Please cite this article in press as: Silva MG, et al. Repertoire of Theileria equi immunodominant antigens bound by equine antibody. Mol Biochem Parasitol (2013), http://dx.doi.org/10.1016/j.molbiopara.2013.03.002 ARTICLE IN PRESS G Model MOLBIO 10746 1–7 Molecular & Biochemical Parasitology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular & Biochemical Parasitology Repertoire of Theileria equi immunodominant antigens bound by equine antibody 1 Marta G. Silva a,b,∗ , Telmo Grac ¸ a b,c , Carlos E. Suarez a , Donald P. Knowles a,b Q1 2 a Animal Disease Research Unit, USDA-ARS, 3003 ADBF, Washington State University, Pullman, WA 99163-6630, USA 3 b Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA 4 c Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164-7010, USA 5 6 a r t i c l e i n f o 7 8 Article history: 9 Received 11 June 2012 10 Received in revised form 15 March 2013 11 Accepted 18 March 2013 12 Available online xxx 13 Keywords: 14 Theileria equi 15 Affinity chromatography 16 Tandem mass spectrometry 17 EMA family proteins 18 Antigens 19 a b s t r a c t Theileriosis in horses and cattle is caused by tick-borne Apicomplexa parasites and results in death or life- long infection in their respective hosts. Transmission risk associated with persistent infection severely limits movement of horses and cattle resulting in economic losses. The recent reemergence of Thei- leria equi infection in U.S. horses demonstrates the continual threat Apicomplexa parasites represent to global animal health. A paucity of data concerning equine immune responses to T. equi, including antigens recognized by antibodies in clinically asymptomatic, persistently infected horses, precludes vaccine development. Therefore, this investigation was initiated to characterize antigens recognized by the equine antibody response to T. equi. This goal was accomplished by defining T. equi merozoite anti- gens that are recognized by antibodies in horses infected with distinct T. equi isolates. Previously it was shown that equine post-infection serum consistently recognized at least five T. equi merozoite antigens, but their precise identity remained unknown. To determine specificity of antibody target identification, T. equi merozoite antigens were first isolated using equine post-infection serum in affinity chromatography. Proteins recognized by the equine antibodies were then isolated from two-dimensional electrophoresis gels, and analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) using the recently available T. equi genome database. Five T. equi antigens were identified and include Equi Merozoite Antigen-2 (EMA-2), EMA-3 and EMA-6, a previously uncharacterized protein annotated as “signal peptide containing protein”, and 40S ribosomal protein S12. © 2013 Published by Elsevier B.V. 1. Introduction 20 Theileria equi is an Apicomplexa tick-borne parasite respon- 21 sible for causing equine theileriosis. Infection occurs in tropical, 22 subtropical and temperate regions of the world, including Asia, 23 Europe, Africa and America, and is associated with geographic dis- 24 tribution and seasonal activity of the ixodid ticks Rhipicephalus sp., 25 Dermacentor sp., and Hyalomma sp. [1]. More recently, Amblyoma 26 cajennense was also identified as a competent vector for T. equi 27 in the USA [2]. Pathogenesis is characterized by initial infection 28 of peripheral blood mononuclear cells by T. equi sporozoites fol- 29 lowed by infection and lysis of erythrocytes by merozoites [3]. T. 30 equi causes acute, sub-acute, and persistent infection disease. Infec- 31 tion of horses with T. equi results in fever and anemia; however, 32 Abbreviations: EMA, equi merozoite antigen; 1-2D, one or two dimensional elec- trophoresis; LC–MS/MS, liquid chromatography tandem mass spectrometry; cELISA, competitive ELISA; nPCR, nested PCR. ∗ Corresponding author at: Animal Disease Research Unit, U.S. Department of Agriculture, Agricultural Research Service, 3003 ADBF, WSU, PO Box 646630, Pull- man, WA 99163-6630, USA. Tel.: +1 5093358721; fax: +1 5093358328. E-mail address: marta@vetmed.wsu.edu (M.G. Silva). lymphocyte proliferation and/or transformation, as occurs in infec- 33 tion of cattle with T. parva, are not detectable in T. equi infection 34 [4]. Acute infection of immunologically naïve horses with T. equi 35 may result in death [1], and available data indicate that horses 36 which survive acute infection become persistently infected and 37 thus lifelong reservoirs for transmission [5,6]. Persistent infection 38 is characterized by fluctuation in the levels of parasitized erythro- 39 cytes between 10 3.3 to 10 6.0 parasites/ml of blood [7]. Persistent 40 infection with T. equi is characterized by nonspecific signs such as 41 weight loss and poor body condition. 42 The recent detection of T. equi infection in 292 out of a total of 43 360 horses in Southern Texas, and the involvement of the newly 44 discovered A. cajennense vector, provides a clear example of the 45 insidious nature of the reemergence of a vector borne infection and 46 the role of parasite persistence in transmission [8]. In the absence of 47 a vaccine, the control of equine theileriosis is performed essentially 48 from two ways: restricting importation of infected horses (mainly 49 in non-endemic regions) and, by the detection of infected horses 50 followed either by euthanasia or treatment with chemotherapeu- 51 tics aimed at elimination of infection and transmission risks, or by 52 maintenance of premunition, the immunity associated by persis- 53 tent infection (mainly in endemic regions). Treatment to eliminate 54 infection and transmission risk of horses persistently infected with 55 0166-6851/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.molbiopara.2013.03.002