Elsevier

Veterinary Parasitology

Volume 227, 30 August 2016, Pages 93-97
Veterinary Parasitology

Short communication
Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay

https://doi.org/10.1016/j.vetpar.2016.07.032Get rights and content

Highlights

  • •

    We established a fluorescence assay for mass drug screening in vitro.

  • •

    5% HCT was the best condition for mass drug screening against B. divergens..

  • •

    Actinonin was the most effective drug against the in vitro growth of B. divergens.

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.

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. 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, including diminazene aceturate, tetracycline hydrochloride, chloroquine diphosphate, pyronaridine tetraphosphate, luteolin, actinonin, and (−)-epigallocatechin-3-gallate from green tea.

Section snippets

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 consisting 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 tetraphosphate, luteolin, (−)-epigallocatechin-3-gallate from green tea, and actinonin (all drugs from Sigma-Aldrich, Japan) were

Linearity assessment and HTS assay validation

Significant linear relationships between the emitted fluorescence signals and parasitemia percentages were observed at 5% and 10% HCTs (Fig. 1).

Statistical parameters for HTS assay were calculated for each HCT to evaluate the quality of the assay for mass drug screening against the in vitro growth of B. divergens. The results showed that the best HCTs were 5% and 10%, as the Z’ factors were within the permissible limit (≥0.5) at both HCTs, and the highest values of S/N ratios were obtained from

Discussion

The current therapies used to treat B. divergens in cattle, including imidocarb dipropionate, remains debatable (Mallick et al., 1987). Therefore, developing more effective drugs against B. divergens has a priority in veterinary treatment research.

Recently, a fluorescence-based method using SGI, which binds to the double-stranded DNA of parasites, was employed for mass drug screening against the in vitro growth of B. bovis, B. bigemina, B. caballi, T. equi, and P. falciparum parasites (

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This study supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Higher Education of Egypt.

References (18)

There are more references available in the full text version of this article.

Cited by (0)

1

These authors contributed equally to this study.

View full text
© 2016 Elsevier B.V. All rights reserved.