Figure 1.
Effect of inoculum size and timing of immunosuppression on the severity of F. oxysporum infection.
(A) BALB/c mice (n = 6) were inoculated intravenously with F. oxysporum microconidia. Immunosuppressive treatment (150 mg cyclophosphamide per kg body weight) was initiated three days prior to infection (day −3) and repeated every 3 days thereafter. Survival was recorded for 20 days. An inoculum dose of 2×106 microconidia caused significantly lower mortality than 2×107 microconidia (p = 0.0016); 106 microconidia caused significant lower mortality than 2×106 microconidia (p = 0.0024). (B) BALB/c mice (n = 6) were inoculated with 2×107 microconidia, and immunosuppressive treatment was initiated either on day −3 or on day 0, and repeated every 3 days thereafter. Survival was recorded over 20 d. (C) Fungal burdens in mice immunosuppressed on day 0 and inoculated with 2×107 microconidia. On each of the indicated days post-infection (dpi), one animal was sacrificed and organ homogenates quantified.
Figure 2.
F. oxysporum displays distinct growth morphologies in different organs.
PAS-hematoxylin-staining of tissue sections from different organs. Mice were subjected to immunosuppressive treatment on day 0, and every 3 days thereafter. Organs were obtained on day 1 (A, D), 4 (B, E, G, H, I) or 14 (C, F) after infection with 2×107 microconidia: heart (A–C), kidney (D–F), lung (G), spleen (H), liver (I). Fungal structures are indicated by arrows. G, microconidial germlings; M, mycelium; C, chlamydospores. Scale bar = 50 µm.
Figure 3.
Invasive growth of F. oxysporum in the kidney.
(A) Fungal biomass on the surface of an aseptically removed kidney from a mouse subjected to immunosuppressive treatment on day 0, and every 3 days thereafter. The kidney was obtained 20 d after infection with 106 microconidia. Visible fungal lesions are indicated by arrows. (B) Kidney cross section from a mouse subjected to immunosuppressive treatment on day 0, and every 3 days thereafter. The kidney was obtained 20 d post-infection with 106 microconidia. Bright areas correspond to fungal biomass stained with the chitin-binding dye Calcofluor White (CFW). The white box indicates the area used for the tissue section shown in C. (C) PAS-hematoxylin staining of a tissue section. The area of invasive mycelial growth is surrounded by arrows. M, mycelium. Scale bars are indicated.
Figure 4.
F. oxysporum causes infection in the tails and eyes of immunosuppressed mice.
Mice were subjected to immunosuppressive treatment on day 0, and every 3 days thereafter. (A) Tail of a non-infected immunosuppressed BALB/c mouse. (B–E) Tails of immunosuppressed BALB/c mice intravenously inoculated with 106 conidia at 10 d post-infection. Macroscopic symptoms are indicated by arrows: swellings (B, D), open lesions (C), necrosis (D, E), and loss of the tip (E). (F, G) CFW staining of tissue samples taken from the lesion shown in (C). Fungal structures are indicated by arrows. H, hyphae; C, chlamydospores. (H–L) Infection of the eye tissue in immunosuppressed mice infected with F. oxysporum. (H) Uninoculated mouse; (I–K) mice inoculated with 106 conidia at 10 dpi. (L) CFW staining of an eye removed from an immunosuppressed mouse infected with F. oxysporum. H, hyphae. Scale bar = 50 µm (F, G); 1 mm (L).
Figure 5.
F. oxysporum disseminates and persists in immunocompetent mice.
(A) Fungal burdens were determined at day 4 post-infection for groups of 3 BALB/c mice, either immunocompetent or immunosuppressed on day 0 and every 3 days thereafter, and intravenously infected with 2×107 microconidia. (B) Fungal burdens in infected immunocompetent mice sacrificed at 11 d post-infection. (C–F) PAS-hematoxylin-staining of tissue sections obtained from different organs of immunocompetent mice sacrificed at 4 d post-infection. (C) Heart, (D) lung, (E) kidney, (F) liver. (G) Spleen sample from an infected immunocompetent mouse sacrificed at 11 d post-infection was homogenized, KOH-treated and stained with CFW to visualize fungal structures (indicated by arrows). G, microconidial germlings; C, chlamydospores. Scale bar = 50 µm.
Figure 6.
F. oxysporum persistence in immunocompetent mice can lead to systemic infection and death upon subsequent immunosuppression.
(A) BALB/c mice (n = 6) were intravenously inoculated with 2×107 microconidia. Immunosuppressive treatment was initiated either on day 3 or 7 post-infection and repeated every 3 days thereafter. Survival was recorded over 20 d. Fungal burdens (B) and PAS-hematoxylin-staining (C–F) of organ sections from a mouse inoculated with 2×107 F. oxysporum microconidia and subjected to immunosuppressive treatment starting at 7 dpi. The mouse that died was culled at 15 dpi. Organs: heart (C), kidney (D), spleen (E), liver (F). M, mycelium; C, chlamydospores. Scale bar = 50 µm.