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Clinical and Vaccine Immunology, September 2008, p. 1316-1321, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00150-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle{triangledown}

Will L. Goff,1* Wendell C. Johnson,1 John B. Molloy,2 Wayne K. Jorgensen,2 Susan J. Waldron,3 Julio V. Figueroa,4 Olivier Matthee,5 D. Scott Adams,6 Travis C. McGuire,7 Ignacio Pino,8 Juan Mosqueda,4 Guy H. Palmer,7 Carlos E. Suarez,1 Donald P. Knowles,1 and Terry F. McElwain7,9

Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164-6630,1 Queensland Department of Primary Industries and Fisheries, Animal Research Institute, Yeerongpilly, Queensland 4105,2 Tick Fever Centre Wacol, Wacol, Queensland 4076, Australia,3 CENID-PAVET, INIFAP, Jiutepec, Morelos 62550, Mexico,4 Department of Parasites, Vectors and Vector-Borne Diseases, Onderstepoort Veterinary Institute, Onderstepoort 0110, Republic of South Africa,5 VMRD, Inc., Pullman, Washington 99163,6 Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7040,7 Veterinary Clinic, Route 349 KM1.0 Interior, Mayaguez, Puerto Rico 00680,8 Washington Animal Disease Diagnostic Laboratory, Pullman, Washington 99163-20379

Received 28 April 2008/ Returned for modification 1 July 2008/ Accepted 3 July 2008

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

* Corresponding author. Mailing address: Animal Disease Research Unit, USDA-ARS, Washington State University, Pullman, WA 99164-6630. Phone: (509) 335-6003. Fax: (509) 335-8328. E-mail: wgoff{at}vetmed.wsu.edu

{triangledown} Published ahead of print on 16 July 2008.

Clinical and Vaccine Immunology, September 2008, p. 1316-1321, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00150-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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