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

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 degrees C or 37 degrees C conditions. Cultures at low temperature (27 degrees 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.