Immune response to Nocardia brasiliensis antigens in an experimental model of actinomycetoma in BALB/c mice

Abstract

Nine- to twelve-week-old BALB/c mice were injected in footpads with 10(7) CFU of a Nocardia brasiliensis cell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-gamma increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.

FIG. 1

Clinical evolution of mycetoma in BALB/c mice. (A) Normal hind footpad before infection; (B) severe edema greater than 7 mm at 15 days of infection; (C) edema abscess and sinus 28 days after infection.

FIG. 2

Typical full-blown mycetoma lesion, with 19-mm edema open abscess, fistula formation, and healing scars, 90 days after infection.

FIG. 3

Histopathological evolution of mycetoma. Paraffin-embedded sections stained with hematoxylin and eosin demonstrate multiple microabscesses (A; magnification, ×40), foamy macrophages limiting the abscess by day 10 (B), polymorphonuclear and mononuclear cells, with giant vacuolar macrophages also visible (C; magnification, ×40), and N. brasiliensis microcolony 90 days after infection (D).

FIG. 4

Western blot identification of immunodominant antigens. The nitrocellulose strips contain soluble crude cellular extract from N. brasiliensis incubated with sera from infected mice at different times after infection. Strip A, negative control before infection; strips B and C, sera from infected BALB/c mice 30 and 90 days; strip D, serum from BALB/c mouse 160 days after infection.

FIG. 5

Anti-N. brasiliensis antibody response in mice, determined by ELISA at various times after infection. ○, IgM response (each point represents the mean value for five mice in each group [SD, ±0.070 and ±0.140]); □, anti-N. brasiliensis IgG response (SD, ±0.647 and ±0.053); ■, total antibody response to N. brasiliensis immunodominant antigens (SD, ±0.036). All serum samples were diluted 1:50.

FIG. 6

Spleen and lymph node cell proliferation. ○, lymphocyte proliferation (stimulation index) from draining lymph node stimulated in vitro with UV-killed whole N. brasiliensis cells; ●, mean value for spleen lymphocytes from five mice stimulated in vitro with the soluble antigen CCE.