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. 2021 Aug 20;11(1):16959.
doi: 10.1038/s41598-021-96365-w.

Isolation of viable Babesia bovis merozoites to study parasite invasion

Hassan Hakimi  1   2 , Masahito Asada  3 , Takahiro Ishizaki  4 , Shinichiro Kawazu  5
Affiliations

Isolation of viable Babesia bovis merozoites to study parasite invasion

Hassan Hakimi et al. Sci Rep. .

Abstract

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces 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 significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Time-lapse imaging of B. bovis. GFP-expressing B. bovis merozoites were observed over 24 h period and events following parasite egress and subsequent invasion were witnessed (n = 10).
Figure 2
Figure 2
Dose–response curve of C2 and ML10 for B. bovis. The parasites were cultured in presence of different concentrations of C2 or ML10. Data are shown as mean ± SEM of triplicate culture.
Figure 3
Figure 3
Impact of ML10 and C2 concentration and length of exposure on egress block and parasite viability. (a) Parasites were cultured in presence of 0.5, 1 or 2 μM of ML10 or 1, 2 or 5 μM of C2 for 4, 12, or 24 h. The initial percentage of iRBCs was ~ 1% and percentages of iRBCs in presence of drugs were calculated. The data are shown as mean ± S.D. of triplicate culture. Statistical comparisons were done between each group and initial percentage of iRBCs. (*P < 0.05; **P < 0.01; ***P < 0.001; determined by unpaired t test). (b) The proportion of ring, binary, and multiple stages in presence of ML10 or C2 for 4, 12, or 24 h. The data are shown as mean ± S.D. of triplicate culture. (c) Cultures that were exposed to different concentrations of ML10 or C2 for 4, 12, or 24 h were washed and allowed to grow in fresh medium for 24 h. The statistical significance of the difference between each group and parasite treated for 4 h of 0.5 μM of ML10 or 1 μM of C2 determined by unpaired t test. (**P < 0.01; ***P < 0.001; ****P < 0.0001). The data are shown as mean ± S.D. of triplicate culture.
Figure 4
Figure 4
Babesia bovis growth and egress following removal of C2 and ML10. (a) The Giemsa-stained smears were prepared before drug removal to validate the effects of drugs on parasite morphology. Scale bar = 10 µm. (b) Parasites that had been arrested in the culture in the presence of 0.5 μM of ML10 or 1 μM of C2 for 4 or 12 h were washed and transferred to fresh medium to allow egress and invasion of new RBC for 36 h. The data are shown as mean ± S.D. of triplicate culture. (c) Proportion of ring, binary, and multiple stages in initial parasites, at the time and following removal of ML10 or C2 were calculated for 36 h (mean ± S.D. of triplicate culture).
Figure 5
Figure 5
Invasion kinetics of B. bovis filter isolated merozoites. (a) The proportion of merozoites that successfully invaded erythrocytes is plotted over time relative to a 60-min maximum incubation (mean ± S.D. of three independent experiments in triplicate culture). (b) Parasite growth over 36 h time course. Smears were prepared every 2 h and data are shown as mean ± S.D. of triplicate culture. (c) Proportion of ring, binary, and multiple stages following invasion (mean ± S.D. of the triplicate experiment).
Figure 6
Figure 6
BbVEAP knockdown did not affect parasite invasion. (a) Western blot analysis of myc-glmS expressing B. bovis in the presence or absence of glucosamine (GlcN). Anti-SBP4 antibody was used to detect SBP4 protein as a loading control. The image is representative of three independent experiments. Full-length blots are presented in Sup. Fig. 3. (b) The myc-glmS expressing merozoites in the presence or absence of GlcN for 24 h were filter isolated and an invasion assay was performed. Percentage of iRBCs was measured at 1 h and 24 h after the invasion. The data are shown as mean ± S.D. of three independent experiments in triplicate culture. (ns, not significant; **P < 0.01 determined by unpaired t test). Scale bar = 10 µm. (c) Proportion of ring, binary, and multiple stages in parasites in the absence or presence of GlcN at 1 h or 24 h following invasion (mean ± S.D. of three independent experiments in triplicate culture. **P < 0.01; ***P < 0.001 determined by unpaired t test).
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