Delayed invasion of the kidney and brain by Borrelia crocidurae in plasminogen-deficient mice

Abstract

Borrelia crocidurae is an etiologic agent of relapsing fever in Africa and is transmitted to humans by the bite of soft ticks of the genus Ornithodoros. The role of the plasminogen (Plg) activation system for the pathogenicity of B. crocidurae was investigated by infection of Plg-deficient (plg(-/-)) and Plg wild-type (plg(+/+)) mice. No differences in spirochetemia were observed between the plg(-/-) and plg(+/+) mice. However, signs indicative of brain invasion, such as neurological symptoms and/or histopathological changes, were more common in plg(+/+) mice. Quantitative immunohistochemical analysis demonstrated infection of spirochetes in kidney interstitium and brain as soon as 2 days postinoculation. Lower numbers of extravascular spirochetes in plg(-/-) mice during the first days of infection suggested a less efficient invasion mechanism in these mice than in the plg(+/+) mice. The invasion of the kidneys in plg(-/-) mice produced no significant inflammation, as seen by quantitative immunohistochemistry of the CD45 common leukocyte marker. However, significant kidney inflammation was observed with infection in the plg(+/+) mice. In brain, inflammation was more severe in plg(+/+) mice than in plg(-/-) mice, and the numbers of CD45(+) cells increased significantly with duration of infection in the plg(+/+) mice. The results show that invasion of brain and kidney occurs as early as 2 days after inoculation. Also, Plg is not required for establishment of spirochetemia by the organism, whereas it is involved in the invasion of organs.

FIG. 1

Dose-dependent coating of B. crocidurae (B.c.) by plasmin. Different concentrations of B. crocidurae were incubated with Plg and uPA, forming active plasmin on the surface of the spirochetes. Some of the borrelial preparations received only Plg, uPA, or HBSS. The sham control received Plg and uPA but no spirochetes. Data are means plus standard deviations for three replicate samples.

FIG. 2

(A) Mean course of spirochetemia of plg^−/− and plg^+/+ mice during infection with B. crocidurae. (B) Mean percentage of spirochetes associated with erythrocyte rosettes during the infection process. Error bars for each point were omitted for clarity.

FIG. 3

Micrographs of kidney and brain tissue from plg^+/+ mice. (A) Immunofluorescence staining showing B. crocidurae spirochetes in kidney interstitium, day 12 p.i. (B) Immunoperoxidase staining of CD45⁺ cells in kidney, day 2 p.i. (C) Spirochetes in plexus choroideus, day 5 p.i. (D) Spirochete associated with vessel wall in cerebrum. The sample was double stained with fluorescein isothiocyanate-conjugated antibody to endothelium (CD31/PECAM-1). (E) Spirochete in the brain parenchyma. Counterstaining with DAPI shows nuclei. (F) Activated microglia.

FIG. 4

Numbers of spirochetes in kidney interstitium (A) and cerebrum (B) of B. crocidurae-infected plg^−/− and plg^+/+ mice, as demonstrated by immunohistochemistry. Analyses included five mice of each category sacrificed on day 0 and three of each category for days 2, 5, 8, 12, and 14 p.i.

FIG. 5

Numbers of leukocytes in kidney interstitium (A) and glomeruli (B) after infection of plg^−/− and plg^+/+ mice with B. crocidurae, as demonstrated by immunohistochemical detection of the CD45 antigen. Day 0 values are means for five mice from each group; others are values for individual mice, with three plg^−/− and plg^+/+ mice represented at each time point.

FIG. 6

Numbers of leukocytes in cerebrum after infection of plg^−/− and plg^+/+ mice with B. crocidurae, as demonstrated by immunohistochemical detection of the CD45 antigen. Day 0 values are means for five mice from each group; others are values for individual mice, with three plg^−/− and plg^+/+ mice represented at each time point.