Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis

Abstract

The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4(+) and CD8(+) T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing the Leishmania infantum D-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.

FIG. 1.

Susceptibility of mice to intravenous infection with L. infantum promastigotes. BALB/c mice were infected intravenously as described in Materials and Method with various numbers of L. infantum promastigotes (10⁴ to 10⁶) as indicated. Parasite burdens were determined by using the limiting dilution analysis method at the times postinfection indicated. It should be noted that at 16 weeks postinfection, parasites were found in the livers of mice infected with 10⁴ L. infantum promastigotes; however, the parasite burden was below the minimal quantifiable level (≤170 parasites/liver). Symbols are as follows, with the numbers of promastigotes noted in parentheses: ○, liver (10⁶); □, spleen (10⁶); ▴, liver (10⁵); ▾, spleen (10⁵); •, liver (10⁴); ▪, spleen (10⁴).

FIG. 2.

Susceptibility of BALB/c mice to intradermal infection with L. infantum promastigotes. Shown are the experimental results for mice infected intradermally with 10⁷ promastigotes of L. infantum. Parasite burdens were determined at the site of infection (skin), draining lymph node, spleen, and liver by limiting dilution analysis. The tissue parasite burdens at various times postinfection are indicated. ♦, lymph node; ○, cutaneous infection site; •, spleen; □, liver.

FIG. 3.

Skin inflammatory responses of BALB/c mice following infection with L. infantum. Frozen foot sections were collected on the indicated day postinfection with 10⁷ promastigotes of L. infantum. Sections were stained with H&E (A to C) or stained with immunoperoxidase and counterstained with methyl green (D to F) at either 1.5 (A, B, and D) or 28 (C, E, and F) days postinfection. At day 1.5 of infection, tissues were composed of a collection of parasitized macrophages (arrows in panel A) and were characterized with massive infiltration of PMNs (arrows in panel B) and low levels of CD4⁺ T cells (D). In contrast, parasite-containing macrophages were rare in tissues at 28 days postinfection (panel C versus panel A) while CD4⁺ (E) and CD8⁺ (F) T cells were readily detected. All images were taken with 40× magnification.

FIG. 4.

Immune response of BALB/c mice intradermally infected with L. infantum. Cytokine responses were examined in the draining lymph node and splenic tissues of BALB/c mice intradermally infected with 10⁷ L.infantum promastigotes. Cells were stimulated with either L. infantum promastigote homogenate antigen or ConA, and cytokines were measured as described in Materials and Methods at various times postinfection. The sensitivities of the IL-4, IL-2, IFN-γ, and IL-10 assays were 2 U/ml, 0.5 U/ml, 0.5 U/ml, and 200 pg/ml, respectively. Background cytokine levels were determined by using the supernatants from unstimulated cell populations. Background levels for IL-2 and IL-4 were undetectable while the levels for IL-10 were consistently 500 to 800 pg/ml; background levels of IFN-γ (18 U/ml) were detected only at day 3 postinfection.