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United States Department of Agriculture

Agricultural Research Service

Title: Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

item Goff, Willard
item Johnson, W Carl - Carl
item Molloy, J
item Jorgensen, W
item Waldron, S
item Figueroa, J
item Matthee, O
item Adams, D
item Mcguire, T
item Pino, I
item Mosqueda, J
item Palmer, G
item Suarez, Carlos
item Knowles, Donald - Don
item Mcelwain, T

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2008
Publication Date: 9/4/2008
Publication URL:
Citation: Goff, W.L., Johnson, W.C., Molloy, J.B., Jorgensen, W.K., Waldron, S.J., Figueroa, J.V., Matthee, O., Adams, D.S., Mcguire, T.C., Pino, I., Mosqueda, J., Palmer, G.H., Suarez, C.E., Knowles Jr, D.P., Mcelwain, T.F. 2008. Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle. Clinical and Vaccine Immunology. 15(9):1316-1321.

Interpretive Summary: Tick-borne diseases (TBD) of livestock are economically important throughout the world. Babesia bigemina is one of three important parasites causing the TBD babesiosis in cattle. It is often misdiagnosed in many areas due to the lack of a good assay that can differentiate this parasite infection from other similar parasites. Since the pathology and treatments associated with each parasite differ, accurate diagnosis is important. In addition, the assay should be formatted so that a large number of samples can be evaluated efficiently thus serving as a tool for use in regional diagnostic laboratories. We described an improved assay for detecting Babesia bigemina infections in cattle and have validated its use and determined its reliability in laboratories in different countries. The assay has the characteristics necessary and meets the regulatory stringency for use as an international standard.

Technical Abstract: A competitive ELISA (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 (ROC) analysis revealed 16% inhibition as the threshold for a negative sample 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, 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 post-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 all above 0.8 indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Last Modified: 10/17/2017
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