RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture

• Ishizaki, Takahiro

• Sivakumar, Thillaiampalam

• Hayashida, Kyoko

• Tuvshintulga, Bumduuren

• Igarashi, Ikuo

• Yokoyama, Naoaki

Abstract

Babesia bovis is an apicomplexan hemoprotozoan that can invade bovine red blood cells (RBCs), where it multiplies asexually. RBC invasion assays using free viable merozoites are now routinely used to understand the invasion mechanism of B. bovis, and to evaluate the efficacy of chemicals and antibodies that potentially inhibit RBC invasion by the parasite. The application of high-voltage pulses (high-voltage electroporation), a commonly used method to isolate free merozoites from infected RBCs, reduces the viability of the merozoites. Recently, a cold treatment of B. bovis in vitro culture was found to induce an effective release of merozoites from the infected RBCs. In the present study, we incubated in vitro cultures of B. bovis in an ice bath to liberate merozoites from infected RBCs and then evaluated the isolated merozoites in RBC invasion and invasion-inhibitions assays. The viability of the purified merozoites (72.4%) was significantly higher than that of merozoites isolated with high-voltage electroporation (48.5%). The viable merozoites prepared with the cold treatment also invaded uninfected bovine RBCs at a higher rate (0.572%) than did merozoites prepared with high-voltage electroporation (0.251%). The invasion-blocking capacities of heparin, a polyclonal rabbit antibody directed against recombinant B. bovis rhoptry associated protein 1, and B. bovis-infected bovine serum were successfully demonstrated in an RBC invasion assay with the live merozoites prepared with the cold treatment, suggesting that the targets of these inhibitors were intact in the merozoites. These findings indicate that the cold treatment technique is a useful tool for the isolation of free, viable, invasion-competent B. bovis merozoites, which can be effectively used for RBC invasion and invasion-inhibition assays in Babesia research. (C) 2016 Elsevier Inc. All rights reserved.

Journal

• Experimental Parasitology

  Experimental Parasitology 166 10-15, 2016-07

  Academic Press

Keywords

• Babesia bovis
• Cold treatment
• Invasion-inhibition assay
• Merozoite isolation
• RBC invasion

Details

• CRID: 1050282812560743168
• NII Article ID: 120006390946
• NII Book ID: AA00641226
• DOI: 10.1016/j.exppara.2016.03.010
• ISSN: 00144894
• Web Site: https://obihiro.repo.nii.ac.jp/records/1209
• Web Site: https://api.elsevier.com/content/article/PII:S0014489416300364?httpAccept=text/plain
• Web Site: https://api.elsevier.com/content/article/PII:S0014489416300364?httpAccept=text/xml
• Text Lang: en
• Article Type: journal article
• Data Source:
  • IRDB
  • Crossref
  • CiNii Articles
  • KAKEN