Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr Cocktail Babesia bovis antigens for global detection of Babesia bovis infection in cattle Shimaa El-Sayed a,b , Mohamed Abdo Rizk a,c,*,1 , MohamadAlaa Terkawi a , Ikuo Igarashi a a National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan b Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt c Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt ARTICLE INFO Keywords: B. bovis Spherical body protein 1 Spherical body protein 4 Cocktail antigen ABSTRACT The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked im- munosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis–infected sera and the lowest OD values with normal bovine sera or B. bigemina–infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis. 1. Introduction Piroplasmosis caused by different species of Babesia and Theileria in various livestock animals affects the health status of the infected hosts with great economic losses (Uilenberg et al., 2006). Babesia bovis and Babesia bigemina are the main etiological agents for bovine babesiosis (Bock et al., 2004). These bovine Babesia parasites are found in tropical and subtropical regions of the world and have a worldwide distribution (Uilenberg et al., 2006). The apical complex, which consists of rhop- tries, micronemes, and a spherical body, is a common characteristic structure within Babesia parasites (Yokoyama et al., 2006). Proteins derived from these organelles are believed to have critical functions in parasite invasion, growth, and survival in erythrocytes (Yokoyama et al., 2006). After a parasite's invasion into erythrocytes, its stabili- zation and survival depend on spherical body proteins (SBPs) (Terkawi et al., 2011a). Among these proteins, B. bovis spherical body protein 4 (BbSBP-4), which is a native protein and is expressed in large amounts during the late stage of parasite division in the cytosol of infected host cells (Terkawi et al., 2011b). On the other hand, shortly after parasite invasion into erythrocytes, B. bovis spherical body protein 1 (BbSBP-1), which is known as an immunodominant 80 kDa merozoite protein lo- cated in the spherical body, has been detected within the cytoplasmic face of the parasitized RBC membrane (Hines et al., 1995Tetzlaff et al., 1992). This detectable protein was found to have the ability to prompt the production of both CD4 + - and CD8 + -T lymphocytes (Hines et al., 1995). A recent study, has demonstrated the successful application of indirect enzyme-linked immunosorbent assay (iELISA) with rBbSBP-4 for epidemiological surveys and the control of bovine babesiosis (Terkawi et al., 2011a). Serodiagnoses by immunofluorescent antibody test and iELISA are https://doi.org/10.1016/j.exppara.2019.107758 Received 12 July 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 * Corresponding author. Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt. E-mail addresses: dr_moh_abdo2008@mans.edu.eg, Rizkma@obihiro.ac.jp (M.A. Rizk). 1 Current address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080–8555, Japan. Experimental Parasitology 206 (2019) 107758 Available online 12 September 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved. T