Veterinary Parasitology 227 (2016) 93–97 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Short communication Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay Mohamed Abdo Rizk a,b,1 , Shimaa Abd El-Salam El-Sayed a,c,1 , Mahmoud AbouLaila d , Bumduuren Tuvshintulga a,e , Naoaki Yokoyama a , Ikuo Igarashi a,∗ a National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan b Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt c Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt d Department of Parasitology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City 32511, Minoufiya, Egypt e Laboratory of Molecular Genetics, Institute of Veterinary Medicine, Ulaanbaatar, Mongolia a r t i c l e i n f o Article history: Received 21 April 2016 Received in revised form 28 July 2016 Accepted 29 July 2016 Keywords: B. divergens Large-scale drug screening Babesia fluorescence assay a b s t r a c t The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the effica- cies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC 50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based meth- ods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin- treated cultures. On contrary, tetracycline hydrochloride and (−)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indi- cated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Babesia divergens appear to be a serious cause of infections in Europe, for both humans and cattle (Uilenberg, 2006). Therefore, there is an urgent need to develop a new and alternative strategy to the currently used microscopy method that is not suitable for mass drug screening. A fluorescence-based assay using SYBR Green I (SGI) stain has been recommended, as it provides more consistent data within a short period with little effort (Smilkstein et al., 2004). Although we have recently developed a novel fluorescence- based assay in our laboratory for large-scale drug screening against Babesia and Theileria parasites (Rizk et al., 2015), the assay has not yet been established for drug screening against B. divergens. ∗ Corresponding author: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan. E-mail address: igarcpmi@obihiro.ac.jp (I. Igarashi). 1 These authors contributed equally to this study. Therefore, in the present study, we have evaluated and confirmed the validation of the assay for mass drug screening against the in vitro growth of B. divergens using seven different drugs, includ- ing diminazene aceturate, tetracycline hydrochloride, chloroquine diphosphate, pyronaridine tetraphosphate, luteolin, actinonin, and (−)-epigallocatechin-3-gallate from green tea. 2. Materials and methods 2.1. Chemical and antibabesial agents SYBR Green I (SGI) nucleic acid stain (Lonza, USA; 10,000x) was stored at −20 ◦ C and thawed before use. A lysis buffer con- sisting of Tris (130 mM; pH 7.5), EDTA (10 mM), saponin (0.016%; W/V), and TritonX-100 (1.6%; V/V) was prepared in advance and stored at 4 ◦ C. Diminazene aceturate (Novartis, Japan), tetracycline hydrochloride, chloroquine diphosphate, pyronaridine tetraphos- phate, luteolin, (−)-epigallocatechin-3-gallate from green tea, and actinonin (all drugs from Sigma-Aldrich, Japan) were prepared as 100 mM stock solutions. http://dx.doi.org/10.1016/j.vetpar.2016.07.032 0304-4017/© 2016 Elsevier B.V. All rights reserved.