Babesiosis is an emerging tick-borne zoonotic disease caused by the intraerythrocytic parasite Babesia, resulting in a malaria-like disease (Dvoraková & Dvorácková, 2007). Babesia rodhaini is a rodent Babesia related to B. microti, the major causative agent of human babesiosis. Both species belong to the “small group” of Babesia, characterized by the relatively smaller size (1.0-2.5 µm) of trophozoites (Homer et al., 2000). Babesia rodhaini has the potential to infect human erythrocytes (Kawabuchi et al., 2005) and inoculation of even a single parasite leads to 100% mortality in mice (Clark, 2001). In contrast, B. microti (Munich strain) causes a self-limiting disease in mice that resolves eventually (Igarashi et al., 1999). Babesia microti causes babesiosis in animals and humans worldwide and has seen increasing interest as an emerging zoonosis  (Leiby, 2011; Vannier & Krause, 2012). Babesia is not only transmitted by tick vectors but also by blood transfusion or during pregnancy (Young et al., 2019; Xue et al., 2021). According to the CDC, approximately 2,000 cases of babesiosis occur every year in the United States (Centers for Disease Control and Prevention, 2012). Babesia is a menace to the supply of blood and is lately the most common transfusion-transmitted infection in the United States (Lobo et al., 2013). Babesiosis can be life-threatening in infants or immunocompromised patients (Gabrielli et al., 2016).

The spleen is the largest secondary lymphoid organ in the body and is involved in hematopoietic functions. It elicits immunological responses against blood-borne pathogens, rendering it indispensable in interrelating the immune system’s response (Cesta, 2006). The spleen is likewise involved in the clearance of parasitized erythrocytes by filtration, including babesiosis (White et al., 1998). Eventually, the fate of the host mainly relies on whether the host has an intact spleen (Roberts et al., 1972). Splenectomized and elderly individuals are at a higher risk of severe symptomatology and may experience hemolytic anemia, splenomegaly, hepatomegaly, renal failure, and even death (Bloch et al., 2019; Young et al., 2019). Hence, as the biggest immune organ, the spleen plays an essential role in protecting against Babesia infection. When infected with Babesia, even though the spleen is seriously injured, it still actively initiates immunomodulatory responses (Xue et al., 2021). The spleen consists of B lymphocytes and macrophages and can also produce immunoglobulins and factors that deploy immune functions (Golub et al., 2018). Some studies indicated that cellular immunity, T cells, and macrophages are critical for the clearance of B. microti in mice (Igarashi et al., 1999; Clawson et al., 2002; Li et al., 2012). Still, these studies are uncertain in establishing the crucial function of each of these factors in the resolution of infection (Skariah et al., 2017). No vaccine has yet proven effective against Babesia infection (Man et al., 2017). More importantly, the limited knowledge of the mechanisms of immunity and pathogenesis in babesiosis causes hindrance to the development of effective preventive and therapeutic interventions (Vos & Bock, 2006; Rathinasamy et al., 2019).

Co-infection, or mixed infection, is a type of infection wherein a host is concomitantly infected with two or more pathogens (Cox, 2001). Co-infections can be inconsequential, deleterious, or even beneficial, and have complex interactions, including modulation of the host response (McArdle et al., 2018). The actual clinical picture of concomitant infections involves diverse microorganisms that have an influence not only on each other but also on the host (Lundqvist et al., 2010). However, existing knowledge is limited on which co-infections are of importance to our health (McArdle et al., 2018). In the context of babesiosis, simultaneous infection with two Babesia species has been recorded in humans (Mayne, 2014), animals (Young et al., 2019), and tick vectors (Steiner et al., 2008). However, information concerning the prevalence of co-infections is scant (Moutailler et al., 2016). Unlike the Plasmodium species, the pathogenesis of Babesia infection and co-infections remains understudied; hence, it is poorly understood despite its significant impact on human health. Previously, immunity to babesiosis had been demonstrated in a phenomenon referred to as heterologous immunity. To the best of our knowledge, only a few studies have investigated B. microti and B. rodhaini co-infections experimentally using mouse models (Cox & Young, 1969; Zivkovic et al., 1984; Inoue et al., 1994; Li et al., 2012; Wang et al., 2016). Babesia microti provided cross-protective immunity against B. rodhaini (Li et al., 2012). During the chronic stage of the Babesia co-infection, significant decrease in antibody levels and blood cytokine levels in mouse sera were observed. In addition, macrophages were identified as the immune cell primarily responsible for the conferment of cross-species protection against a subsequent lethal challenge (Li et al., 2012). However, whether the same immune dynamics occur during acute Babesia co-infection is not known.

In the present study, we highlighted the importance of investigating concomitant acute co-infections and a better understanding of immune dynamics involved in the protective immunity induced post-B. rodhaini lethal challenge during acute primary babesiosis in mice.

To date, the immune mechanisms responsible for protection against Babesia are not well characterized (Aguilar-Delfin et al., 2003). Due to a co-infecting agent, an immune response to one organism may either synergistically or antagonistically induce trickle-down effects related to the infection process (Homer et al., 2000). By using a murine co-infection model (Li et al., 2012), we have described the immune response during the acute phase of Babesia infection. In this study, we investigated the influence of B. microti acute co-infection on B. rodhaini on the development of parasitemia, immune response, disease manifestations, hematology, the role of splenic immune effector cells, and cytokine kinetics.

At first, we aimed to confirm the previously established protection seen during Babesia co-infection and zoom into specific clinical and hematologic characteristics. We illustrated that B. microti primary infection attenuated parasitemia peak and slightly exacerbated anemia in co-infected mice. In contrast, the Br mice which received no primary infection manifested a rapid increase in parasitemia and exacerbated anemia (Wang et al., 2016). Thus, our current findings verified that the previously demonstrated cross-protection exhibited during the chronic B. microti-B. rodhaini co-infection (Li et al., 2012) also occurs and commences at a specific time point during the acute stage of co-infection. The current results demonstrate that cross-species protection against B. rodhaini arising from a prior B. microti infection can be observed in challenge-infected mice as early as day 4 post-primary infection. Within the host, B. microti infection exists in three phases: establishment, progression, and resolution (Knapp & Rice, 2015). After the establishment of infection, Babesia damages the host’s red blood cells (Dvoráková & Dvorácková, 2007). During the progression phase, damage to the erythrocytes is caused by the parasite’s egress (Sevilla et al., 2018), followed by the lysis of the host cell eventually leading to anemia and reticulocytosis (Homer et al., 2000; Kjemtrup & Conrad, 2000), which were both observed in infected mice from this study. The density of erythrocyte decreases as a consequence of the egress (Sevilla et al., 2018). It is postulated that the rupture of the plasmatic membrane of the erythrocyte is potentially accompanied by the release of hemoglobin during the course of the egress process. This may explain the hemolysis and hemoglobinuria in babesiosis (Zintl et al., 2003; Gray et al., 2010; Hildebrandt et al., 2013; Narurkar et al., 2017).

Previous studies of malaria established that parasites show tropism for mature erythrocytes. In the same way, B. microti also shows obvious tropism for mature erythrocytes (Borggraefe et al., 2006), as evident in the current IFA results where B. microti and B. rodhaini parasites infecting reticulocytes were rarely observed. This decline in the mature erythrocyte population during parasite infection leads to curtailed parasitemia (Okada et al., 2015), which may have contributed to the parasitemia levels plummeting right after its peak in Babesia co-infected mice. This tropism towards mature erythrocytes might be due to parasite-infected reticulocytes being preferably killed than those infecting mature erythrocytes (Skariah et al., 2017). Similarly, we found the remains of dead B. microti (crisis forms) appeared inside erythrocytes (Clark et al., 1975), as revealed by our results. Crisis forms of parasites are punctuated and degenerating parasites are induced by crisis form factor (CFF) and immune response (Sow et al., 2015). The presence of crisis forms of Babesia and the occurrence of reticulocytosis, with the reduced mature peripheral erythrocytes population, may be a potential mechanism for controlling Babesia infection.

In this study, mice infected with B. rodhaini produced significantly higher autoreactive IgGs compared with co-infected groups at day 6 pci, which coincided with severe hemolytic anemia. During the establishment stage, antibodies play a role in preventing erythrocyte infection either by binding the free sporozoites (Homer et al., 2000; Leitner et al., 2020) or by promoting phagocytosis of merozoites during the resolution phase, facilitating clearance of infected RBCs by phagocytic cells through a mechanism called antibody cell-dependent inhibition (ADCI) (Marsh and Kinyanjui, 2006). Severe anemia, which developed from extravascular hemolysis and enhanced erythrocyte accumulation in the spleen, may have been partially brought about by anti-erythrocyte autoantibodies. However, it is not clearly understood how these autoantibodies directly affect the pathogenesis of babesiosis (Chiou et al., 2014). Our results also showed a decline in antibody production during the acute co-infection phase, similar to a previous study (Li et al., 2012). Likewise, subversion of the adaptive immune response against B. microti, as seen in various co-infected Babesia models (Djokic et al., 2018b; Djokic et al., 2019; Efstratiou et al., 2020), may have been implicated in the specific immune response recorded in this study. In contrast, a study by Yi et al. (2018) demonstrated that despite disorganized splenic architecture during infection, B. microti-infected mice were able to elicit a strong adaptive immune response. It is proposed that cells of the innate immune system regulate the growth rate of the merozoites and thus, it affects the rate of parasitemia (Homer et al., 2000). Although antibodies block the invasion of merozoites, they do not play any significant part in opsonophagocytosis of Babesia- or Plasmodium-infected erythrocytes (Djokic et al., 2021).

Spleen is the prime location where regulation of detrimental immune responses take place (Bronte and Pittet, 2013). It is a vital organ involved in clearing infected RBCs, and malaria infection can result in the rupture of the spleen (Lacerda-Queiroz et al., 2017). Previous B. microti-B. rodhaini co-infected models did not provide any information regarding the analysis of spleen histopathology and splenocyte composition. Therefore, it was imperative to shed light on the situation of the spleen during the acute stage of Babesia co-infection. In humans and murine malaria, disorganization of splenic architecture, as well as splenomegaly, were attributed to innate immune activation, extension of monocytic cells, and elimination of infected RBCs (Weiss, 1989; Weiss et al., 1989; Villeval et al., 1990). The alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during malaria infection and could impact the development of immunity to malaria (Cadman et al., 2008). As B. microti initiates early phase activation of macrophages, this eventually leads to curbing of the replication of parasites (Terkawi et al., 2015). Subsequent studies established that B. microti confers immunity against other Babesia (Li et al., 2012) and Plasmodium (Efstratiou et al., 2020), which is primarily based on macrophages. Our FACS analysis results support this finding as depicted by the significant increase in splenocyte macrophage population and were confirmed by observations from the histopathology of spleen sections of co-infected mice. On the other hand, the decline in B and T cell populations noted in this study is consistent with other studies (Djokic et al., 2018a; Djokic et al., 2019). These results emphasize the pivotal role of innate immunity in the acute phase of Babesia co-infection in mouse models. During acute infection, there is vigorous activation of mononuclear cells resulting in apoptosis of monocytes, dendritic cells, B and T cells, leading to apoptosis of the thymus and ultimately slumping the output of naive T cells (Rénia and Goh, 2016). Contrarily, during reinfection, chronically activated T cells can induce a state of anergy and exhaustion, which may have affected the T and B cells splenocyte population in co-infected mice in the present study.

Cytokines drive the symptoms, pathological remodeling, and the consequences of the infection, which rely on the reciprocal regulation of the pro- and anti-inflammatory cytokines. Previously, B. microti-infected mice during the acute stage had decreased cytokine production in response to lethal B. rodhaini infection (Li et al., 2012). Our current findings indicate that the timing of B. rodhaini challenge during B. microti acute infection stage is crucial in the inflammatory cytokine response post-challenge, as evidenced by the cytokine levels in co-infected groups bm/br4 and bm/br6. Higher levels of cytokines could potentially allow parasite clearance during peak parasitemia (Bronte & Pittet, 2013; Djokic et al., 2018a). Thus, the role of cytokines as savior or foe in the pathogenesis of babesiosis is a time-dependent phenomenon (Ahmed, 2002).

IFN- γ (Koch et al., 2005) and IL-10 (Kumar et al., 2019), which have the ability to act as mediators of immunity, are classic examples of double-edged sword cytokines. Cross-protected mice showed lower levels of IFN- γ and IL-10, whereas Br mice showed higher levels. During blood-stage malaria, one of the pathological roles of IFN-γ is its contribution to anemia by suppressing erythropoiesis (Okada et al., 2015). As observed in both humans and mice, early production of IFN-γ leads to protection from experimental cerebral malaria (King & Lamb, 2015), while lack of early production of IFN-γ results in fatal infection (Stevenson & Riley, 2004). In both Br and bm/br4 groups, peak parasitemia and severe anemia were observed at day 6 pci. At the same time, B. rodhaini infection extensively damaged the spleen of mice from both groups, with dramatic reduction of cell population, especially in the marginal zone. These high pathologies may have arisen from elevated levels of circulating IFN-γ during infection. Despite comparable levels of IL-10 serum levels in Br group and bm/br4 group at day 6 pci, Br mice succumbed to the infection, while the bm/br4 group survived. IFN- γ and TNF-α overproduction is limited by IL-10 during B. microti infection (Djokic et al., 2018a). This scenario was also observed in our study, wherein at day 6 pci, the elevated IL-10 levels may have contributed to the relatively lower levels of IFN- γ and TNF-α in bm/br4 compared with the Br group, which became central in the survival of co-infected mice. Similarly, lethality of malaria is thought to be due to the overproduction of IL-10 in the early stage of infection (Li et al., 2012). Regardless of the origin of IL-10, it represses the function of macrophage and dendritic cells, thereby restricting Th1 and Th2 effector responses. Additionally, the production of IL-12 by monocytes is repressed by IL-10 (D’Andrea et al., 1992), which is necessary for induction of a protective immune response against malaria and shifting to a pro-inflammatory cytokine response (Crutcher et al., 1995; Xu et al., 2001). Nonetheless, the consequence of IL-10 depends on time and location and is influenced by the type of cell producing it (Kumar et al., 2019). Thus, it is possible that in the bm/br4 group, lower production of IFN-γ at day 6 pci may have slightly lowered the parasitemia, and ultimately prevented lethality. IFN-γ is recognized as a crucial cytokine that mediates splenic cell death and prompts host lethality after Plasmodium infection (Lacerda-Queiroz et al., 2017). Although Br and bm/br4 both groups had proportional levels of IFN-γ, decreased loss of splenic cells in the bm/br4 group may be attributed to the cross-protection immunity conferred by B. microti. Notably, FACS analysis showed the lowest T cell population in the bm/br6 group (12.82%), which may explain the low levels of IFN-γ at day 6 pci as Th1 cells are one of the major sources of IFN- γ.

It was suggested that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-γ, and induction of macrophage-derived effector molecules like NO (Aguilar-Delfin et al., 2003). IL-12 plays an indispensable role in enhancing Th-1-associated immunity (Trinchieri, 1993). Early production of IFN-γ by NK cells is dependent upon IL-12 from DCs. Additionally, early induction of IFN- γ and IL-12 mediates survival and leads to protection against babesiosis (Li et al., 2012; Efstratiou et al., 2020). Moreover, Th-1-associated immunity is important in controlling blood-stage replication of Babesia and Plasmodium parasites (Li et al., 2012; Djokic et al., 2018a). Thus, IL-12 is a potent mediator of host-defense mechanisms in various experimental malaria models (Luty et al., 2000; Malaguarnera et al., 2002). Our results corroborate previous findings wherein IL-12 levels were lowest in control Br mice, while highest in bm/br6 mice, reflecting its role in the provision of immunity against fatality.

For the activation of immune cells, intracellular ROS is one of the messengers in the signaling pathway. Ty et al. (2019) showed a mechanism wherein activation of macrophages occurs by oxidative stress leading to the release of inflammatory cytokines. On the other hand, Li et al. (2012) and Johnson et al. (1996) also proposed that macrophages may kill parasites via reactive oxygen and nitrogen intermediates, and depending on the quantity and site of production, they may insinuate favorable or detrimental influence on malaria. Reactive oxygen and nitrogen intermediates are shown to be responsible for the resultant crisis forms of Plasmodium and Babesia. A previous study demonstrated that inoculation of NO caused lysis of erythrocytes instead of killing P. berghei, concluding that Plasmodium parasites are protected from ROS by hemoglobin released by hemolysis, but the parasite probably contains intrinsic defense mechanisms against ROS (Sobolewski et al., 2005). Macrophages produce a huge amount of ROS and RNS triggering oxidative stress (Percário et al., 2012). However, there is uncertainty regarding the role of oxidative stress during malaria. According to some authors, oxidative stress exerts protective effects, whereas others stated that it contributes to the pathophysiology of malaria. Reactive oxygen and nitrogen species (ROS and RNS), in association with oxidative stress, are responsible for systemic complications caused by malaria. When residing in host cells, Plasmodium parasites are prone to elevated levels of oxidative stress. In order to defend themselves, these parasites evolved ways to evade this stress in certain ways; one of them is the antioxidant defense mechanism (Percário et al., 2012). Malaria causes the induction of a “cytokine storm” with the detection of high levels of cytokines in the bloodstream (Clark, 2007; Clark et al., 2008). The mechanism by which ROS triggers inflammation during malaria is by the xanthine oxide-produced ROS giving rise to elevated levels of IL-1β, while the parasites activate caspase-1, causing the activation of the NLRP3 inflammasome (Ty et al., 2019). Hence, a new function of extracellular ROS was proposed for the induction of inflammatory cytokines and complementing with an infectious parasite for the activation of the inflammasome in macrophages. Curbing this pathway via hampering ROS may open new avenues for designing anti-disease remedies where pathology is contributed by oxidative stress-driven inflammation (Ty et al., 2019). As depicted by our results, the highest levels of ROS in Br mice could have possibly backfired on the host, contrary to protecting by fighting against the parasite, resulting in lysis of erythrocytes which contributes to the pathology of B. rodhaini.

The current study holds some weaknesses as well. In our research, we did not perform the immunohistochemistry analysis of the spleen and immunophenotyping of immune cells in blood to corroborate the splenic cell population results, which would have further strengthened our concept about immune effector cells. Furthermore, the involvement of ROS in erythrocyte lysis needs to be confirmed. As ROS may adversely affect the kidneys, it is necessary to check how this organ is affected during babesiosis. In the backdrop of this missing information, we shall have a more accurate and deeper understanding of the immune mechanism involved during co-infection, which may be helpful to design effective vaccines in the future. The way forward is to address these shortcomings in future studies.

Our study demonstrated that during co-infection of mice with Babesia at certain time points in the acute stage, activation of the innate immune response by B. microti attenuated B. rodhaini parasitemia and led to the survival of co-infected mice. Our findings revealed that B. microti undermined the adaptive immune response through decreased splenic B and T cell populations, which was mirrored by the curtailed humoral immunity against both parasites. As a consequence, Th1 immune response dominated, with IL-10 and IFN- γ appearing to play major roles in the pathology of B. rodhaini.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

All the experimental animal procedures were carried out in strict compliance with the protocols approved by the Research Ethics Review Committee of the Obihiro University of Agriculture and Veterinary Medicine (animal experiment permit: 21-133; pathogen experiment permit: 201709-7; recombinant protein protocol: 1723-5), following the guidelines of the Standards Relating to the Care and Management of Experimental Animals promulgated by Obihiro University of Agriculture and Veterinary Medicine.

IZ, EMG, and XX designed the study. IZ and EMG performed all experiments. DK conducted the histopathological analysis. JL, SJ, ML, YL, and YH assisted in the laboratory experiments. IZ and EMG analyzed the results and drafted the manuscript. JZ and XX revised the manuscript. All authors proofread, reviewed, and approved the final version of the manuscript.

EMG is supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science (JSPS), Japan (20J20134). This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18F18089), the JSPS Core-to-Core program, and a grant from the Strategic International Collaborative Research Project (JPJ008837) promoted by the Ministry of Agriculture, Forestry, and Fisheries of Japan.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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