Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is
on the increase, resulting in renewed attentiveness to this potentially life threatening emerging
zoonotic disease. The molecular mechanisms underlying the pathophysiology and intraerythrocytic
development of these parasites are poorly understood. This impedes concerted
efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell
biological and molecular functional genomics tools, we describe the intra-erythrocytic development
cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated
paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic
Babesia spp. This is further correlated for the first time to nuclear content increases during
intra-erythrocytic development progression, providing insight into the part of the life cycle
that occurs during human infection. High-content temporal evaluation elucidated the contribution
of the different stages to life cycle progression. Moreover, molecular descriptors
indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation.
Additionally, differential expression is observed as the parasite equilibrates its developmental
stages during its life cycle. Together, this information provides the first temporal evaluation
of the functional transcriptome of B. divergens parasites, information that could be
useful in identifying biological processes essential to parasite survival for future antibabesiacidal
S1 Text. Explanation of the development of a fluorescent cell biological evaluation technique
to detect intra-erythrocytic B. divergens parasites.
S1 Fig. Flow cytometric analysis of intra-erythrocytic B. divergens parasites. Uninfected
erythrocytes were analysed in parallel to B. divergens infected erythrocytes, either fixed
(0.025% glutaraldehyde for 45 min) or unfixed. Cells were subsequently stained with either
1:100 and 1:1000 SYBR Green I (30 min, dark, room temperature). (A) Dotblot analysis and
(B) histograms of 1) uninfected, unfixed, stained erythrocytes; 2–5) B. divergens infected erythrocytes
either unfixed (2 & 3) or fixed (4 & 5). In panels 2 and 4 cells were stained with 1:100
SYBR Green I and in panels 3 and 5 with 1:1000 SYBR Green I. Erythrocytes infected with P.
falciparum parasites were comparatively analysed in panel 6 (glutaraldehyde fixed and stained
with 1:1000 SYBR Green I. (C) Effect of SYBR Green I concentrations on parasitemia determined
from both unfixed and fixed B. divergens infected erythrocytes. Results are the mean of
three independent experiments each performed in triplicate (± S.E.). Significance is indicated
at P<0.001 ( ) (unpaired Student-t test). (D) Linear correlation analysis (R2 value of 0.98) of B.
divergens parasitemia detection between light microscopy and flow cytometry. Data are the
mean of three independent experiments each performed in triplicate (± S.E.). Confidence levels
(95%) indicated by dashed lines.
S1 Table. Differentially affected transcripts identified in the initiate culture before culture