Background

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia. Like the other apicomplexan parasite Plasmodium falciparum, the causative agent of human malaria, Babesia parasites also invade human red blood cells (hRBCs) to cause the pathological symptoms associated with human babesiosis, with clinical outcomes ranging from mild to severe and, in some cases, leading to death. More than 100 species of Babesia are known to cause infection in a wide range of mammalian hosts, including livestock, with significant health and economic impacts (Renard and Ben Mamoun, 2021). Human babesiosis is an emerging worldwide concern; although humans are not the natural host of Babesia. Over the past 50 years there has been a rapid increase in cases of human babesiosis caused by different Babesia species (Renard and Ben Mamoun, 2021). While B. microti is the most common species causing human babesiosis, other species such as B. divergens and B. duncani have also been shown to lead to severe and sometimes lethal clinical outcomes (Persing et al., 1995; Rożej-Bielicka et al., 2015; Vannier et al., 2015; Kumar et al., 2021; Renard and Ben Mamoun, 2021). The first case of human babesiosis was identified in a splenectomized patient in Europe, but most cases of babesiosis found in northeastern and midwestern United States had no history of immune impairment (Skrabalo and Deanovic, 1957; Hunfeld et al., 2008; Vannier et al., 2015). Although humans are a dead-end host of Babesia parasites, cases of accidental transmission through blood transfusion from Babesia-infected individuals have been reported, and some rare cases of transplacental transmission have also been documented (Fox et al., 2006, Walker et al., 2022). The first case of transfusion-transmitted babesiosis (TTB) was reported in 1979, ten years after the first reported clinical case of B. microti human babesiosis in the United States (Scholtens et al., 1968, Jacoby et al., 1980). Although B. microti is the most common agent of TTB, cases caused by B. duncani have also been reported (Kjemtrup and Conrad, 2000; Kjemtrup et al., 2002). As the recipients are often immunocompromised, TTB could be fatal (Herwaldt et al., 1997; Claycomb et al., 1998; Conrad, 2000; Leiby, 2011; Renard and Ben Mamoun, 2021). The blood donors carrying Babesia parasites are often asymptomatic, which highlights the necessity for generating tools for efficient diagnosis of parasite infection and for the development of a vaccine and new therapies for the prevention and treatment of human babesiosis. For a long time, the life cycle and biology of Babesia parasites have been poorly elucidated, mainly due to the lack of suitable tools for continuous in vitro propagation of the parasites and/or lack of animal models to study their pathogenesis and virulence. The in culture and in mouse (ICIM) model for Babesia infection described herein will assist in answering some key questions about Babesia biology, pathogenesis, and survival in human red blood cells, namely how these parasites interact with the host and modulate its immune response. The ICIM model has also been important in the discovery and development of novel antibabesial drugs (Lawres et al., 2016; Chiu et al., 2021; Pal et al., 2022). In previous studies (Abraham et al., 2018), the continuous in vitro culture of B. duncani in hRBCs was reported in commercially available Claycomb (Sigma) and HL1 (Lonza) media. The HL1 medium has been discontinued since March 2021; the Claycomb medium often suffers supply shortages, is relatively expensive, and contains several mammalian proteins (bovine albumin, fetuin, transferrin, human insulin, long R3IGF-1, and long EGF) that, while important for HL-1 cardiomyocytes, are of no importance to Babesia intraerythrocytic development (Claycomb et al., 1998; White et al., 2004; Singh et al., 2022). We have recently reported that the DMEM/F-12 is an alternative growth medium for continuous in vitro culture of B. duncani in hRBCs (Singh et al., 2022). It differs from a standard DMEM medium, which does not support the growth of the parasite, by the presence of six amino acids (alanine, asparagine, aspartic acid, cysteine, glutamic acid, and proline), two vitamins (biotin and cobalamin), and four inorganic salts (cupric sulfate, ferric sulfate, magnesium chloride, and zinc sulfate) as well as hypoxanthine, thymidine, linoleic acid, lipoic acid, and putrescine (Singh et al., 2022). Furthermore, an optimized animal model of B. duncani infection that allows a consistent and reproducible evaluation of parasite development and virulence in mice has been recently described (Pal et al., 2022). The ICIM model, which combines the in vitro propagation of the parasite in human red blood cells and parasite virulence in mice, will usher a new era of advanced research on Babesia by facilitating the use of genetic tools and resources to conduct large scale functional analysis to link gene expression and function to disease progression and parasite virulence. This model will greatly enhance our understanding of this disease, as well as help in developing novel therapeutic strategies with improved efficacy.