title: Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.
abstract: The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies 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 IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, 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 indicated 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.
authors: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan; Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
authors: Mohamed Abdo
authors: MA
authors: Rizk
authors: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan; Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
authors: Shimaa Abd El-Salam
authors: SA
authors: El-Sayed
authors: Department of Parasitology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City 32511, Minoufiya, Egypt.
authors: Mahmoud
authors: M
authors: AbouLaila
authors: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan; Laboratory of Molecular Genetics, Institute of Veterinary Medicine, Ulaanbaatar, Mongolia.
authors: Bumduuren
authors: B
authors: Tuvshintulga
authors: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan.
authors: Naoaki
authors: N
authors: Yokoyama
authors: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan. Electronic address: igarcpmi@obihiro.ac.jp.
authors: Ikuo
authors: I
authors: Igarashi
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001403:Babesia / Q000145:classification / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000379:methods*; D061848:Optical Imaging; D015203:Reproducibility of Results
chemical_list: D000981:Antiprotozoal Agents
keywords: B. divergens; Babesia fluorescence assay; Large-scale drug screening