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 intra-erythrocytic 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 anti-babesiacidal discoveries.
Human babesiosis is a rapidly emerging, zoonotic, infectious disease causing potentially life-threatening malaria-like symptoms in humans. It is caused by intra-erythrocytic protozoan parasites of the genus Babesia [1] and it is transmitted to humans via an ixodid tick vector or through a blood transfusion from asymptomatic carriers [2]. Bovine babesiosis is well regarded as one of the most important diseases of livestock, especially in the tropical and sub-tropical regions of the world [3]. However, human babesiosis disease prevalence has escalated over the past 50 years from a few isolated cases to global endemic areas now being recognized [4,5]. In Europe, cattle associated B. divergens is the most common causative agent of human babesiosis, especially throughout regions with extensive cattle industries, as the distribution geographically correlates with both pathogen infected host species and tick-vector infested regions, allowing for zoonotic transmission potential [6]. Disease burden outside North America and Europe is currently poorly described but considering the worldwide distribution of Babesia parasites, improved surveillance is required. Since the symptoms of human babesiosis resemble that of malaria and diagnosis is predominantly reliant on microscopic evaluation of blood smears, this disease may be misdiagnosed as a malaria infection, especially in areas of co-endemicity. Early disease detection, diagnosis and treatment with effective anti-babesiacidal compounds are therefore vital for both human and animal health [7]. In humans, Babesia parasites can be cleared by anti-malarials including atovaquone (with azithromycin) or quinine (plus clindamycin) but highly immuno-compromised individuals respond poorly to these treatments. As early reports of resistance against these combinations have been noted in the past few years, the need for alternative treatments is evident [8,9]. Against this background of potential zoonotic human babesiosis medical emergencies, it is quite surprising that our understanding of the basic biological processes underlying Babesia pathophysiology is still poorly understood, even with the recent application of genetic manipulation for transfection of Babesia parasites as well as the sequencing of the B. bovis genome [10,11]. Particularly intriguing is the fact that the precise progression and duration of the intra-erythrocytic, asexual developmental cycle (IDC) has not been clarified. During its IDC, Babesia parasites undergo asexual replication by binary fission (budding) of trophozoites to form 2–4 merozoites [12]. Each merozoite is thought to undergo a single cycle of division and then escape via cell lysis to re-infect new erythrocytes [13]. This establishes a perpetual, asynchronous asexual parasitic growth cycle, which is thought to last approximately 8 hours [14] and encompasses several developmental stages all present at the same point in time within the hosts’ bloodstream [15]. However, the description of the IDC and its different stages are fraught with uncertainties: historically different stages were described only based on light microscopy; little attention has been paid to their sequence of development and descriptions of the various Babesia parasitic in vitro stages do not share a consensus in literature and display considerable morphological pleiomorphism. Moreover, fundamental biological questions remain unanswered, particularly concerning the molecular descriptors governing the IDC of Babesia parasites. In this study, the in vitro IDC of the human pathogen B. divergens was comprehensively evaluated by employing various high-content cell biological and molecular strategies as has been previously applied to the more widely studied but related hemoprotozoan malaria parasite, Plasmodium falciparum. The study particularly focused on B. divergens as human pathogen and model organism for Babesia since it is amenable to in vitro cultivation. This is to our knowledge the first quantitative description and temporal evaluation of intra-erythrocytic B. divergens development and enabled clear characterization of the stage-specific development, based on nuclear proliferation in these parasites. The information is novel not just from a biological perspective, but will also be essential in future prioritization of anti-babesiacidal compounds.
Based on the findings presented here that correlate the linear increase of DNA content to its associated nuclear content and morphological classification, the progression of intra-erythrocytic B. divergens parasites in its development from one IDC stage to another could be proposed. Temporal evaluation of in vitro proliferation for each developmental form (ring, paired piriforms, tetrads and/or multiple infections) was performed over a 16-hour period. Parasitemia steadily increased to 15% over the 16-hour period monitored in the asynchronous culture (Fig 3). However, between 4–6 hours of development, the ring formation population doubled (from a 2% to a 4% contribution to parasitemia, P<0. 05, n = 3) (Fig 3). This was additionally associated with an increase in paired piriforms (2% increase in contribution to total parasitemia for paired piriforms). Similar observations were made between 8–10 hours of development, where a significant increase in paired piriforms was again observed (8% to 10%, P<0. 05, n = 3) (Fig 3). Overall, the population distribution in the mixed culture remained predominantly paired piriforms (9. 04 ± 1. 03% contribution to total parasitemia that ranged from 10. 3–15. 3%) followed by ring formations (3. 33 ± 0. 68% contribution to total parasitemia) (Fig 3). The tetrad (or multiple infection) population is the most stable throughout the temporal evaluation with no significant variation in population size (average 1. 08 ± 0. 28% contribution to total parasitemia maintained throughout). The increase in ring and piriform populations between 4–6 hours of development additionally contributed to a 3% increase in total parasitemia (from 10 to 13% total parasitemia) and again a 2% increase in parasitemia between 8–10 hours (from 13 to 15% total parasitemia). This is indicative of life cycle progression through re-infection of erythrocytes (contributing to new rings formed) and parasite developmental maturation (contributing to new paired piriforms). If significant increases in the ring populations are taken as an indicator of merozoite invasions of erythrocytes and initiation of new development cycles, it appears therefore that the in vitro development of B. divergens is typified by a 4 hour progression window and that, after two parasitic life cycles and under the culture conditions employed, equilibrium could be established and maintained. As such, a multiplication index of 3-fold was subsequently observed for continued B. divergens in vitro development. There are currently no data describing the molecular events associated with B. divergens intra-erythrocytic development, information that is essential to understanding the nature of stage-specific progression of this parasite’s IDC. Since clear contributions of morphologically distinct life cycle compartments were observed to contribute to the IDC progression of B. divergens parasites, we set out to describe the global transcriptome of these parasites through its IDC as an indicator of the physiological processes involved. Transcriptome analysis (mRNA abundance determination) was subsequently conducted on asynchronous, newly initiated cultures with a custom designed DNA microarray containing 15744 target features covering 97% of the B. bovis genome (3703 independent ORFs) and using a reference pool design strategy (Fig 4). B. divergens parasites seem to adapt to culturing under unlimited growth conditions (4% increase in parasitemia, P<0. 05, n = 3) (Fig 4A). Again, this adaptation was resultant of a significant increase in both ring and paired piriform parasites (from 2. 5 to 5% and 4. 75 to 7. 5%, respectively, P<0. 05, n = 3); after this the parasite population distribution equilibrated. These observations were further evident from correlation data, indicating that the transcriptome of equilibrated parasites showed the best correlation (r = 0. 19) between parasites that have been in culture for 9 and 16 hours (C9 vs. C16, Fig 4B). Comparatively, initiate cultures showed complete disconnect from established cultures (C0 vs. C16 anti-correlated at -0. 03). This clearly indicates a physiological adaptation event underscored by a predominant transcriptional repression in the initiate culture (931 undefined transcripts, 387 transcripts with increased abundance, compared to 2385 repressed transcripts). Differential expression between the initiate culture and the parasites 4 hours after inoculation resulted in 164 transcripts significantly affected (82 decrease and increase in abundance, respectively) (S1 Table). Of these, only 50 could be annotated using the dCAS annotation system [18] based on each transcript’s COG classification (E-value cut-off of less than 1x10-4) (Table 1). The processes mostly affected include transcription (26%), translation (8%), protein turnover (21%), cellular (30%) and metabolic (11%) processes, and stress defense mechanisms (2%). All activated transcripts may be associated with the changes in parasite population distributions (increases in ring and paired piriforms). Clustering of transcripts based on co-expression profiles indicated the expected similarity in expression profiles across the transcriptome (Fig 4C). Moreover, transcripts in some of these co-expressed clusters additionally showed clear chromosomal synteny (e. g. BBOV_III006240 and BBOV_III007280 on chromosome III; BBOV_IV007380 and BBOV_IV001730 on chromosome IV) implying transcriptional level regulation for these transcripts. Further temporal evaluation of the B. divergens transcriptional landscape during its IDC indicated correlation across the transcriptional landscape (Fig 5). However, clear transcriptional activation to a permissive state was observed as the parasites progressed in their life cycle, particularly evident for parasites in culture for at least 9 hours (e. g. in C4,45% of the transcripts showed increased abundance; this increased to 60% at C9) (Fig 5A). However, after 16 hours in cultivation, the transcriptome seems more unbiased and show a more equal distribution of abundances in transcripts with an inclination towards transcriptional repression. This may be correlated to a slight increase in the paired piriform population in the morphological profiles observed between parasites in culture for 9 or 16 hours. Further comparison of the transcriptomes of the parasite populations indicated the presence of differential expression patterns across the 4–16 hour evaluations. To distinguish only processes associated with IDC progression and minimize adaptation responses as a result of culture initiation (time 0–4 h), transcriptional profiles were evaluated between parasites in culture for 4,9 and 16 hours, respectively (Fig 5B). Functional annotation of transcripts associated with the alternative expression patterns identified several transcripts associated with normal biological, cellular and functional pathways throughout the investigated period. This included 11 major functional categories (catalytic activity 16%; energy production and conversion 5%; lipid transport metabolism and synthesis 2%; membrane protein components 6%; mitochondrial components 3%; proteolysis 13%; ribosomal components 17%; transcription and translation 25%; transport 6%, and variant surface antigen expression 9%), which were present across all clusters and time points. However, certain biological processes proved more variable over the temporal evaluation, particularly protein turnover, transcription and translation with increased levels of activity at 9 hours in culture. Comparatively, energy production is, as expected, maintained throughout the temporal evaluation.
Our current understanding of the intra-erythrocytic developmental cycle of B. divergens has been clouded by imprecise and conflicting classifications, mostly due to historical analyses relying for the most part only on morphological microscopic observations. For instance, discrepancies in morphological evaluation of different life cycle forms are reported, where trophozoites are sometimes referred to as merozoites [12]. Additionally, one of the major challenges with studying the stage-specific development of B. divergens intra-erythrocytic development is the inability to synchronize these parasites in vitro to a single stage. A well-developed synchronization strategy induced by either sorbitol or mannitol is widely applied in P. falciparum research and is mainly associated with changes in membrane permeability and buoyant density of erythrocytes parasitized by this organism [19]. However, these techniques were evaluated and found to be ineffective against B. divergens in vitro, resulting in parasite death within 24 hours. The data obtained by combining advanced cell biological and molecular strategies allow for the first time objective and clear evaluation of the chronology and stage-specificity of intra-erythrocytic development of B. divergens parasites. These are sensitive and quantitative and allow various developmental stages to be distinguished. Flow cytometry, light and fluorescent microscopy allowed for the accurate detection of intra-erythrocytic B. divergens parasites as well as determination of dynamic proliferation, developmental stage assessment and isolation of asynchronous B. divergens parasites, based on morphology as well as nuclear content. Asynchronous, independent nuclear division occurs during intra-erythrocytic P. falciparum development, where daughter merozoites follow a non-geometric expansion and parasitic multiplication consequently deviates from what is expected from equal numbers of binary divisions [20]. Similar findings were observed with light microscopy and flow cytometry for the asynchronous in vitro B. divergens cultures in this study and enabled the isolation of specific developmental stages over a 16-hour period. The increase in MFI values observed between the three infected populations corresponds to the fluorescent microscopy images, which ultimately indicate an increase in DNA nuclear content from one developmental stage to the next. Based on the DNA measurements, which underlie the morphological findings of the present study, asynchronous in vitro Babesia dynamics was further evaluated. With primary parasitology classifications in mind, intra-cellular and actively metabolizing parasites are usually classified as trophozoites, which divide asexually (merogony) with the resultant formation of daughter merozoites. Erythrocytes infected with a ring formation, contain a single parasitic nucleus (1N) and erythrocytes infected with either paired piriforms, tetrads or multiple infected erythrocytes, contain two or more nuclei (2N or >2N). These findings correlate nuclear content to a particular isolated cell population (based on morphology); previously unclear for Babesia parasites. Here we define intra-erythrocytic B. divergens parasites directly after invasion as mono-nucleated rings (1N nuclear content), which then rapidly progress to metabolically active but still mono-nucleated, haploid trophozoite populations. These forms are only morphologically distinguishable based on anaplasmoidy in rings compared to the more rounded / ovoid trophozoites and not on differences in nuclear content; further specific classification would require metabolic flux data. With the associated nuclear content information provided in this paper, we were able to indicate the subsequent progression of mono-nucleated ring / trophozoites to bi-nucleated paired piriforms (2N nuclear content) during binary fission in which a single parasite undergoes a single nuclear division event resulting in two daughter merozoites. During this nuclear division event, the nucleus becomes typically V-shaped (as observed with fluorescent microscopy). The formation and origin of multi-nucleated tetrads is less clear. These parasites may undergo two nuclear division cycles that may result in the formation of four daughter merozoites. Binary fission dictates the duplication of nuclear content in a cell, followed by DNA segregation and finally cytokinesis. The formation of tetrads would therefore imply either that (a) a duplicate binary fission event occurred simultaneously from a single ring, visible as the characteristic cross morphology prior to cytokinesis or (b) that a minor proportion of paired piriforms would not undergo cytokinesis after DNA replication but rather undergo a second round of DNA replication, resulting in the tetrad formations with a 4N nuclear content prior to cytokinesis and ultimately to the formation of 4 daughter merozoites. High-resolution real-time microscopy evaluation is needed to address these possibilities. Since tetrad forms are infrequently observed in culture compared to the other morphological forms, tetrad formation is either relatively rare (only 10–25% of the total parasite population) or occurs rapidly (with quick kinetics of cytokinesis) such that these forms are rarely observed. With an optimal multiplication index of 2–3, it does not make a major contribution to B. divergens parasite proliferation in vitro. All merozoites of B. divergens parasites are typically piriform and joined by their pointed ends and also do not fill the complete erythrocyte, as expected of ‘small babesiae’. The kinetics of invasion (and re-invasion) after initial contact between the parasite and the erythrocyte, proceeds rapidly, between 45 seconds and 10 minutes [21]. We therefore hypothesize a parasitic propagation diagram based on the morphological observations, DNA measurements, temporal distribution and transcriptome expression dynamics (Fig 6). If an increase in parasitemia and associated increased ring populations is taken as indicators of new infections, then the significant increase in newly infected erythrocytes (ring formations) observed here during the first 4–6 hours in culture provides indications of the kinetics of the B. divergens in vitro IDC merogony. This is markedly quicker than previous reports where the in vitro life cycle of B. divergens parasites was claimed to last around 8 hours under the culture conditions used in that study [14]. However, the 2 hourly evaluation performed in our study enabled a finer analysis of the IDC progression kinetics that was not probed previously. The asexual Babesia parasitic life cycle has only been described within the erythrocytes of their vertebrate hosts and the salivary glands of the tick vector, with limited data currently available concerning the Babesia sexual life cycle [22]. Only two studies have, however, reported on the visualization of intra-erythrocytic gametocytes in B. divergens [12,23]. Our temporal evaluation of B. divergens revealed contrasting results to that achieved with the optimized flow cytometry strategy during the interrogation of mixed infected erythrocyte populations. The overall population distribution (associated with temporal evaluation) remained predominantly paired piriforms followed by rings, tetrads and multiple infections. Comparatively, the overall population distribution observed with flow cytometry during the interrogation of mixed infected erythrocyte populations was predominantly ring formations followed by paired piriforms, tetrads and multiple infections. The inability to accurately visualize and characterize gametocytes with light microscopy within mixed B. divergens cultures may have contributed these contrasting results. However, we analyzed our transcriptional data set for genes characterizing gametocytes (i. e. bdccp 1; bdccp 2 and bdccp 3) [24]), but these were not differentially affected in our data set. This can be interpreted as: 1) we did not observe gametocytes in B. divergens cultures in vitro (supporting our morphological descriptors characterising merozoites) or 2) the parasites are not under undue physiological stress, which have been implicated to result in increased gametocytogenesis. The transcriptional response that we do observe therefore mimics typical parasite physiology. Flow cytometry measurements may therefore include ring forms as well as gametocytes, thereby increasing the DNA content. Morphological findings suggest that a trend in parasitic development from one stage to the next can be observed and that all stages reach a plateau in their developmental process. Transcriptome analyses of the initiate culture revealed an expression pattern for B. divergens parasites. The draft B. divergens genome (recently deciphered) in combination with the information presented here, can facilitate future Babesia related studies and improve our understanding of the parasites biology, host-parasite interaction as well as improve control and treatment strategies [25,26]. Additionally, the information presented here represents a preliminary catalog for B. divergens gene expression during its IDC, measured over time. The differential transcript abundance analysis identified several activated and repressed transcripts associated with parasitic growth and development, which may have contributed to changes in parasite population distributions. The transcriptional analysis was subsequently linked to the morphological findings. The initial stress response (between 0–4 hours) potentially induced by the addition of complete culture media, may have influenced the expression pattern. To minimize the possible stress response effects, an alternative expression pattern was suggested for the hierarchical clustered data set, which ranged between 4–16 hours. Functional annotation of the visualized transcripts and transcriptional response revealed predominantly activated and repressed components associated with transcription, translation, protein turnover, cellular and metabolic response as well as stress defense mechanisms, confirming the observed change in parasite population distributions (increases in ring and paired piriforms). The transcriptome mirrors the Babesia genome with glycolysis and components of the TCA cycle, glycerolipid and glycerophosphospholipid metabolism, pyrimidine and associated nucleotide synthesis, amino acid synthesis and certain components associated to apicoplast metabolism [11]. Moreover, almost a tenth of the differential transcriptome is associated with expression of transcripts in the ves1 family (expressing VESA1). Similar to the malaria parasite, Babesia spp use antigenic variation to escape detection by the host’s immune system [26]. The hierarchical clustering employed here indicated that some of these biological processes do however show differential expression (e. g. protein expression and transport). Overall, the transcriptionally permissive state of the B. divergens IDC resembles that of the IDC transcriptome of P. falciparum parasites (with the exception of silenced virulence genes) [27]. In the latter, phase-ordering indicated a ‘just-in-time’ transcriptional activation, with transcripts finely associated with highly synchronized specific stages in the IDC. Even through the data presented here for B. divergens implies molecular control factors involved in the IDC, fine resolution temporal analysis of this parasite will be confounded as the population is composed of mixed stages, making stage-specific analysis difficult. More comprehensive identifications of novel compounds against veterinary Babesia species have only recently gained attention [28]. However, for screening platforms to identify effective anti-babesiacidal compounds, several lessons may be learnt from highly advanced screening strategies from other thoroughly investigated diseases like malaria. As such, anti-malarial screening strategies have been clearly delineated and require early decision making based on target product and target candidate profiles. Particularly for in vitro hit identification, the ability of novel compounds to target specific (or all) stages of malaria parasite development as well as their speed of action is used to classify these in vitro specific profiles, thereby enabling their further prioritization [29,30]. Similar strategies are required for screening of anti-babesiacidal drugs, as evaluation of the stage-specific nature of compounds would be imperative in understanding it’s mode-of-action and examine new treatment and dosage strategies. Current evaluation of growth-inhibiting effects of potential anti-babesiacidal compounds rely on either microscopic examination of Giemsa-stained smears and / or the evaluation of incorporation of isotopes into in vitro Babesia cultures [31], with neither of these allowing stage-specific or temporal evaluation of compound action. Light microscopy is time-consuming, subjective, labor intensive and operator dependent with poor quantitative robustness. Although isotopic techniques overcome the latter, this is being replaced by cost effective, reliable and non-radioactive fluorescence-based assays. Apart from the use of such dyes in plate-reader formats (enabling high-throughput screening platforms) [28] these assays are usually performed without evaluation of stage-specificity. However, the possibility to combine these dyes with techniques such as flow cytometry will allow for an additional level of characterization that has proved useful to address these caveats. One such example has been the successful application to the stage and temporal evaluation of anti-malarial compounds [17,32]. The expansion of our biological knowledge of B. divergens parasites’ intra-erythrocytic development through the molecular blueprint of its complete transcriptome provided in this paper should enhance future discovery of novel anti-babesiacidal drugs.
Approval for the importation and in vitro cultivation of B. divergens was obtained from the South African Department of Agriculture, Forestry and Fisheries. Human blood and sera was collected from volunteer donors and used for cultivation with ethical approval (University of Pretoria Faculty of Natural and Agricultural Sciences Ethics Committee approved the project protocol with identification number EC120821-077). Volunteer donation was based on written informed consent from only adult donors at a registered phlebotomy facility. No minors were allowed to donate blood in this study. Parasite cell culturing was based on CDC criteria for such research. No animals were used in this study. All parasites were grown under in vitro conditions only. The Rouen 1987 strain of B. divergens was kindly provided by Dr. Stephane Delbecq (Laboratoire de Biologie Cellulaire et Moleculaire UFR Pharmacie, Montpellier, France). Asynchronous B. divergens parasite cultures were maintained in human erythrocytes (type O+) suspended in complete culture medium [RPMI-1640 medium supplemented with 25. 2 mM HEPES, 22. 2 mM D-glucose, 50 mg/l hypoxanthine, 21. 4 mM sodium bicarbonate, 48 mg/l gentamycin (Sigma) ] further supplemented with 10% human serum [33]. Cultures were maintained at 37°C in a gaseous environment of 90% N2,5% O2 and 5% CO2 on a rotary platform (60 rpm) at ~5% hematocrit and 10–15% parasitemia, with daily media replacement. Comparatively, intra-erythrocytic P. falciparum (3D7 strain) parasites were obtained from the MR4 (www. mr4. org) and were maintained at a 5% hematocrit, 2–5% parasitemia in complete culture medium [RPMI-1640 medium supplemented with 25. 2 mM HEPES, 22. 2 mM D-glucose, 50 mg/l hypoxanthine, 21. 4 mM sodium bicarbonate, 48 mg/l gentamycin (Sigma) ] further supplemented with 0. 5% Albumax II (Invitrogen) [34] in the same gaseous environment under shaking conditions as above. For both intra-erythrocytic B. divergens and P. falciparum parasites, Giemsa-stained thin smears light microscopy was used for the daily determination of both the parasitemia and morphology. Thin blood smears were prepared, air dried, fixed with methanol and stained for 5 minutes prior to examination. The percentage parasitemia was determined as the number of infected erythrocytes per 100 cells, with a minimum of 1000 erythrocytes counted [35]. Asynchronous intra-erythrocytic B. divergens parasites (5% hematocrit, 10–15% parasitemia) were examined every two hours over a 16-hour time period using Giemsa-stained smears and light microscopy. The asexual developmental life stages were classified as rings, paired piriform and tetrad and/or multiple infection formations. Fluorescent microscopy (Zeiss Axiovert 200 fluorescent microscope) was also used to examine intra-erythrocytic B. divergens parasites using the Axiovision release 4. 8. 2 software for analyses. A custom designed DNA microarray slide was used for gene expression analysis. The base composition probe design strategy and probe selection parameters were selected according to Agilent Technologies eArray 60-mer platform specifications. Available B. bovis sequences were downloaded and retrieved from the National Center for Biotechnology Information (NCBI) (http: //www. ncbi. nlm. nih. gov/) and supplemented with additional published sequence data [5]. Selected probes were randomly distributed across the array using the 8x 15K design format. All arrays were ordered from Agilent Technologies (https: //earray. chem. agilent. com/earray/).
Vector-borne parasitic diseases are still the major cause of morbidity and mortality in both humans as animals. Some of these parasites have been well studied, including the malaria parasite, Plasmodium falciparum, since these cause major fatalities in humans. However, other parasites like Babesia divergens, who closely resemble malaria parasites, are currently becoming a major concern since these are zoonotically transmitted to humans from their natural hosts (e. g. cattle) by ticks. Their rising levels indicate the possibility of human medical emergencies occurring. Unfortunately, basic biological processes in these parasites are poorly understood due to their neglected status. In this manuscript, we describe for the first time high-content analysis of the development of B. divergens parasites in vitro over time. We applied cell biological and advanced functional molecular strategies to also provide the first descriptors of the parasite’s transcriptome during life cycle development. This information is unique and to not only dramatically expand our information base on Babesia biology, but also provide information that can be exploited for future drug discovery endeavors.
Morphological and Molecular Descriptors of the Developmental Cycle of Babesia divergens Parasites in Human Erythrocytes