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8/3/2019 Morrow Present

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2006 IDEXX Laboratories, Inc. All rights reserved.

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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2 2006 IDEXX Laboratories, Inc. All rights reserved.

QUALIFYING STATEMENTS

The results from non-US submissions were excluded from analysis.

The results of all known infection studies were excluded fromanalysis.

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 yearreview 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 yearreview period were Maine, New Hampshire, New Mexico, SouthDakota, Utah, Vermont and Wyoming.


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Serum POSITIVE Results for Central Region

63%

N= 1252

31%

N = 180

24%

N = 17 47%

N = 471

73%

N = 338

70%

N= 975

72%

N = 1113

72%

N = 434

80%

N = 759

68%

N = 2687

61%

N = 2632

CENTRAL REGION RESULTS


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NORTHEAST REGION RESULTS

Serum POSITIVE Results for Northeast Region

30%

N = 107

NH32%N = 186NH

VT

VT32%N = 169

MA MA47%N = 742CT

CT49%N = 1065

56%N = 3808

57%N = 2172

81%N = 100 62%

N = 1850

NJ NJ48%N = 2100

DE DE

65%N = 221

MD

MD46%N = 1404


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35%N =5518

31%N=483

55%N =291

38%N = 1014

36%N = 73

21%N = 42

33%N =137

33%N = 745

33%N = 168

31%N = 105

WEST REGION RESULTS

Serum POSITIVE Results for West Region

36%N = 112


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75%N= 548

68%N = 4961

80%N = 913

75%N = 558

80%N = 718

85%N = 318

SOUTHWEST REGION RESULTS

Serum POSITIVE Results for Southwest Region

80%N = 731


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57%N=2066

72%N = 500

76%N = 270

65%N=540

75%N = 1083

73%N=758

SOUTHEAST REGION RESULTS

Serum POSITIVE Results for Southeast Region


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Seroprevalence

21-40%41-60%

61-85%


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Seroprevalence

21-40% 41-50%

51-60% 61-70%

71-85%


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Seroprevalence

41-40% 41-55%

56-65% 66-75%

76-85%


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States with the highest # serum submissions and seroprevalence:

State #submitted %positive

California 5518 35%

Connecticut 1065 49%

Florida 2066 57%

Illinois 1113 72%

Kentucky 2687 68%

Maryland 1404 46%

Nebraska 1252 63%

New Jersey 2100 48%

New York 3808 56%

Ohio 2632 61%

Pennsylvania 2172 57%

Tennessee 1083 75%

Texas 4961 68%

Virginia 1850 62%

Washington 1014 38%


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PUBLISHED SEROPREVALENCE STUDIES

EBI EBI

State Reference #Horses %Positive #Tests %Positive

California Vet Parasit (2001) 95:273-282 93 27% 5518 35%

Colorado J Equine Vet Sci (1999) 19:122-126 608 34% 745 33%

Florida Vet Parasit (2001) 95:273-282 40 28% 2066 57%

Michigan JAVMA (2001) 48:113-128 1121 60% 434 72%

Missouri Vet Parasit (2001) 95:273-282 39 54% 718 80%

Montana Vet Parasit (2001) 95:273-282 15 0% 168 33%

Ohio JAVMA (1997) 210:519-524 1056 54% 2632 61%

Oklahoma J Vet Diag Invest (2003) 15:597-600 798 89% 913 80%

Oregon JAVMA (1997) 210:525-527 334 45% 291 55%

Pennsylvania JAVMA (1997) 210:517-518 117 45% 2172 57%

Wyoming J Parasit (2003) 89:716-720 276 7% 112 36%


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SUMMARY

The overall seroprevalence from US submissions was 58%.

The seroprevalence ranged from a low of 31% in Arizona toa high of 85% in Arkansas.

The seroprevalence tended to be lower in the western statesand the far northeast while higher in the southeastern centralstates.

In states with moderate seroprevalence rates, 22-42% testnegative and even in those with higher seroprevalence, up to20% test negative.

The western blot continues to be a useful diagnostic tool witha negative result essentially ruling out S. neuronacausedEPM.


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Commercially Available Sarcocystis neurona IgG Tests

Source Since Reference Validation Samples Test Format Antigen Used Variation Interpretation

EBI/IDEXX June 1995 (1) J Vet Diagn Invest (1993) 5:88-90 (1) 7 necropsy positive horses, serum western blot merozoite lysate none 14.5 kDa

(2) Vet Parasit (1997) 68:199-213 (2) 5 infected and 2 uninfected foals, serum and csf

(3) Proceedings International Equine (3) 295 necropsy cases, serum and csf ~40% were

Neurol Conf (1997) p.4 positive for EPM

Neogen November 1996 not published not published western blot merozoite lysate quantification of csf IgG 17 kDa

based on serum levels

Mic higan St ate ~fall 1998 J Vet Diagn Inves t (2000) 12: 28-32 Serum only: 6 necropsy positive wes tern blot meroz oite lysate pre-block ing of blot with 16 and 30 kDa

University 45 from India anti-S. cruzi IgG12 from Germany

UC Davis ~ early 2004? (1) J Vet Diagn Inves t (2003) 15:8-13 (1) 48 sera: 7 nec rops y pos it ive, neurologic IFA whole meroz oit es indirec t fluores cent ant ibody endpoint tiit er= las t

(2) J Parasit (2004) 90:379-386 2 necropsy positive, non-neurologic dilution showing whole

(2) 20 OSU infected horses, serum and csf parasite fluorescence

(4 non-infected horses not tested) titer gives "probability"

8 UFL infected horses, serum and csf of EPM

2 non-infected control horses

20 vaccinated (several pre-exposed) and

6 non-vaccinated horses, s erum and csf110 Necropsied horses, serum and csf

8 EPM positive

8 EPM suspect

94 EPM negative

E llis on/Antec h ~ late 2004 J App Res Vet Med (2003) 1:318-327 6 hors es infec ted wit h E llis on model, s erum and c sf ELISA rec ombinant SAG1 ELISA c orrelates a given t it er


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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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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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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. neuronaIgG when samples collected

from the same horse at different times (for example, pre and post treatment) are analyzed side-by-

side on the same blot. This service is provided (at no extra fee) when requested.


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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 1 2 3 4

Pos

WkPos

Neg

Pre

Post

Pre

Post

Pre

Post

Pre

Post

The arrows on the left side define bands of immunoreactivityto S. neuronaantigens.>> point out two crossreactive (nonspecific) bands of ~ 65

kDa and 30 kDa> point out three S. neuronaspecific bandsKnown positive, weak positive and negative control sera arein the far left lanes.Pre and Post designate the vaccination status.Representative results from four horses are:

Horse # Pre Post1 negative negative2 negative positive3 positive equivalent

positive4 positive increased

positive

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>>

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