Initiated Babesia ovata Sexual Stages under In Vitro Conditions Were Recognized by Anti-CCp2 Antibodies, Showing Changes in the DNA Content by Imaging Flow Cytometry.

Thu-Thuy Nguyen,Rika Umemiya-Shirafuji,Juan Mosqueda,Shin-Ichiro Kawazu,Junya Yamagishi,Minh-Anh Dang-Trinh,Luna Higuchi,Masahito Asada,Hassan Hakimi

doi:10.3390/pathogens8030104

Abstract

Sexual stage induction under in vitro conditions is useful for biological and molecular studies of Babesia parasites. Therefore, in the present study, we induced B. ovata tick stages using the chemical inducers: xanthurenic acid (XA), dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine (TCEP) at 27 °C or 37 °C conditions. Cultures at low temperature (27 °C) or treated with XA/TCEP induced a large number of extra-erythrocytic merozoites, which transformed into round shape cells at 12–24 h post-induction (pi). However, typical forms of tick stages (aggregation forms and the spiky forms/ray bodies) were only observed in the cultures treated with 40 mM or 60 mM of DTT during 3–6 h pi. The induced cells were recognized by anti-CCp2 rabbit antisera. DNA content of the cell population treated with 40 mM of DTT was analyzed by imaging flow cytometry at 0, 12 and 48 h pi. The results indicated that the parasite population with diploid-like double DNA content increased at 48 h pi. Our observations on morphological and changes in the DNA content provide useful information for understanding the life cycle of B. ovata under in vitro conditions, which will facilitate further studies on basic biology and the development of transmission blocking vaccines against bovine babesiosis.

Highlights

Babesiosis is a tick-transmitted disease of animals manifested by anemia and occasional hemoglobinuria caused by the protozoan parasites of the genus Babesia [1]
B. ovata cultures were exposed to either xanthurenic acid (XA), DTT or tris (2-carboxyethyl) phosphine (TCEP) and incubated at two different temperatures: 37 ◦ C in the presence of 5% CO2 or 27 ◦ C in air (Supplementary Table S1)
XA and TCEP induced the morphological changes of the parasites similar to that observed in the control at 27 ◦ C

Summary

Introduction

Babesiosis is a tick-transmitted disease of animals manifested by anemia and occasional hemoglobinuria caused by the protozoan parasites of the genus Babesia [1]. Several Babesia spp. are known to infect cattle, with an estimation of 1–2 billion cattle worldwide currently exposed to one or more babesiosis pathogens [2,3]. Bovine babesiosis has considerable economic impact related to losses in milk and meat production, abortions, and a general impact on the global cattle trade industry [4]. Babesia bigemina and B. bovis are the most important species in terms of economic and Pathogens 2019, 8, 104; doi:10.3390/pathogens8030104 www.mdpi.com/journal/pathogens. B. ovata is among the low pathogenic species, infection may lead to severe conditions in cattle when co-infected with Theileria orientalis [5].

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Conclusion