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Title: Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

Author
item CHUNG, CHUNGWON - Washington State University
item WILSON, CAREY - Washington State University
item B. MUDIYANSELAGE, CHANDIMA - Washington State University
item KANG, EUNAH - Chungnam National University
item ADAMS, SCOTT - Washington State University
item Kappmeyer, Lowell
item Knowles Jr, Donald
item MCELWAIN, TERRY - Washington State University
item EVERMANN, JAMES - Washington State University
item Ueti, Massaro
item Scoles, Glen
item LEE, STEPHEN - University Of Idaho
item MCGUIRE, TRAVIS - Washington State University

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2013
Publication Date: 1/26/2014
Citation: Chung, C., Wilson, C., B. Mudiyanselage, C.B., Kang, E., Adams, S.D., Kappmeyer, L.S., Knowles Jr, D.P., Mcelwain, T., Evermann, J., Ueti, M.W., Scoles, G.A., Lee, S.S., Mcguire, T.C. 2014. Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen. Journal of Veterinary Diagnostic Investigation. 26(1):61-71.

Interpretive Summary: Anaplasma marginale is a tick-borne bacterial pathogen that affects cattle worldwide and causes economic losses for the livestock industry. Animals that survive the disease become persistently infected and are the reservoirs from transmission via the tick-vector. Control of A. marginale is improved by identification of carrier animals using a specific and sensitive serodiagnostic assay. In this study, we developed a new recombinant protein for the diagnostic assay to improve the detection of cattle infected with A. marginale. Therefore, the new serological diagnostic assay will be essential for epidemiological monitoring of infection and help to devise control strategy to prevent the spread of A. marginale to naive animals.

Technical Abstract: This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 139 when tested using recombinant MSP5-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the cELISA, a new recombinant antigen designated rMSP5–glutathione S-transferase was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (=30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested PCR. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera.