Serum Antibodies from a Subset of Horses Positive for Babesia caballi by Competitive Enzyme-Linked Immunosorbent Assay Demonstrate a Protein Recognition Pattern That Is Not Consistent with Infection

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence ofTheileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identifiedBabesia caballi -seropositive horses using rhoptry-associated protein 1 (RAP-1)–competitive enzyme-linked immunosorbent assay (cELISA), despiteB. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1–cELISA as a single serological test to determine the infection status ofB. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted withB. caballi lysate and purifiedB. caballi RAP-1 protein. Antibody reactivity toB. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a nativeB. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions whereB. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected withB. caballi and from areas whereB. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence ofB. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis ofB. caballi .