Babesia microti primarily invades mature erythrocytes in mice

Abstract

Babesia microti is a tick-borne red blood cell parasite that causes babesiosis in people. Its most common vertebrate reservoir is the white-footed mouse. To determine whether B. microti invades reticulocytes, as does the canine pathogen B. gibsoni, we infected the susceptible inbred mouse strains C.B-17.scid and DBA/2 with a clinical isolate of B. microti. Staining of fixed permeabilized red blood cells with 4',6'-diamidino-2-phenylindole or YOYO-1, a sensitive nucleic acid stain, revealed parasite nuclei as large bright dots. Flow cytometric analysis indicated that parasite DNA is primarily found in mature erythrocytes that expressed Babesia antigens but not the transferrin receptor CD71. In contrast, CD71-positive reticulocytes rarely contained Babesia nuclei and failed to express Babesia antigens. Accordingly, the frequency of YOYO-1-positive, CD71-negative cells strongly correlated with parasitemia, defined as the frequency of infected red blood cells assessed on Giemsa-stained blood smears. Importantly, the absolute numbers generated by the two techniques were similar. Parasitemia was modest and transient in DBA/2 mice but intense and sustained in C.B-17.scid mice. In both strains, parasitemia preceded reticulocytosis, but reticulocytes remained refractory to B. microti. In immunocompetent C.B-17 mice, reticulocytosis developed early, despite a marginal and short-lived parasitemia. Likewise, an early reticulocytosis developed in resistant BALB/cBy and B10.D2 mice. These studies establish that B. microti has a tropism for mature erythrocytes. Although reticulocytes are rarely infected, the delayed reticulocytosis in susceptible strains may result from parasite or host activities to limit renewal of the mature erythrocyte pool, thereby preventing an overwhelming parasitemia.

FIG. 1.

Babesia antigens and DNA colocalize to mature erythrocytes but not to reticulocytes. Blood was obtained from an infected C.B-17.scid mouse. Cells were fixed in glutaraldehyde, permeabilized in Triton X-100, and treated with 100 μg/ml DNase-free RNase A. Cells were stained for the transferrin receptor CD71 (green), DNA (blue) with DAPI, and Babesia antigens (red) with a polyclonal antibody obtained from a DBA/2 mouse 3 months after it was infected with B. microti. Images were captured using the Spot Advanced software.

FIG. 2.

Nucleic acid staining is sensitive to RNase in reticulocytes but not in Babesia microti-infected erythrocytes. Blood was obtained from an infected C.B.-17.scid mouse. Upon fixation and permeabilization, whole blood cells were treated with increasing concentrations (from 3 to 300 μg/ml) of DNase-free RNase A. Cells were stained for nucleic acids with YOYO-1 and for the transferrin receptor CD71. For each RNase A concentration, control cells were exposed to an irrelevant monoclonal antibody directed against keyhole limpet hemocyanin (not shown). Since the fluorescence of control cells remained unchanged despite increasing concentrations of RNase A, thresholds (solid lines) were set at 1% of control cells untreated with RNase A. Dashed arrows mark the most intense YOYO-1 staining in CD71-negative cells untreated with RNase A. Note that the intensity of YOYO-1 staining in CD71-negative cells (lower right quadrants) was not affected by RNase A treatment. In contrast, the intensity of YOYO-1 staining in CD71-positive cells decreased as the RNase A concentration was increased. Data are representative of two separate experiments.

FIG. 3.

Babesia microti primarily resides in mature erythrocytes. Blood was obtained from C.B-17.scid mice 3 months after infection with 10⁵ pRBCs. Whole blood cells were fixed, permeabilized, and treated with 100 μg/ml DNase-free RNase A. Cells were stained for nucleic acids with YOYO-1 and for CD71. YOYO-1⁺ cells were fractionated by fluorescence-activated cell sorting (central panel). CD71⁻ cells were sorted into fractions (marked by vertical rectangles) according to their content in nucleic acids. CD71⁺ cells were sorted as a single fraction, represented by the horizontal rectangle. Fractionated cells were stained for the pan-erythroid surface marker TER119. Each fraction was examined under fluorescent microscopy, and the number of nuclei per cell (ranging from 0 to 8) was recorded for 100 cells (see inserts). The median number of nuclei per cell in CD71⁻ cells ranged from one in the fraction with the lowest YOYO-1 staining (F1) to two and three in the fractions with higher intensity, namely F2 and F3, respectively. CD71⁺ cells were rarely positive for nuclei (F4). Data are means ± SEM for cells from three mice.

FIG. 4.

Budding and multiple infections in Babesia microti-infected erythrocytes. Blood cells from Babesia-infected C.B-17.scid mice were fractionated on the basis of CD71 surface expression and nucleic acid content (see Fig. 3). Nucleic acids were stained in green (YOYO-1), whereas the surface marker TER119 was red (Alexa 594). In CD71⁺ cells (A), the uniform distribution of numerous tiny dim green dots reflected the residual RNA content despite RNase treatment. These tiny dots were not seen in CD71⁻ cells (B to G). In these cells, parasite nuclei appeared as bright large green dots. As YOYO-1 staining became brighter, the number of nuclei per cell increased (B to G). In CD71⁻ cells with intense YOYO staining, nuclei varied in number (C to G) and size (C, E, and F). Some nuclei were in close proximity, suggestive of binary fission (C to E). Other nuclei were distant from each other, suggestive of multiple infections per cell (D to F). The majority of cells in the brightest YOYO-1 staining (far right fraction) contained four or more nuclei (data not shown), thereby increasing the chances of visualizing four daughter cells arranged in a tetrad or Maltese cross (G). Images were captured using the Spot Advanced software.

FIG. 5.

Reticulocytes remain refractory to Babesia microti despite severe host susceptibility. Mice from the DBA/2 (n = 4) and C.B-17.scid (n = 3) strains were infected by intraperitoneal injection of 10⁵ pRBCs (day 0). One additional mouse of each strain served as an uninfected control. Blood samples were obtained at 2- to 4-day intervals from day 10 to day 31. (A and C) On each of these days, a drop of blood was placed on a glass slide, a thin blood smear was obtained, and nuclear material was revealed by Giemsa stain. A second drop of blood was placed in heparinized PBS. Blood cells were stained for Babesia antigens (Bab pAb), nucleic acids (YOYO-1), and CD71. (B and D) CD71⁺ cells were analyzed for nucleic acid content and Babesia antigen expression during the course of infection. For each day, staining for the uninfected mouse was subtracted from the staining for each infected mouse. Data are means ± SEM of stained cells as percentages of total counted cells. Note that virtually all CD71⁺ reticulocytes in DBA/2 and C.B-17.scid mice failed to express Babesia antigens. Despite RNase A treatment, YOYO-1 stained residual RNA in reticulocytes, as illustrated in Fig. 4A.

FIG. 6.

Frequency of YOYO⁺ CD71⁻ cells is an accurate measure of parasitemia in Babesia microti-infected mice. DBA/2 (A) and C.B-17.scid (B) mice were infected with B. microti (see the legend to Fig. 5). Infection was monitored from day 10 to day 31. Parasitemia defined as the frequency of infected red blood cells assessed by microscopic analysis of Giemsa-stained blood smears was tested for an association with the frequency of YOYO⁺ CD71⁻ cells (open squares) or Babesia antigen-positive cells (filled triangles) determined by flow cytometry. Coefficients of correlation are reported as r². Slope and origin are reported for each linear regression. Bab pAb, polyclonal antibody against Babesia antigens.

FIG. 7.

Early reticulocytosis in resistant mice but delayed and sustained reticulocytosis in the absence of adaptive immunity. C.B-17.scid (A) (n = 4), C.B-17 (B) (n = 4), BALB/cBy (C) (n = 4), and B10.D2 (D) (n = 7) mice were infected by intraperitoneal injection of 10⁵ pRBCs (day 0). One additional mouse of each strain served as an uninfected control. A drop of blood was collected in heparinized PBS at 2- to 4-day intervals from day 7 until day 91 (A to C) or day 79 (D). Blood cells were stained for nucleic acids (YOYO-1) and for CD71. For each day, staining for the uninfected mouse was subtracted from the staining of cells from each infected mouse. Data are means ± SEM of stained cells as percentages of total counted cells. Note that the scale of the y axis is larger in panel A than in panels B to D, as C.B-17.scid mice have high levels of parasitemia.