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Journal of Clinical Microbiology, July 1999, p. 2285-2290, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

Lowell S. Kappmeyer,1 Lance E. Perryman,2 Stephen A. Hines,3 Timothy V. Baszler,3,4 Jonathan B. Katz,5 Steven G. Hennager,5 and Donald P. Knowles1,3,*

Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164-70301; Department of Microbiology, Pathology, and Parasitology, North Carolina State University, Raleigh, North Carolina 276062; Department of Veterinary Microbiology and Pathology3 and Washington Animal Disease Diagnostic Laboratory,4 Washington State University, Pullman, Washington 99164-7040; and National Veterinary Services Laboratory, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa 500105

Received 8 December 1998/Returned for modification 3 March 1999/Accepted 7 April 1999

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

* Corresponding author. Mailing address: 331 Bustad Hall, ARS-USDA, Washington State University, Pullman, WA 99164-7030. Phone: (509) 335-6022. Fax: (509) 335-8328. E-mail: dknowles{at}vetmed.wsu.edu.

Journal of Clinical Microbiology, July 1999, p. 2285-2290, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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