Babesia microti Infection Changes Host Spleen Architecture and Is Cleared by a Th1 Immune Response

Abstract

Babesia microti is a malaria-like parasite, which infects ∼2000 people annually, such that babesiosis is now a notifiable disease in the United States. Immunocompetent individuals often remain asymptomatic and are tested only after they feel ill. Susceptible C3H/HeJ mice show several human-like disease manifestations and are ideal to study pathogenesis of Babesia species. In this study, we examined parasitemia of B. microti at different time points and assessed its impact on hemoglobin levels in blood, on spleen pathology and overall immune response in C3H/HeJ mice. Peak parasitemia of 42.5% was immediately followed by diminished hemoglobin level. Parasitemia at 21 days of infection was barely detectable by microscopy presented 5.7 × 10⁸ to 5.9 × 10⁹B. microti DNA copies confirming the sensitivity of our qPCR. We hypothesize that qPCR detects DNA released from recently lysed parasites or from extracellular B. microti in blood, which are not easily detected in blood smears and might result in under-diagnosis of babesiosis in patients. Splenectomized patients have been reported to show increased babesiosis severity and result in high morbidity and mortality. These results emphasize the importance of splenic immunity in resolution of B. microti infection. Splenomegaly in infected mice associated with destruction of marginal zone with lysed erythrocytes and released B. microti life forms in our experiments support this premise. At conclusion of the experiment at 21 days post-infection, significant splenic B and T cells depletion and increase in macrophages levels were observed in B. microti infected mice suggesting a role of macrophage in disease resolution. Infected mice also showed significantly higher plasmatic concentration of CD4 Th1 cells secreted cytokines such as IL-2 and IFN-γ while cytokines such as IL-4, IL-5, and IL-13 secreted by Th2 cells increase was not always significant. Thus, Th1 cells-mediated immunity appears to be important in clearance of this intracellular pathogen. Significant increase in IL-6 that promotes differentiation of Th17 cells was observed but it resulted in only moderate change in IL-17A, IL-17F, IL-21, and IL-22, all secreted by Th17 cells. A similar immune response to Trypanosoma infection has been reported to influence the clearance of this protozoan, and co-infecting pathogen(s).

Keywords: Babesia microti; babesiosis; blood-borne pathogen; immunosuppression; protozoan pathogenesis; tick-borne infection.

FIGURE 1

Comparison of diagnostics by IFA, FISH and Microscopy for babesiosis with qPCR using molecular beacon probes for BmTPK in human blood samples. For all patients samples, qPCR was repeated three times and results of each repeat are marked by ‘+’ or ‘–’ sign. Thus, +/+/+ indicates that qPCR was positive in all three repeats of the qPCR for an individual sample. There is a high correlation between all three tests when samples were negative by qPCR. Higher sensitivity of detection of B. microti by qPCR is demonstrated by a significant number of positive samples that were diagnosed negative for babesiosis by Indirect IFA, FISH and microscopic examination of Giemsa-stained blood smears.

FIGURE 2

Course of B. microti infection and detection of parasites in blood of susceptible C3H/HeJ mice. (A) Increase in parasitemia resulted in a dramatic drop in hemoglobin levels reaching the lowest levels 1 day after peak B. microti parasitemia was obtained. Recovery of hemoglobin was rapid after decline in parasitemia. Parasitized erythrocytes were barely detectable by microscopy on 21st day of infection. (B) High copy numbers of B. microti DNA were present in blood of the infected animals at this stage of infection. (C) Several extracellular, released parasites can be observed in Giemsa stained blood smear of infected mice supporting our qPCR results. (D) The presence of external, released green fluorescent B. microti (marked by arrows) as well as intracellular parasites (marked by arrowheads) were observed by IFA of the blood smears confirming Giemsa stained blood smears results shown in (C).

FIGURE 3

Impact of B. microti infection on spleen of the infected mice. (A) B. microti infection resulted in a significant increase in spleen sizes of mice compared to uninfected, naïve mice. (B) White and red pulp zones were significantly enlarged while demarcation zone was not apparent in the spleen of B. microti infected mice. Numbers of lysed erythrocytes (marked by arrowheads) as well as various free parasitic forms (marked by arrows) were also observed in infected mouse spleen. (C) In IFA conducted the spleen section, red color indicates auto-fluorescence of RBCs, blue shows nuclear staining of cells while green fluorescence marks B. microti probed with infected human plasma followed by detection with Alexa fluor 488 conjugated secondary antibodies. Apparent lysed infected erythrocytes presence is marked by a circle. Several free, released parasitic forms were also detected (marked by arrows) among erythrocytes. Bars represent 25 μm.

FIGURE 4

Increased cytokine levels in mice after 21 days of infection. (A) Highest difference between B. microti infected and uninfected, naïve mice was observed in concentration of IL-2 (99.7 pg/ml compared to 80 pg/ml, p < 0.0005). (B–D) Concentrations of TNF-α (95.2 pg/ml), IL-6 (119.8 pg/ml) and IL-10 (207.1 pg/ml) were significantly higher in infected mice relative to control group (p < 0.005, <0.01, <0.01, respectively). (E–I) IFN-γ, IL-4, IL-13, IL-17F, and IL-22 of B. microti infected mice, showed modest but statistically significant increase in infected mice plasma relative to the naïve animals (p < 0.05 each; 95% CI). (J–L) Concentrations of IL-5, IL-17A, and IL-21 were not significantly different between infected and uninfected mice. Comparisons were made between groups using unpaired student two-tailed t-tests for unequal variance at 95% Confidence Interval (CI) (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005).