Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

Abstract

Background: Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.

Methods: In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.

Results: Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.

Conclusion: Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite's inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

Figure 1

Molecular characterization of L. infantum isolates. (A) Amplification of the k26 gene. See Additional file 3: Figure S2 for coding DNA sequences and alignment. (B) RFLP patterns for L. infantum isolates after HaeIII digestion of an amplified fragment of the minicircles kDNA. Profiles for DNA extracted from axenic promastigotes are shown, though DNA from experimentally infected murine tissues delivered comparable restriction patterns. For each strain, left lane corresponds to the digested DNA and right lane to undigested product. Gels were prepared with 2% (A) or 2.5% (B) agarose and stained with ethidium bromide. MW - molecular weight marker with bp units assigned in the left of the figure.

Figure 2

In vitro development of L. infantum strains. (A) Growth curves of Bibiano (light grey), E390M (dark grey), HL (black) and ST (white) L. infantum strains in NNN. Cultures were started with 10⁶ promastigotes of each strain per mL and growth was followed for a week, with a daily counting of parasite numbers in a Neubauer chamber. (B) Parasite viability measured by flow cytometry as percentage of AnnV^-/7AAD^- cells. The mean of three independent experiments is plotted; bars represent SD. (C) Cell cycle analysis of HL during growth period. Data show means of one representative experiment of three independent assays. (D) Illustrative phenotype of L. infantum promastigotes with 4 days of culture in NNN captured by flow cytometry.

Figure 3

Indirect measurement of metacyclogenesis. Quantification of (A)Meta-1, (B)SHERP and (C)histone H4 transcription by RT-PCR over the time of culture in NNN medium for the four strains. Bars represent the mean fold change relative to day 1 with SD of two independent experiments. Statistical significant differences between day 1 and the following days were determined with One-way ANOVA and Dunnett’s multiple comparison test.

Figure 4

In vitro differential infectivity of L. infantum strains. BMMo were infected with promastigotes of each strain after 4 days of culture in NNN in a ratio of 10:1 (parasites:macrophage). The kinetics of infection was followed counting (A) the infected cells and (B) the number of parasites per infected cell on fluorescent microscope. To account to the overall parasite load, an infection index (C) was calculated multiplying the individual data from (A) by (B). Statistically significant differences between ST and the other strains were determined with One-way ANOVA followed by Tukey’s multiple comparison test.

Figure 5

Quantitative distribution of L. infantum in BALB/c mice 2 and 6 weeks after infection. The parasite burden was quantified on the (A) spleen, (B) liver, (C) bone marrow, (D) lymph node and (E) blood. Data represent means ± SD of 3–5 animals per group of a representative experiment of two independent assays. T test was used to determine statistically significant differences between 2 and 6 weeks of infection with each strain; * p < 0.05. With one-way ANOVA followed by Tukey’s multiple comparison test we calculated statistically significant differences between strains at each time point; # p < 0.05. Dashed line indicate limit of detection for quantification for each tissue.

Figure 6

Cell populations in spleens of naive and Leishmania-infected mice in the acute and chronic phases. (A), (F) Total cells were counted and stained for identification of major splenic populations by flow cytometry. (B), (G) CD4⁺ T cells. (C), (H) CD8⁺ T cells. (D), (I) B cells. (E), (J) Monocytes/macrophages. (A)-(E) Absolute number of cells per spleen. Data represent means ± SD of 3 to 5 animals per group of one experiment representative of two. (F)-(J) Fold modification of cell numbers in the infected mice in relation to naïve. Boxes and whiskers with 5–95 percentile and mean (showed by “+”) of 3 to 5 animals. Dashed and solid lines indicate a 2- or 4-fold modification, respectively, relative to naïve mice. One-way ANOVA followed by Dunnett’s multiple comparison test was performed to calculate statistically significant differences between naive and infected mice at 2 and 6 weeks after infection.

Figure 7

Leishmania-specific humoral response.Leishmania-specific sera reactivity of naive and infected animals 2 and 6 weeks post-infection was analyzed by ELISA. Specific (A) IgG2a and (B) IgG1 were quantified and are depicted as means ± SD of one representative experiment of 2 independents. Statistically significant differences are pointed out as given by one-way ANOVA followed by Dunnett’s multiple comparison test.