer Morrow, PhD 5/31/06
Developed in the research laboratory of Dr. David Granstrom while on the faculty of Gluck Equine Research Center, University of Kentucky Commercialized by the University of Kentucky as Equine Biodiagnostics, Inc. in 1995 (acquired by IDEXX Laboratories in October 2003) References on method and validation: J Vet Diagn Invest (1993) 5:88-90 Vet Parasit (1997( 68:199-213 Proceedings International Equine Neurol Conf (1997) p.4

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Equine Biodiagnostics/IDEXX Sarcocystis neurona IgG western blot ACVIM EPM Society SIG panel, Louisville KY Jennifer Morrow, PhD 5/31/06
Original validation samples (respective to above publications): 7 necropsy positive EPM horses, serum 5 infected and 2 uninfected foals, serum and csf

295 necropsy cases, paired serum and csf:

123 were EPM necropsy positive 3 western blot false negatives were due to acute onset 6 western blot false positives were due to cases in which blood brain barrier breach was likely

specificity = 89% for CSF and 71% for serum sensitivity = 89% for CSF and 89% for serum

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Equine Biodiagnostics/IDEXX Sarcocystis neurona IgG western blot ACVIM EPM Society SIG panel, Louisville KY Jennifer Morrow, PhD 5/31/06
The test format is a standard western blot and uses whole merozoite lysate as the antigen. The interpretation of a positive result is based on the presence of a 14.5 kDa antigen specific for S. neurona. Although the western blot format is not meant to be a quantitative test format, it is possible to visually compare the level of reactive specific S. neurona IgG when samples collected from the same horse at different times (for example, pre and post treatment) are analyzed side-byside on the same blot. This service is provided (at no extra fee) when requested.

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Equine Biodiagnostics/IDEXX Sarcocystis neurona IgG western blot ACVIM EPM Society SIG panel, Louisville KY Jennifer Morrow, PhD 5/31/06
This assay has been used to test several hundred thousand sera and csfs, from clinical submissions, at least five different infection studies and clinical trials for all the currently FDA approved EPM drugs. The test is performed daily M-F with 24 hour turnaround time. Our staff has a combined ~40 work years experience performing and interpreting this test. Each result is read by two independent observers with greater than 95% agreement. A third person resolves any discrepant reads (usually those with equivocal immunoreactivity) and a repeat test may be performed to further confirm the result. Contact info: (800) 621-8378 or at www.ebiky.com

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EQUINE BIODIAGNOSTICS/IDEXX SERUM WESTERN BLOT

Controls Pos WkPos Neg 1 Pre Post 2 Pre Post 3 Pre Post 4 Pre Post

The arrows on the left side define bands of immunoreactivity to S. neurona antigens. >> point out two crossreactive (nonspecific) bands of ~ 65 kDa and 30 kDa > point out three S. neurona specific bands Known positive, weak positive and negative control sera are in the far left lanes. Pre and Post designate the vaccination status. Representative results from four horses are: Horse # 1 2 3 4 Pre negative negative positive positive Post negative positive equivalent positive increased positive

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