Babesia duncani, a Model Organism for Investigating Intraerythrocytic Parasitism and Novel Antiparasitic Therapeutic Strategies

Abstract

Pathogens such as Plasmodium, Babesia, and Theileria invade and multiply within host red blood cells, leading to the pathological consequences of malaria, babesiosis, and theileriosis. Establishing continuous in vitro culture systems and suitable animal models is crucial for studying these pathogens. This review spotlights the Babesia duncani in culture-in mouse (ICIM) model as a promising resource for advancing research on the biology, pathogenicity, and virulence of intraerythrocytic parasites. The model offers practical benefits, encompassing well-defined culture conditions, ease of manipulation, and a well-annotated genome. Moreover, B. duncani serves as a surrogate system for drug discovery, facilitating the evaluation of new antiparasitic drugs in vitro and in animals, elucidating their modes of action, and uncovering potential resistance mechanisms. The B. duncani ICIM model thus emerges as a multifaceted tool with profound implications, promising advancements in our understanding of parasitic biology and shaping the development of future therapies.

This review focuses on the B. duncani ICIM model, which serves as a promising resource for advancing research into the biology, pathogenicity, and virulence of intraerythrocytic parasites, as well as a tool to evaluate new drugs, vaccines, and diagnostic tests.

Intraerythrocytic parasitism defines the intricate relationship between the red blood cell as a host and an organism that resides within this cell and diverts its resources for its growth, reproduction, and survival. Vertebrates are hosts to a wide range of intraerythrocytic parasites including bacteria and eukaryotic protozoa [1]. Mammals and birds are particularly vulnerable to these infections with some species of Plasmodium, Babesia, and Theileria responsible for severe and often fatal disease outcomes in humans, wildlife, and livestock (Table 1). While these parasites have each evolved distinct mechanisms for growth and replication in host erythrocytes, they predominantly share common pathways and survival strategies, adapted to the often nutrient-poor environment within red blood cells. Understanding how these cellular processes lead to disease manifestation and the associated pathology necessitates the establishment of a system wherein genetic changes could be made in vitro and their impact on virulence subsequently examined in an animal model.

Table 1.

Overview of Key Resources Available for the Study of Various Babesia, Plasmodium, and Theileria Parasites

Species	Vectors	Mammalian Hosts	In Vitro Culture	Mouse Model	Genetic Manipulation
Babesia species
B. microti	Ticks	Deer, mice, humans	Short term	+	Yes
B. divergens	Ticks	Cattle, deer, mice, humans	Long term	−	No
B. duncani	Ticks	Deer, mice, humans	Long term	+	Yes
B. bovis	Ticks	Cattle	Long term	+	Yes
B. bigemina	Ticks	Cattle	Long term	−	Yes
B. ovata	Ticks	Deer, cattle	Long term	−	Yes
B. caballis	Ticks	Horses	Long term	−	No
B. canis	Ticks	Dogs	Short term	+ (chimera)	No
B. gibsoni	Ticks	Dogs	Long term	+	No
Plasmodium species
P. falciparum	Mosquitoes	Humans	Long term	+ (humanized)	Yes
P. vivax	Mosquitoes	Humans	Short term	+ (humanized)	No
P. malariae	Mosquitoes	Humans	−	−	No
P. ovale	Mosquitoes	Humans	−	+ (humanized)	No
P. knowlesi	Mosquitoes	Humans, primates	Long term	−	Yes
P. chabaudi	Mosquitoes	Mice	Short term	+	Yes
P. berghei	Mosquitoes	Mice	Short term	+	Yes
P. yoelii	Mosquitoes	Mice	Short term	+	Ye
Theileria species
T. equi	Ticks	Horses	Long term	−	Yes
T. annulata	Ticks	Cattle	Long term	+ (chimera)	Yes
T. parva	Ticks	Cattle	Long term	+ (chimera)	Yes

Information about the in vitro culture conditions, animal models, vectors, reservoir hosts, and availability of genetic tools for manipulation of the parasites can be found in references [2–35].

Red blood cells in host organisms play a critical role in the asexual reproductive cycles of intraerythrocytic apicomplexan parasites. Merozoites, the free form of these parasites capable of invading erythrocytes, enter these cells to initiate asexual reproduction. Subsequently, these parasites progress through various developmental stages, starting with the ring form, which further matures into trophozoites and then undergo division to form tetrads in the case of Babesia species and schizonts in the case of Plasmodium and Theileria species. The rupture of the infected erythrocytes results in the release of a new batch of invasive merozoites, thus initiating a new intraerythrocytic life cycle.

The establishment of in vitro culture systems for the continuous propagation of several intraerythrocytic apicomplexan parasites has played a pivotal role in advancing our understanding of the biology and pathogenesis of these parasites (Figure 1). The development of the first continuous in vitro culture system for the human malaria parasite Plasmodium falciparum in 1976 by Trager and Jensen [36] represents a significant breakthrough in the study of the parasite and a major driver of malaria research and development. This system has been crucial in advancing investigations into the biology of P. falciparum, including the mechanisms underlying its invasion, development, egress, sexual differentiation, as well as its modes of communication with the host cell. Furthermore, this system has served a critical function in advancing therapeutics strategies for the treatment of malaria through screening of new chemical libraries, rapid implementation of structure-activity relationships to identify lead inhibitors, and for evaluation of new vaccine candidates. The zoonotic parasite Plasmodium knowlesi, which has been linked to cases of malaria in humans [37], has also been successfully adapted for continuous culture in vitro [38]. However, other Plasmodium species that infect humans such as P. vivax, P. ovale, and P. malariae have been more challenging to culture continuously in vitro due to various factors, including the need for nonhuman primate hosts for continuous supply of reticulocytes in the case of P. vivax and P. ovale or in general the need for more complex culture conditions [39, 40].

Figure 1.

Advantages of the in culture-in mouse (ICIM) model for understanding Babesia duncani biology and pathogenicity. The ICIM model serves as a comprehensive platform, seamlessly integrating in vitro and in vivo approaches. This model offers a versatile toolkit for dissecting the biology, pathogenicity, and virulence of Babesia duncani. The combination of cell culture propagation, 96-well plate format studies, and the C3H-HeJ animal model provides a robust foundation for a wide array of investigations, ranging from molecular and cellular analyses to drug screening and optimization studies. Propagation in cell culture facilitates detailed cell biological analyses, exploration of metabolic and metabolomic changes that occur during the parasite's life cycle, and supports biochemical analyses and genetic manipulation studies. The 96-well plate format enables efficient and high-throughput studies, including screening of chemical libraries, structure-activity relationship studies for lead compound optimization, and toxicity profiling for identifying drugs with an ideal therapeutic profile. The animal model (C3H-HeJ) provides an in vivo system for studying parasite virulence and allows investigation into the role of parasite genes in parasite development under physiological conditions.

In the case of piroplasmids, tick-borne parasites that infect blood cells or endothelial cells [41], continuous in vitro culture has been achieved for the Theileria species, T. annulata and T. parva as well as several Babesia species including B. divergens, B. bovis, B. bigemina, B. ovata, B. duncani, B. canis, B. gibsoni, and B. caballi [2–8, 42–46] (Table 1). Babesia microti, the predominant causative agent of human babesiosis, has so far been difficult to propagate continuously in vitro in human erythrocytes despite multiple attempts. Short-term in vitro culture of B. microti has been used to get early insights into the activity of clinically approved antibabesial drugs in vitro and to identify early lead drugs [47].

While continuous in vitro culture has been successful for several hemoparasites, only a few of these have a mouse model of infection where the same species and genetically modified variants of these species can be propagated in vitro and subsequently injected into wild-type or transgenic mice to study the role of specific genes in pathogenesis and virulence. Compared to other mammals, such as nonhuman primates, mice are particularly attractive as experimental models in scientific research due to their genetic similarity to humans, short reproductive cycle, cost-effectiveness, and ease of handling. Moreover, the wide array of transgenic lines across various genetic backgrounds further enhances their utility in research. Advancements in the creation of humanized mouse models and mice chimeras has enabled certain Plasmodium species to be studied in vivo, albeit for a short time, for example to assess the efficacy of new drugs in vivo. A new model, named the human immune system human erythrocyte (HIS-HEry) model, has been shown to support efficient human erythropoiesis and enabled long-lasting multiplication of inoculated cryopreserved P. vivax parasites and their sexual differentiation, including in the bone marrow [48]. Other humanized mice, including human liver chimeric FRGN huHep mice, have been shown to support P. vivax liver stage and subsequent blood stage development, including gametocytes in vivo [49]. Humanization of immunodeficient mouse strains has also enabled blood and liver stage development of P. falciparum [50].

To overcome these challenges, most studies of the biology, immunobiology, and pathogenesis of malaria have relied on well-established experimental murine models of infections with species that naturally infect mice. While murine models of malaria have been invaluable for studying various aspects of the disease, these models have limitations in replicating human malaria infection. One significant challenge is the species-specific differences between rodent and human parasites, especially in their biology and pathogenesis.

In the case of Theileria species, chimeric severe combined immunodeficiency (SCID) mice have been used to host Theileria-infected bovine cell lines [9]. In the case of Babesia species, the fact that mice can be infected by some species that also infect humans makes the study of the biology, pathogenesis, and immunobiology of these parasites possible in various mouse strains, including transgenic mice, thus allowing investigations into the role of host factors in natural infection. This has been the case for the human babesiosis parasites B. microti and B. duncani. Babesia venatorum has also been shown to propagate in SCID mice [51] but long-term propagation of this species in mice could not be achieved, and the pathological effects of this infection in animals remains unknown. Other important human pathogens, such as B. divergens and B. MO1, do not infect mouse red blood cells and thus lack a mouse model of infection. However, the CE2 strain of B. divergens has been propagated in a gerbil model of cerebral babesiosis [52].

In this review, we spotlight B. duncani in culture-in mouse (ICIM) model, which has emerged as an optimal system for exploring the evolutionary dynamics of intraerythrocytic parasitism and the adaptive strategies employed by intraerythrocytic parasites for survival within host erythrocytes both in vitro and in vivo. B. duncani is one of the agents responsible for human babesiosis, a malaria-like illness, with potentially fatal consequences, particularly for individuals with a compromised immune system [53]. The parasite can be continuously propagated in vitro in human erythrocytes, which can then be injected into C3H mice where they continue their development within mouse erythrocytes, leading to a fatal outcome. The ICIM model thereby serves as a pertinent platform not only for elucidating fundamental aspects of parasite biology, pathogenesis, and virulence, but also for evaluating new therapies and therapeutic strategies, diagnostic tests, and vaccine candidates, both in vitro and in animals [54].

In 2018, Abraham and colleagues reported successful continuous in vitro culture of the B. duncani WA1 isolate in human erythrocytes [43]. This was achieved using commercially sourced HL1 or Claycomb media to which 20% fetal bovine serum (FBS) was added [43]. Subsequently, in 2019, McCormack and colleagues achieved continuous culture of the B. duncani WA1 isolate in hamster erythrocytes using HL-1 medium supplemented with 20% FBS [55]. Due to the complex composition, limited compositional information, and scarcity of the HL1 medium during the beginning of the coronavirus disease 2019 (COVID-19) pandemic, an alternative culture medium, DMEM-F12, was found to support the continuous propagation of the parasite in human erythrocytes in the presence of 20% FBS [10, 56]. The composition of DMEM-F12 proved crucial in identifying several nutrients essential for B. duncani's survival within host erythrocytes [57]. Interestingly, some of these nutrients, such as putrescine and lipoic acid, are also vital for the development of other intraerythrocytic parasites, enabling the transfer of knowledge from B. duncani to other Babesia species and even to P. falciparum, shedding further light on their mechanisms of survival within human erythrocytes. In 2023, Jiang and colleagues reported another addition to the list of media supporting the continuous growth of B. duncani in vitro in serum-free conditions [58]. The authors demonstrated long-term culture of B. duncani in hamster erythrocytes using VP-SFM AGT medium supplemented with AlbuMax I [58]. However, similar to HL1 medium, information about the composition of the VP-SFM AGT medium is not provided by the manufacturer.

Initial insights into the pathogenicity and virulence of B. duncani were gleaned from monitoring infection in mouse and hamster models. Early comparisons between B. duncani (WA1) and B. microti infection in golden hamsters (Mesocricetus auratus) revealed that B. duncani is particularly virulent in hamsters, leading to elevated parasitemia, acute disease, and fatality within 10 days of intraperitoneal inoculation [59, 60]. Murine B. duncani infection experiments demonstrated strain-dependent degrees of pathogen resistance. C57BL/6 and C57BL/10 mice exhibited higher levels of resistance and infection recovery, while BALB/cJ, CBA/J, and 129/J mice showed intermediate levels of resistance. A/J, AKR/N, DBA/J, and C3H mice displayed the greatest susceptibility, with fatal infections [61, 62]. Various types of knockout mice were employed to assess the roles of immune cells and inflammatory cytokines in the immune response against B. duncani. TNFRp55^−/− mice were utilized to elucidate the role of tumor necrosis factor-α (TNF-α) in B. duncani infection, revealing that disruption of the TNF-α pathway prevented infectivity, and CD4 T cells were implicated in the antiparasite immune response [61]. Infections of IFNGR2KO mice (deficient in interferon-γ [IFN-γ]-mediated responses) and Stat4KO mice (deficient in interleukin 12 [IL-12]-mediated responses) showed increased susceptibility [63], highlighting the importance of IL-12 and IFN-γ in defense against babesiosis. C57BL/6 mice deficient in different immune cell populations (B cells, CD4⁺ T cells, natural killer [NK] cells, and macrophages) and SCID BALB/c mice exhibited impaired resistance to B. duncani [63, 64], emphasizing the pivotal role of innate immunity, especially macrophages and NK cells, and the secondary role of acquired immunity.

The optimization of the mouse model of B. duncani infection using intravenous inoculation was reported by Pal and colleagues [65]. The study showed that C3H/HeJ mice infected with either B. duncani-infected murine erythrocytes or free merozoites succumbed to infection caused by a cytokine storm. Notably, while low-dose intravenous inoculation of up to 10⁵ infected erythrocytes resulted in 100% mortality in C3H/HeJ mice, it caused 50% mortality in Balb/c mice and 0% mortality in C57BL6 mice [65]. Conversely, higher doses (10⁶ or 10⁷-infected erythrocytes) led to mortality in all 3 mouse strains due to high parasitemia [65].

The B. duncani ICIM model has played a crucial role in evaluating the safety and efficacy of various classes of antiparasitic drugs in vitro and in mice, including those currently used for human babesiosis treatment as well as promising novel classes with potential therapeutic value, such as endochin-like quinolones (ELQs), tafenoquine, fosinopril, and antifolates [47, 65–68]. Furthermore, the model has been pivotal in evaluating the viability of monotherapies for babesiosis treatment. Parasite recrudescence in mice after treatment with either atovaquone, ELQs, or tafenoquine monotherapies underscored the necessity of combination therapies as a critical strategy for achieving radical cure. Subsequent in vitro studies revealed a highly synergistic effect with the combination of several ELQ prodrugs and atovaquone [65, 68], and an additive effect with the combination of tafenoquine and atovaquone [67]. These findings guided animal studies, where these drug combinations successfully eliminated infections without recurrence [65, 67, 68]. Equally interesting, the tafenoquine and atovaquone combination not only cleared parasites but also induced protective immunity, as treated animals became immune and survived subsequent challenges with otherwise lethal infections [67].

The amenability of the B. duncani in vitro culture system for high-throughput chemical screening has allowed for the screening of Food and Drug Administration-approved drugs to identify new antiparasitic agents [67]. Moreover, the ICIM model has facilitated a deeper understanding of the mechanisms of action of these drugs and strategies used by the parasite to mount resistance to them in vitro and in vivo. Parasites resistant to treatment, either selected in vitro or in mice, can be sequenced, with mutations mapped to specific genes using the recently reported genome sequence, assembly, and annotation of B. duncani [66]. For instance, a recent study by Vydyam and colleagues identified a mutation in the parasite esterase gene BdFE1, which hinders the conversion of the ACE inhibitor fosinopril to its active molecule fosinoprilat, thus significantly altering its antibabesial activity [67]. This discovery was pivotal in elucidating the essential role of parasite mediated activation for Fosinopril to exert its antiparasitic activity.

The ability to assess the biological activity of compounds both in vitro and in mice underscores the significance of this system as a proxy for evaluating the efficacy of drugs against other important human pathogens, including B. microti and P. falciparum. Several well-known antimalarials including pyrimethamine, WR99210, atovaquone, ELQs, and tafenoquine have been found to be effective against B. duncani in vitro and against B. duncani and B. microti in mice, and exert their activity through similar modes of action [53, 54, 65, 66, 68]. As new drugs and prodrugs continue to be developed against new parasite targets, we anticipate that the B. duncani ICIM model could serve as a platform to assess their efficacy both in vitro and in vivo, while also investigating their mechanisms of action. This is especially beneficial for compounds targeting metabolic and cellular processes conserved among these parasites. This system can also be instrumental in evaluating new candidate vaccine antigens by examining the impact of antibodies against these antigens on parasite development in vitro and further assessing their ability to confer protection following challenge in animals. Additionally, the ICIM model can be used to evaluate the importance of specific parasite genes in parasite development, survival, and virulence in animals. Similarly, by leveraging available transgenic mice with defined gene knockouts or knockdowns, we can start to explore the role of specific host factors in Babesia virulence [66].

Unlike P. falciparum, which has a notably low GC content (approximately 19%), B. duncani has a GC content of approximately 37%, which is similar to that of Saccharomyces cerevisiae, making cloning and gene expression in bacteria and yeast straightforward, and facilitating the creation of stable vectors and targeting cassettes for the genetic manipulation of this organism. The recent development of stable transfection in B. duncani [69] promises to shed more light on the role of specific genes in parasite development both in vitro and in vivo, advancing our knowledge of virulence, antigenic variation, sexual differentiation, and transmission of this parasite—a wealth of information with potential implications for research on closely related pathogens with similar infection, survival, and propagation mechanisms.

In summary, the ability to propagate B. duncani in vitro in human red blood cells and in mice makes this model unique among other intraerythrocytic parasites. The advantages of the B. duncani ICIM model encompass straightforward and well-defined culture conditions, ease of handling, suitability for large-scale screening to assess the efficacy and safety of novel antiparasitic drugs, an optimized mouse model for infection, genetic tractability, and the availability of comprehensive genomic resources. Moreover, the conservation of many cellular processes and pathways, such as folate metabolism, lipid biosynthesis, pantothenate utilization, and mitochondrial electron transport chain, across diverse hemoparasites enhances the significance of studying B. duncani, offering insights into fundamental cellular mechanisms relevant to apicomplexan parasites. Thus, the B. duncani ICIM model emerges as a valuable tool for gaining a comprehensive understanding of broader aspects of parasite biology.

Notes

Acknowledgment. We thank Dr Pratap Vydyam and Hannah Wang for their assistance with the design of Figure 1.

Financial support. This work was supported by the National Institutes of Health (grant numbers RO1-AI123321, RO1-AI138139, RO1-AI152220, and RO1-AI136118); the Steven and Alexandra Cohen Foundation (grant number Lyme 62 2020); and the Global Lyme Alliance.