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

Authors: AWINDA, PETER - Washington State University; MEALEY, ROBERT - Washington State University; WILLIAMS, LAURA - Washington State University; CONRAD, PATRICIA - University Of California; PACKHAM, ANDREA - University Of California; REIF, KATHRYN - Washington State University; GRAUSE, JUANITA - Animal And Plant Health Inspection Service (APHIS); PELZEL-MCCLUSKEY, ANGELA - Animal And Plant Health Inspection Service (APHIS); CHUNG, CHUNGWON - Washington State University; REGINALDO, BASTOS - Washington State University; Kappmeyer, Lowell; HOWE, DANIEL - University Of Kentucky; NESS, SALLYANNE - Cornell University; Knowles Jr, Donald; Ueti, Massaro

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2013
Publication Date: 11/20/2013
Citation: Awinda, P.O., Mealey, R.H., Williams, L.B., Conrad, P.A., Packham, A.E., Reif, K.E., Grause, J.F., Pelzel-Mccluskey, A.M., Chung, C., Reginaldo, B., Kappmeyer, L.S., Howe, D.K., Ness, S.L., Knowles Jr, D.P., Ueti, M.W. 2013. 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. Clinical and Vaccine Immunology. 20(11):1752-1757.

Interpretive Summary: Babesia caballi is a parasite that affects horses worldwide and causes significant economic losses for the equine industry. The United States is considered free of B. caballi. However, an epidemiological survey of the U.S. has identified positive horse sera suggesting the presence of infection in the U.S. Since the biological vectors are present in the U.S. there is a concern regarding the risk of transmission to the susceptible U.S. horse population. This study evaluated the suitability of a single serological test to determine the infection of U.S. horses. We demonstrated that serological U.S. positive horses were not infected with B. caballi. We also concluded that a combination of two serological tests (RAP1-cELISA and western blot) is required for the accurate serodiagnosis of B. caballi.

Technical Abstract: 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 re-emergence of Theileria equi in the U.S. prompted widespread national surveillance testing for disease identification and control purposes and led to the determination of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi seropositive horses using RAP1-cELISA, despite the U.S. mainland being considered non-endemic for B. caballi, another tick-borne agent responsible for EP. The purpose of the present study was to evaluate the suitability of RAP1-cELISA as a single serological test to determine the infection status of B. caballi in native U.S. horses. Immunoblotting indicated that serum antibodies from U.S. horses reacted with B. caballi lysate as well as purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ~50 kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from endemic regions 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 with B. caballi and from endemic areas had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naïve horses demonstrated no evidence of B. caballi transmission confirming that antibody reactivity in cELISA positive U.S. horses were not consistent with infection. Therefore, we conclude that a combination of RAP1-cELISA and immunoblot is required for the accurate serodiagnosis of B. caballi.