Borrelia burgdorferi Exposure in a Semi-feral Herd of Ponies in Chester County Pennsylvania and Comparison of ELISA, Western Blot, and Canine SNAP 3Dx Assays

Equine Behavior Laboratory - New Bolton Center
School of Veterinary Medicine, University of Pennsylvania
¹Animal Health Diagnostic Laboratory, Cornell University

     Lyme borreliosis or Lyme disease, caused by the spirochetal bacterium, Borrelia burgdorferi, is endemic in Chester County Pennsylvania. The primary vector for transmission of B. burgdorferi in this area is Ixodes scapularis or the blacklegged tick (previously known as Ixodes dammini or the deer tick). Infection rates of 10 to 75% have been reported for asymptomatic equines in the northeastern United States. Testing for exposure to B. burgdorferi can be done by multiple methods, but currently is typically done using an enzyme-linked immunosorbent assay (ELISA) and/or a Western blot (WB) to detect antibodies for B. burgdorferi. A preliminary study reported that an in-house enzyme immunoassay (SNAP 3Dx) developed for canine testing was successful use for equine samples (n=164) (Chandrashekar and Daniluk, ACVIM Annual Forum Abstracts, 2004) The objectives of our study were to (1) assess the level of natural exposure to B. burgdorferi in a semi-feral herd of ponies maintained at the University of Pennsylvania, New Bolton Center, using current standard laboratory testing methods of (ELISA and WB) and (2) compare results with those of the canine in-house SNAP 3Dx test.

     Our herd of Shetland-type ponies (n=60) has been maintained continuously at pasture in Chester County Pennsylvania since 1994, with no known Lyme disease and not vaccinated against Lyme disease. Another group of ponies (n=13) living continuously in other pastures at the same facility were also tested.

     A total of 73 individual animals were sampled in October of 2004. Serum was collected and frozen for kinetic ELISA (KELA) and Western blot (WB) testing at Cornell Diagnostic Laboratory, at the College of Veterinary Medicine at Cornell University in Ithaca, NY. Included for testing at Cornell Diagnostic Laboratory were 8 split-sample duplicates. Whole blood in a heparinized collection tube was refrigerated until use (within 24 hours) with the IDEXX Laboratories SNAP 3Dx assay for simultaneous detection of canine heartworm (Dirofilaria immitis) antigen, antibody to Borrelia burgdorferi, and antibody to Ehrlichia canis.

Results:   

Kinetic ELISA (KELA) Assay Results
·  16 of 73 (22%) KELA results were in the negative range (0-129)*
·  55 of 73 (75%) KELA results were in the equivocal range (130-379)*
·  2 of 73 (3%) KELA results were in the positive range (380+)*

* range is relevant to the reagent performance, and maybe adjusted periodically

WB Assay Results
·  10 of 73 (14%) negative WB
·  19 of 73 (26%) equivocal WB
·  44 of 73 (60%) positive WB

SNAP 3Dx Assay Results
· 12 of 73 (16%) negative SNAP 3Dx
· 61 of 73 (84%) positive SNAP 3Dx

KELA and WB Assay Comparison
·  10 of 16 (63%) KELA negatives were negative on WB
·  6 of 16 (38%) KELA negatives were equivocal on WB
·  13 of 55 (24%) KELA equivocals were equivocal on WB
·  42 of 55 (76%) KELA equivocals were positive on WB
·  2 of 2 (100%) KELA positives were positive on WB

WB and SNAP 3Dx Assay Comparison
· 63% (46 of 73) agreement of SNAP 3Dx and WB
· 3 of 44 (7%) WB positives, were negative on SNAP 3Dx
· 20 of 29 (69%) WB negatives or equivocals, were positive on SNAP 3Dx

Specificity (correct negatives): SNAP 3Dx versus WB, of 10 WB negatives, 5 (50%) were negative on SNAP 3Dx.

Sensitivity (identification of true positives): SNAP 3Dx versus WB, 41 of 44 (93%) WB positives were SNAP 3Dx positive. 41 of 61 (67%) SNAP 3Dx positives, were also positive on WB. Of 10 WB negatives, 5 (50%) were positive on SNAP 3Dx (false-positives, if WB is the gold standard).

The 8 split-sample duplicates had an average within-animal difference of 16 units on the KELA assay. For 7 of 8 of the duplicate samples within-animal WB results agreed. For the remaining sample, 1 aliquot was equivocal on WB and 1 aliquot was positive on WB.

Conclusions:  Evidence of exposure to Borrelia burgdorferi within this herd is as high as expected for semi-feral conditions.  Comparisons of these KELA, WB and the canine SNAP 3Dx require further study to determine best method of testing.
 

This is a Dorothy Russell Havemeyer Foundation project.