Long-term survival of Babesia microti and Borrelia burgdorferi in C3H/HeJ mice and their effect on Lyme arthritis and babesiosis manifestations

Abstract

Babesia microti is increasingly co-transmitted with Lyme disease-causing Borrelia burgdorferi by ticks in the endemic regions of the United States. Infection in mice by this parasite mirrors human babesiosis, such as anemia, splenomegaly, alterations in splenic leukocyte balance, and diminished humoral immunity associated with enhanced Lyme disease manifestations at the acute phase. To evaluate the long-term survival of B. burgdorferi and B. microti in mice and their effects on pathogenesis, we conducted a 16-week infection experiment in C3H/HeJ mice. All mice infected with B. microti, irrespective of the co-infection, displayed a low-level, albeit microscopically detectable, parasitemia in both male and female mice even after 16 weeks post-infection. Splenomegaly was detected at this stage in both male and female mice and was significantly higher in females infected solely with B. microti compared to co-infected mice, likely due to a greater peak parasitemia at the acute phase of infection and persistent splenic manifestations in these mice. Interestingly, B. microti disrupted the blood-brain barrier (BBB) in mice, as reported during cerebral malaria caused by Plasmodium falciparum. Furthermore, B. burgdorferi colonization could be detected until 16 weeks of infection, while more pronounced Lyme inflammatory arthritis was observed at 4 weeks post-infection. This study underscores the complex interactions between B. microti and B. burgdorferi to affect each disease, highlighting the potential implications for vaccine development against Lyme spirochetes and therapeutic management of co-infected individuals.IMPORTANCETick-borne co-infections are becoming increasingly prevalent worldwide due to simultaneous or sequential acquisition and transmission by Ixodes species ticks during their blood meal. We reported that B. burgdorferi and B. microti co-infection reciprocally affects these pathogens during the acute phase of infection; however, the effect of co-infections on microbial long-term persistence in the murine model was not previously investigated. In this study, we have filled a critical lacuna in understanding the interactions between these two pathogens at different stages of infection and their effects on the host and disease manifestations in mice. Our investigation provides insights into their pathogenicity to allow the development of effective vaccines and successful antimicrobials against these tick-borne co-infections.

Keywords: Apicomplexan protozoan; Babesia microti; Borrelia burgdorferi; Lyme arthritis; co-infection; spirochete.

Fig 1

Co-infection experiment plan in C3H/HeJ mice and euthanasia time points. We carried out an infection experiment using 5-week-old C3H/HeJ mice for 4 months to determine if B. burgdorferi or B. microti (Bm) persists long term in co-infected rodent hosts and to ascertain their reciprocal effects on the disease’s pathogenesis at short- and long-term infection. C3H/HeJ female and/or male mice were injected with 100 µL containing 10⁴ Bm-iRBC subcutaneously (sc) in the left flank and/or infected with 10³ N40 spirochetes sc in the right flank and organized into four experimental groups: (i) N40; (ii) N40 + Bm co-infection; (iii) Bm; (iv) Naïve. Parasitemia determination by microscopy and IVIS were used at different time points to determine B. microti and N40 burdens, respectively. This experiment was conducted three times, first in both sexes of mice and then repeated two more times only in female mice to confirm the results.

Fig 2

Comparative analysis of B. microti parasitemia and splenomegaly and B. burgdorferi colonization in male and female C3H/HeJ mice. (A) C3H/HeJ female and male mice were inoculated sc in the left flank with 10⁴ Bm-infected red blood cells (iRBCs) alone or together with 10³ N40 spirochetes (inoculated sc in the right flank). B. microti parasitemia was determined in blood smears stained with Giemsa stain and by calculation of the average percentage of iRBC of total RBCs observed in 25 microscopic fields using oil immersion at 1,000×. (B) After terminal bleeding, mice were euthanized by CO₂ asphyxiation at 16 weeks post-infection. Spleens were aseptically harvested and weighed to determine splenomegaly. (C) The effect of Bm infection on N40 colonization was determined in both sexes of mice infected with N40 and/or Bm using IVIS-200 after intraperitoneal injection of 200 µL of 30 mg/mL D-luciferin at 2 weeks post-infection. The presence of B. burgdorferi N40 strain was visualized in different tissues measured by bioluminescent radiance as a semi-quantitative indicator of colonization by spirochetes. Parasitemia and splenomegaly data are represented by mean ± s.e.m. and were processed using GraphPad Software version 9.5.1. Significant differences between the two groups were tested by using two-tailed unpaired Student t-tests for unequal variances for parasitemia data and one-way ANOVA, followed by Tukey’s post hoc for analysis to evaluate splenomegaly. Statistical differences with P < 0.05 were considered statistically significant at 95% confidence interval (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Fig 3

Babesiosis-associated splenomegaly and colonization by B. burgdorferi detected by live imaging at different stages of infection in C3H/HeJ female mice. (A) C3H/HeJ female mice were euthanized by CO₂ asphyxiation at 2, 4, or 16 weeks of infection. Spleens were aseptically harvested and weighed for the determination of splenomegaly. The data represented by mean ± s.e.m. of spleen weights were processed using GraphPad Software version 9.5.1. Significant differences between the two groups were tested by using one-way ANOVA, followed by Tukey’s post hoc test. Statistical differences with P < 0.05 were considered significant at 95% CI (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (B) To determine the effect of the Bm infection on N40 colonization, live imaging of C3H/HeJ female mice groups infected with N40 and/or B. microti was determined using IVIS-200 (PerkinElmer) after intraperitoneal (ip) injection of 200 µL of 30 mg/mL D-luciferin at 2, 4, 8, or 16 weeks pi. Based on the net radiance photon measurement, one mouse was selected from each group to represent the average bioluminescence.

Fig 4

Effect of B. microti co-infection on B. burgdorferi N40 colonization levels in female C3H/HeJ mice. (A) Live imaging of C3H/HeJ female mice infected with B. burgdorferi N40 and/or B. microti was conducted to detect bioluminescence. Quantification of radiance associated with stably bioluminescent N40 colonization is presented by measuring net radiance (photons/s/cm²/sr) in five mice from each group obtained after deducting values from naïve mice. (B) Burden of B. burgdorferi was determined in joints of infected mice by employing duplex qPCR using recA gene of spirochetes and nidogen gene of mice for quantification using specific molecular beacon probes for each gene. In both panels A and B, data were averaged, and standard errors determined the range of radiance obtained at different time points. Significant differences between the two groups were tested by using two-tailed unpaired Student t-tests for unequal variances. Statistical differences were considered statistically significant at 95% CI and are marked by asterisks (NS, not significant, *P < 0.05).

Fig 5

B. microti infection disrupts BBB integrity and B. burgdorferi colonization in dura mater persists irrespective of co-infection with B. microti. (A) To evaluate the integrity of the BBB, penetration of Evans blue dye was evaluated at 2 weeks pi. The BBB disruption is indicated by blue color remaining in the brain even after perfusion with PBS. The brains were then immersed in formamide solution for 48 hours at 37°C to release Evans blue dye, which was quantified in 96-well plates by optical density measurement at 620 nm with a spectrophotometer. A standard curve for Evans Blue with known values was determined to calculate the weight of dye (mg)/formamide (mL). The graph bars at the right side of panel A represent mean ± SD of the dye (mg/mL) released in the formamide solution. Significant differences between the two groups were determined by one-way ANOVA, followed by Tukey’s post hoc test. Statistical differences with P < 0.05 were considered statistically significant at 95% CI (NS, not significant, ****P < 0.0001). (B) At 2 weeks post-infection, bioluminescent images of brains from C3H/HeJ female mice infected with N40 alone or together with B. microti were determined using IVIS-200. Mice were anesthetized with isoflurane and intracardially injected with 200 µL 30 mg/mL D-luciferin substrate. After 5 minutes, the animals were euthanized, and skulls opened for acquisition of images for bioluminescence detection in dura mater and brain.

Fig 6

B. microti co-infection enhances inflammatory Lyme arthritis at the early stages of infection. Joints from Naïve or C3H/HeJ female mice infected with B. microti, N40 alone or together with B. microti were harvested at 4 and 16 weeks of infection and prepared for histopathological analysis. Joints were first fixed with formalin, processed using the 10% EDTA-decalcification protocol, and 5-μm-thick sections were cut by a microtome and then mounted on slides for hematoxylin and eosin (H&E) staining. The joints from co-infected mice had higher leukocyte infiltration (indicated by red arrows) compared to N40 singly infected mice, which was more apparent at 4 weeks pi; however, overall histopathology scoring rates were comparable. Inflammation almost completely resolved in all N40-infected and co-infected mice at 16 weeks pi (Table 2).