Isolation of viable Babesia bovis merozoites to study parasite invasion

Abstract

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induce alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG) and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no role in invasion. Given the importance of invasion for establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

Comparative single-cell transcriptional atlases ofBabesiaspecies reveal conserved and species-specific expression profiles

“…Cells were orthogonally projected onto the curve and ordered to mimic pseudo-time ( Fig 2A ). The start point was arbitrarily picked and set to 0 and the end point was set to 12 hr (55, 56), representing the approximate time of a division cycle in Babesia spp (37, 56). We partitioned the projected cells along the pseudo-time into 20 minutes time intervals.…”

“…Data on the division cycle for B. bovis in asynchronous culture suggests that cell division is complete in approximately 5 hr, yet this does not describe the time from invasion to egress (104). However, in a more recent study on synchronized B. bovis , this process is approximately 12 hr (56). In contrast, B. divergens has a much more complex replicative cycle (55).…”

“…In all studies of the dynamics of B. divergens division dynamics, there is a significant range of time for a single division, highlighting the difficulty in generating an exact measurement (55, 105, 106). This variability leads to difficulty in synchronizing Babesia parasites, which currently relies on mechanical release of parasites from RBCs using filtration (55, 56), and we have recently shown that parasites may be egress competent at various times in their intraerythrocytic development (37). Unfortunately, no such detailed data on the division time in B. bigemina exists, however the replication rate in culture is similar to that in B. bovis , and the two parasites follow a similar pattern of dividing from single to double parasites prior to egressing.…”