pathogens Article Development of an Indirect ELISA to Detect Equine Antibodies to Theileria haneyi Reginaldo G. Bastos 1 , Kelly P. Sears 1 , Kelcey D. Dinkel 1 , Lowell Kappmeyer 2 , Massaro W. Ueti 1,2 , Donald P. Knowles 1 and Lindsay M. Fry 1,2, *   Citation: Bastos, R.G.; Sears, K.P.; Dinkel, K.D.; Kappmeyer, L.; Ueti, M.W.; Knowles, D.P.; Fry,L.M. Development of an Indirect ELISA to Detect Equine Antibodies to Theileria haneyi. Pathogens 2021, 10, 270. https://doi.org/10.3390/ pathogens10030270 Academic Editor: Siddhartha Das Received: 29 January 2021 Accepted: 23 February 2021 Published: 27 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Veterinary Microbiology & Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA; reginaldo_bastos@wsu.edu (R.G.B.); kellyp.sears@wsu.edu (K.P.S.); kelcd23@hotmail.com (K.D.D.); massaro.ueti@usda.gov (M.W.U.); dknowles@wsu.edu (D.P.K.) 2 Animal Disease Research Unit, USDA-ARS, Pullman, WA 99164, USA; Lowell.kappmeyer@usda.gov * Correspondence: Lindsay.fry@usda.gov 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. The current equi merozoite antigen 1 (EMA1)-based competitive enzyme-linked immunosorbent assay (ELISA)used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi merozoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactivity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi 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 demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world. Keywords: equine theileriosis; Theileria haneyi; enzyme-linked immunosorbent assay (ELISA), serology 1. Introduction Theileria haneyi is an apicomplexan hemoparasite and one of two known causative agents of equine theileriosis. T. haneyi appears to have a global distribution, with infected equids having been identified in North America, South America, and Africa [1–5]. The organism causes milder clinical disease (variable fever, anemia) than T. equi in experimen- tally infected horses, and is capable of superinfection with T. equi [3,6]. Horses remain persistently infected following the acute stage of disease, and these asymptomatic horses are presumed to be reservoirs of infectious organisms for competent tick vectors. Unfortu- nately, while the antiparasitic drug imidocarb diproprionate (ID) resolves the majority of equine infections with U.S. strains of T. equi, T. haneyi does not appear to be susceptible to ID, and co-infection of horses with T. equi and T. haneyi reduces the efficacy of ID against T. equi [7]. Initial investigation into the serologic immune response to T. haneyi revealed that sera from T. haneyi-infected horses react with affinity purified, T. equi (Florida isolate) equi merozoite antigens (EMA) 1 and 2 [6]. Interestingly, genomic analysis revealed that the T. haneyi genome lacks the ema1, 3, and 4 genes, but contains three novel EMA family members, designated ema11-13 [3]. Antigenic cross-reactivity is attributed to high amino acid identity within the EMA family, both within the T. equi genome and between the T. equi and T. haneyi genomes [3,8]. The EMA family has garnered significant attention in the veterinary diagnostic community, and regulatory T. equi serologic assays approved by The World Pathogens 2021, 10, 270. https://doi.org/10.3390/pathogens10030270 https://www.mdpi.com/journal/pathogens