A novel animal model of Borrelia recurrentis louse-borne relapsing fever borreliosis using immunodeficient mice

Abstract

Louse-borne relapsing fever (LBRF) borreliosis is caused by Borrelia recurrentis, and it is a deadly although treatable disease that is endemic in the Horn of Africa but has epidemic potential. Research on LBRF has been severely hampered because successful infection with B. recurrentis has been achieved only in primates (i.e., not in other laboratory or domestic animals). Here, we present the first non-primate animal model of LBRF, using SCID (-B, -T cells) and SCID BEIGE (-B, -T, -NK cells) immunocompromised mice. These animals were infected with B. recurrentis A11 or A17, or with B. duttonii 1120K3 as controls. B. recurrentis caused a relatively mild but persistent infection in SCID and SCID BEIGE mice, but did not proliferate in NUDE (-T) and BALB/c (wild-type) mice. B. duttonii was infectious but not lethal in all animals. These findings demonstrate that the immune response can limit relapsing fever even in the absence of humoral defense mechanisms. To study the significance of phagocytic cells in this context, we induced systemic depletion of such cells in the experimental mice by injecting them with clodronate liposomes, which resulted in uncontrolled B. duttonii growth and a one-hundred-fold increase in B. recurrentis titers in blood. This observation highlights the role of macrophages and other phagocytes in controlling relapsing fever infection. B. recurrentis evolved from B. duttonii to become a primate-specific pathogen that has lost the ability to infect immunocompetent rodents, probably through genetic degeneration. Here, we describe a novel animal model of B. recurrentis based on B- and T-cell-deficient mice, which we believe will be very valuable in future research on LBRF. Our study also reveals the importance of B-cells and phagocytes in controlling relapsing fever infection.

Figure 1. B. duttonii spirochetemia in mice.

BALB/c (A), NUDE (B), SCID (C), and SCID BEIGE (D) mice. B. duttonii established infection in all four mouse strains. Spirochetemia was significantly (p = 0.02) higher in B-cell-deficient mice (C, D). No differences were observed between wild-type and T-cell-deficient mice (A vs. B) or between SCID mice and NK-cell-deficient SCID BEIGE mice (C vs. D). Data are presented as medians with 25^th and 75^th percentiles. Notice that the scales of the y-axes in this figure differ from those in Figures 2 and 4.

Figure 2. B. recurrentis spirochetemia in SCID BEIGE and SCID mice.

B. recurrentis established a relatively low-grade (compared to B. duttonii, see Figure 1C, D), stable infection in SCID BEIGE and SCID mice, but not in BALB/c or NUDE animals. Strain A17 (panel A) caused a higher (p<0.01) level of spirochetemia than did strain A11 (panel B). No difference in B. recurrentis spirochetemia was observed between SCID BEIGE and SCID mice. Data are presented as medians with 25^th and 75^th percentiles.

Figure 3. Splenomegaly in infected mice.

Although expansion of B- and T-cells is the major cause of splenomegaly in wild-type animals, SCID BEIGE mice infected with B. recurrentis A17 displayed significant (p<0.01) splenomegaly,which indicates a T-, B-, and NK-cell-independent immune response.

Figure 4. Effect of clodronate-induced macrophage depletion on spirochetemia.

Mice were injected with clodronate liposomes in PBS to deplete macrophages (and other phagocytic cells) in the blood and in organs in contact with the blood (e.g., the spleen and liver). PBS was used as a negative control. The injections (indicated by black triangles) were given every fifth day, starting one day before infection. A. BALB/c mice infected with B. duttonii developed very high spirochetemia, and all except one individual (dotted line) died at day 6 p.i. B. B. duttonii-infected SCID BEIGE mice treated with clodronate developed uncontrolled spirochetemia, and they all died before day 8 p.i. Control SCID BEIGE and BALB/c mice injected with PBS had much lower spirochetemia compared to their macrophage-depleted counterparts. C. SCID BEIGE mice treated with clodronate and infected with B. recurrentis A17 developed high but not lethal spirochetemia. The maximum spirochetemia in any of the control SCID BEIGE and BALB/c mice injected with PBS was 7.3×10⁶/ml. Even between the two peaks shown for the B. recurrentis-infected, clodronate-treated mice, the median spirochetemia was never below 8×10⁶/ml. B. recurrentis was unable to establish detectable spirochetemia in macrophage-depleted BALB/c mice.