Tick-borne diseases have been increasing steadily in the last two decades in North America and Europe that include bacterial, viral and parasitic pathogens. Transmission of multiple pathogens by a single tick bite has been reported to occur (Dunn et al., 2014; Rizzoli et al., 2014; Moutailler et al., 2016). Babesia species are tick-transmitted parasites with the most cases of babesiosis attributed to Babesia microti in the United States, with some patients show infection with B. duncani in Western states, while B. divergens is primarily responsible for babesiosis in Europe (Centers for Disease Control and Prevention, 2012). Approximately 2000 cases of babesiosis occur in the United States every year, which resulted in its declaration as a notifiable disease by CDC in 2011. B. microti infection affects the Northeastern United States; Connecticut, Massachusetts, New Jersey, New York, and Rhode Island along with the Midwestern states of Wisconsin and Minnesota (Knapp and Rice, 2015). Together they represented 94% of babesiosis cases in the United States in 2014.

Babesia species are intracellular parasites of red blood cells (RBCs), where they multiply until erythrocytes burst. Clinical disease ranges from asymptomatic or mild flu-like symptoms in immunocompetent people, including fever and myalgia, to acute or sometimes fatal disease in immunocompromised or splenectomized individuals (Genda et al., 2016). Patients are often tested for babesiosis only after observation of hemolytic anemia. Babesia species can also transmit through transplacental route and can cause jaundice, anemia and neutropenia in children (Joseph et al., 2011, 2012; Luckett et al., 2014). Immunocompetent individuals can establish Babesia carriage state without any clinical manifestations for long periods of time. Due to survival of these parasites during cold storage, infected donated whole blood and blood products can cause blood transfusion transmitted babesiosis (TTB) that have severe consequences in the immunocompromised patients (Herman et al., 2010; Herwaldt et al., 2011; Sinski et al., 2011; Cushing and Shaz, 2012; Cursino-Santos et al., 2014; Fang and McCullough, 2016). Babesiosis is the most prevalent transfusion transmitted disease in the United States such that FDA has recently recommended that blood donors should be screened for Babesia infection (Lobo et al., 2013; United States Food and Drug Administration, 2014).

Current confirmatory test for babesiosis is microscopic examination of Giemsa-stained blood smears. However, this method is time consuming and requires a specific expertise for correct diagnosis because pleomorphic, non-synchronous trophozoites and ring forms can make Babesia identification difficult. This method is also very labor intensive. Therefore, it cannot be used for large scale testing of donated blood samples. Some diagnostic laboratories employ FISH for diagnosis of babesiosis that also requires microscopic examination. Serologic tests are more sensitive and efficient; however, these tests cannot detect acute disease before the adaptive immune response is established and are also unable to distinguish active disease from the past infections or reinfections, thus, creating a major diagnostic problem particularly in the endemic regions (Magnarelli et al., 1998; Foppa et al., 2002; Hunfeld et al., 2002; Yoshinari et al., 2003; Fox et al., 2006; Vannier et al., 2008; Lempereur et al., 2015; Mayne, 2015; Wormser et al., 2015). Nucleic Acid Tests have been used for examination of both humans and ticks and have more promise for diagnosis of babesiosis including blood donors (Bose et al., 1995; Krause et al., 1996a; Cushing and Shaz, 2012; Michelet et al., 2014; Jahfari et al., 2016). We found that our, molecular beacon probes based real-time quantitative Polymerase Chain Reaction (qPCR) test, identifies patient samples with babesiosis with greater sensitivity, in a tick-borne diseases endemic state, New Jersey.

White footed mouse is natural reservoir of B. microti in the United States. Susceptible C3H mice show several manifestations like humans and offer ideal animal model system to investigate pathogenesis of this protozoan parasite (Ruebush and Hanson, 1979). Observation of pronounced splenomegaly in B. microti infected C3H/HeN mice as well as higher parasitemia levels and delayed clearance of B. microti from blood stream of splenectomized mice indicate importance of spleen immunity in resolution of babesiosis even in mice (Coleman et al., 2005). B. microti parasitemia also reduced in Severe Combined Immunodeficient (SCID) mice by adoptive transfer of splenocytes from naïve immunocompetent mice, further emphasizing the role of adaptive immune response facilitated by splenic cells (Vannier et al., 2004). However, a systematic study to assess the consequence of B. microti infection on mice together with both plasmatic and splenic immunity has not been carried out until now. Therefore, B. microti pathogenesis and immunological response to this intraerythrocytic pathogen remain poorly understood. Involvement of innate immunity determined by macrophage during B. microti infection was suggested to play the primary role in babesiosis resistance in mice (Aguilar-Delfin et al., 2001; Terkawi et al., 2015). Adoptive transfer of primed T cells was reported to control mice reinfection by B. microti likely due to also simulating primed B cell response (Meeusen et al., 1985). Furthermore, CD4 and not CD8 cells were shown to participate in elimination of B. microti in mouse infection model (Hanafusa et al., 1998; Igarashi et al., 1999; Hemmer et al., 2000a). Interestingly, human Babesia WA1 parasites infection in mice resulted in upregulation of both TNFα and IFNγ resulting in severe pathogenesis and fatal disease (Hemmer et al., 2000b). Surprisingly, anti-IFNγ monoclonal antibodies partially reduced protection of mice immune against B. microti and IFNγ-deficient mice did not offer any protection against B. microti infection (Igarashi et al., 1999).

We followed the multiplication cycle of B. microti in blood of C3H/HeJ mice and assessed the impact of parasitemia increase on hemoglobin levels in blood, on spleen pathology and on overall splenic and plasmatic immune response. Our studies demonstrate that innate immune response particularly due to macrophage participation and adaptive immune response triggered by stimulation of CD4 cells play critical roles in elimination of parasitized erythrocytes during infection of mice with B. microti. Furthermore, we also describe potential reason for under detection of B. microti during human infection.

Babesia microti infection of humans has become very prominent in the last decade, both in North America and Europe (Hersh et al., 2014; Rizzoli et al., 2014; Knapp and Rice, 2015; Diuk-Wasser et al., 2016). In majority of immunocompetent individuals, infection remains unseen, with mild, flu-like symptoms or diminished hemoglobin levels and anemia. However, in combination with other tick-transmitted diseases such as Borrelia burgdorferi even in healthy person this parasite can intensify clinical disease and its severity (Krause et al., 1996b; Knapp and Rice, 2015). Babesiosis has tremendous consequences in the immunocompromised individuals and infants and can even be fatal (Usmani-Brown et al., 2013; Menis et al., 2015; Akoolo et al., 2017). Equally dangerous is newly identified route of transmission of B. microti, via blood transfusion. In these cases, immunocompetent donors are undiagnosed and remain Babesia carriers such that blood recipients who are often immunocompromized individuals can exhibit high morbidity and mortality (Herwaldt et al., 2011; Cushing and Shaz, 2012; Kitt et al., 2016).

Several diagnostic tests are available for diagnosis of babesiosis; however, blood smear test is still considered gold standard by CDC and WHO (Clinical and Laboratory Standards Institute, 2000; Akoolo et al., 2017). One reason is that antibodies can persist even more than a year after the clearance of Babesia infection (Vannier and Krause, 2012). On the other hand, immunocompromised or immunodeficient individuals can remain seronegative, although they could be carrier of infectious parasites, in some cases for more than 2 years (Raffalli and Wormser, 2016). PCR, as a diagnostic test for B. microti infection, was first described by Persing et al. (1992), which could detect as little as three merozoites per microliter of blood (Persing et al., 1992). Since then, method has been improved greatly. Our laboratory in 2013 developed multiplex PCR method based on molecular beacons probe for BmTPK amplicon, which significantly improved specificity and sensitivity of the assay (Chan et al., 2013) We later also employed this test to examine human samples from endemic zone for tick-borne diseases (Akoolo et al., 2017). For majority of human samples, we were unable to determine time of the start of infection and thus, failed to determine the stage of infection in patients.

During B. microti infection, different strains of mice display majority of symptoms noticed in human patients (Vannier et al., 2004; Coleman et al., 2005; Skariah et al., 2017). Therefore, the major objective of this study was to examine if B. microti infection cycle in TLR4 deficient C3H/HeJ mice follows the same pattern and assess the impact of parasitemia on the mammalian host pathology and immune response. Our qPCR result showed very high levels of parasitic DNA in blood of mice 21 days post infection while examination of blood smears by microscopy demonstrated barely detectable parasitemia. A follow-up careful examination of slides indicated that although parasitized erythrocytes were not detected readily, released parasite representing various life stages of B. microti could be observed, thus explaining our qPCR results. These findings are similar to culture of B. divergens after growth synchronization by Cursino-Santos et al. (2016). In vitro culture of B. microti and B. duncani is not established yet (Skariah et al., 2017). Although we succeeded in B. microti in vitro culture for some passages when active parasites were used for infection, parasites in the absence of ring-forms in RBCs, depicting active B. microti, failed to infect erythrocytes. However, by reviewing blood smears in available literature, different life stages of Babesia species could be observed outside the erythrocytes (Healy and Ruebush, 1980; Shih et al., 1997; Kjemtrup and Conrad, 2000; Saito-Ito et al., 2000; Vannier et al., 2008; Sun et al., 2013). These extra-erythrocytic parasites could be mere remnants of passing parasites or exhibit still active infection. Infectivity of these parasites was not determined.

Involvement of spleen in resolution of babesiosis in humans is well established (Vannier et al., 2004; Coleman et al., 2005; Genda et al., 2016). In our experiments, spleens of all infected mice were significantly enlarged, likely due to its hyperactivity during B. microti infection because of increased hematopoietic support and macrophages activity resulting in erythrophagocytosis (Homer et al., 2000). In B. microti infected mice, various parasitic forms were distinguishable even in spleen sections stained with Hematoxilin-Eosin. Although parasitemia was undetectable by microscopy at 21 days of infection, detection of B. microti DNA by our qPCR supports possibility of continuing low-level infection. Our analysis of white blood cells populations in spleen at this time point showed that both CD19+ B, and CD3+ T cell populations were depleted significantly while proportion of macrophages increased. Despite subversion of splenic adaptive immune response, mice were able to clear B. microti parasitemia in blood. Supporting our results, contribution of macrophage in clearance of B. microti in mice has been noted previously (Terkawi et al., 2015).

To determine the existence of potentially active infection by B. microti during apparent convalescent period when certain level of immune response is maintained, we analyzed concentrations of plasmatic cytokines. We found higher concentration of IL-6, IFN-γ, and TNF-α that have been shown to be the major pro-inflammatory cytokines produced in response to B. microti infection (Igarashi et al., 1999; Hemmer et al., 2000b). Increased cytokines production during babesiosis in patients could be responsible for febrile illness and even myalgia. High concentrations of IL-4 and IL-10, likely produced by regulatory B cells, play a major role in disease resolution but could also indicate still active B. microti infection (Hemmer et al., 2000a; Jeong et al., 2012). In fact, production of anti-inflammatory IL-10 in B. microti infected mice was suggested to be important for survival of the host (Hemmer et al., 2000a). Increase in cytokines levels agree with our qPCR results that detected parasites in blood. Thus, inability to detect parasitemia in patients based upon infected RBCs presence appears to erroneously leave out infectious but released and free B. microti parasites. Thus, our studies follow the infection cycle of B. microti and provide both direct (qPCR and microscopy) and indirect (increase in the specific plasmatic cytokines) evidence of active infection in host despite apparent resolution of babesiosis as detected by parasitemia measurement.

Involvement of CD4 T cells, and IFNγ cytokine has been shown to confer protective immunity against various protozoan pathogens during mammalian infection (Favre et al., 1997; Igarashi et al., 1999; Hemmer et al., 2000a; Hoft et al., 2000; Wang et al., 2004; McCall and Sauerwein, 2010). Previous studies showed that although not essential, CD4 T cells and IFNγ also play important roles in protective immunity against B. microti infection in mice (Igarashi et al., 1999; Hemmer et al., 2000a,b; Skariah et al., 2017). Significantly high levels of IL-2, TNFα, and IFNγ in infected mice could be produced by activated CD4 helper (Th), primarily Th1 cells potentially in response to antigens of B. microti they encounter, indicating that cell mediated immunity is very important for clearance of the parasites from blood. Previously reported persistence of B. microti parasitemia in CD4-/- mice supports this hypothesis (Skariah et al., 2017). Increase in the plasmatic levels of IL-4, IL-5, and IL-13 produced by Th2 cells was not as significant. These results suggest relatively less critical role of humoral immunity in resolution of babesiosis because Th2 cells support stimulation of antibody production. Interestingly, the levels of IL-6 and IL-10 cytokines, which are also produced by Th2 cells, were found to increase significantly and to a much higher levels in the infected mice. However, IL-6 and IL-10 are also produced by activated macrophage and B cells also produce IL-10 (Williams et al., 2000). High levels of IL-10 in serum of B. microti-infected mice have also been reported previously (Jeong et al., 2012). Increase of 5–10-fold levels in TNFα production in response to human Babesia WA1 strain later in infection, suggested to be produced by CD8+ T cells, and concomitant decrease in IL-10 levels was occurred together with fatal disease in mice (Hemmer et al., 2000a). We observed resolution of babesiosis caused by B. microti despite significant increase in plasmatic TNFα level but it was not as high as reported for WA1 strain, which could be attributed to simultaneous increased production of IL-10 and its anti-inflammatory activity. IL-10 may also suppresses overexpression of both IFNγ and TNFα while allowing enough immune response to build that is sufficient to eradicate B. microti infection (Trinchieri, 2007; Couper et al., 2008a,b; Redpath et al., 2014).

Our results further emphasize the importance of macrophage in immunity against B. microti infection. Significant increase in IL-6 that promotes differentiation of Th17 cells (McGeachy et al., 2007) was not found to be associated with downstream increase in cytokines production by Th17 cells, a unique CD4 cells. Th17 cells produce both IL-17 and IL-21, which are implicated in various inflammatory responses (Miossec et al., 2009). Unlike previous studies with Helicobacter pylori where infection causes adenocarcinoma and produces high levels of IL-17 and IL-21 (Amedei et al., 2014), only moderate increase in signature cytokines secreted by Th17 cells, such as IL-17, IL-17F, IL-21, and IL-22 (Volpe et al., 2008; Korn et al., 2009), was observed in our experiments. These results suggest that Th17 cells play only a minor role during B. microti infection and do not trigger a pronounced inflammatory response, as observed during Babesia WA1 infection. This is not surprising because Th17 cells usually are found to be more important players during diverse immune-mediated diseases. In summary, our studies confirm the roles of CD4 cells and macrophage in clearance of parasitized erythrocytes during B. microti infection and thus, contributing to resolution of babesiosis (Igarashi et al., 1999, #10007; Hemmer et al., 2000a,b; Aguilar-Delfin et al., 2003; Skariah et al., 2017).

Our studies with patients and mice samples indicate that qPCR could be effective for detection of both parasitized erythrocytes as well as free parasites released after lysis of infected RBCs. We confirm that spleen is important for resolution of babesiosis in mammalian hosts. Furthermore, increase in pro-inflammatory plasmatic cytokines, IL-6, IFN-γ, and TNF-α together with the presence of activated macrophage in both blood and spleen could help in resolution of infection with B. microti.

All authors contributed significantly to this work. NP conceived the study and VD and LA designed and conducted all experiment. LA started this study and VD analyzed data and wrote first draft of the manuscript. All authors have read and approved the manuscript for submission.

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.