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Title: COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY BASED ON A RHOPTRY-ASSOCIATED PROTEIN 1 EPITOPE SPECIFICALLY IDENTIFIES BABESIA BOVIS-INFECTED CATTLE

Author
item Goff, Willard
item MC ELWAIN, TERRY - WASHINGTON STATE UNIV
item Suarez, Carlos
item Johnson, Wendell
item BROWN, W - WASHINGTON STATE UNIV
item NORIMINE, J - WASHINGTON STATE UNIV
item Knowles Jr, Donald

Submitted to: Clinical and Diagnostic Laboratory Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2002
Publication Date: 1/20/2003
Citation: Goff, W.L., McElwain, T.F., Suarez, C.E., Johnson, W.C., Brown, W.C., Norimine, J., Knowles, D.P. Competitive enzyme-linked immunosorbent assay based on a rhoptry-associated protein-1 epitope specifically identifies Babesia bovis-infected cattle. Clinical and Diagnostic Laboratory Immunology. 2002. v. 10. p. 38-43.

Interpretive Summary: Babesia bovis is a tick-transmitted microorganism, causing disease in cattle throughout much of the tropical and subtropical world. Other related species of Babesia also cause disease, but each in a slightly different way. Thus, control programs require different approaches for each species present in a particular area. In many areas more than one species may occur. For this reason, it is important that accurate diagnoses be made. Assays for differentiating between different babesial species are not available in formats that allow for easy and quick diagnosis. In this study, we detail the use of a recently characterized protein associated with B. bovis to detect antibody present in cattle that are infected with this parasite. Animals that are suffering from acute disease or that are carriers of B. bovis can now be confirmed quickly and reliably. The assay includes reagents that are easily standardized for use throughout the world.

Technical Abstract: The competitive ELISA (cELISA) format has proven to be an accurate, reliable, easily standardized, and high throughput method to detect hemoparasite infections. In this study, a specied-specific, broadly conserved, and tandemly repeated B-cell epitope within the C-terminus of the rhoptry associated protein-1 of the hemoparasite, Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion protein and used an antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope specific monoclonal antibody, BABB75A4. The cELISA accurately differentiated animals with B. bovis specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (specificity = 98.7%). In addition, B. bovis specific sera from Australia, Argentina, Bolivia, Puerto Rico and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days post infection, and using sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiology and disease surveillance tool.