Development of an indirect ELISA to detect equine antibodies to Theileira haneyi

Authors: BASTOS, REGINALDO - Washington State University; SEARS, KELLY - Washington State University; DINKEL, KELCEY - Washington State University; Kappmeyer, Lowell; Ueti, Massaro; KNOWLES, DONALD - Washington State University; Fry, Lindsay

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/23/2021
Publication Date: 2/27/2021
Citation: Bastos, R.G., Sears, K.P., Dinkel, K.D., Kappmeyer, L.S., Ueti, M.W., Knowles, D.P., Fry, L.M. 2021. Development of an indirect ELISA to detect equine antibodies to Theileira haneyi. Pathogens. 10(3). Article 270. https://doi.org/10.3390/pathogens10030270.
DOI: https://doi.org/10.3390/pathogens10030270

Interpretive Summary: Theileria haneyi is an emerging, tick-borne disease of horses in the United States. It was first discovered in horses at the southern border near Eagle Pass, TX in 2016, and was determined to be a new species of Theileria in 2018. Critically, this organism is resistant to current, gold-standard therapies for equine theileriosis, and also interferes with drug-clearance of the related, more virulent pathogen Theileria equi. The prevalence of T. haneyi within U.S. horses, and thus the severity of the thread posed by this parasite to the U.S. horse industry, is currently unknown. Commercial serologic assays used by the USDA to detect T. equi in horses presented to the U.S. border for importation fail to detect T. haneyi. While molecular diagnostic assays have been developed and utilized in academic settings, these tests are expensive and cumbersome, which precludes widespread use. The goal of this work was to develop a serologic assay to detect T. haneyi-infected horses. In this manuscript, we describe antigenic selection and design of the assay, as well as initial optimization and validation steps. We report that our assay has both high sensitivity and specificity for T. haneyi, and will greatly enhance future work to determine the prevalence of this parasite in U.S. horses.

Technical Abstract: The apicomplexan parasite, Theileria haneyi, is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. Critically, while the antiparasitic drug imidocarb diproprionate (ID) consistently resolves infections with U.S. strains of T. equi, T. haneyi is resistant to ID, and co-infection of horses with T. equi and T. haneyi abrogates the anti-T. equi efficacy of ID. Furthermore, serologic diagnostic assays currently used by U.S. regulatory agencies to diagnose T. equi are not able to detect T. haneyi infection, and the cost and complexity of current molecular assays preclude widespread use for regulatory purposes and epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi in the U.S., the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi. In order to achieve this objective, equine merozoite antigen (EMA)-11, a protein exclusive to T. haneyi (ThEMA11), was recombinantly expressed in eukaryotic cells and purified. Sera from T. haneyi-experimentally infected and uninfected horses were utilized for assessment of antigenicity of ThEMA11. Confirmation of sera reactivity enabled design and optimization of an indirect enzyme-linked immunosorbent assay. Specificity of the ELISA was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrates the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world.