1. Establishment of a physiologically relevant mouse model of Leishmania–Plasmodium co-infection - A robust and physiologically relevant murine model was established to investigate the interactions between visceral leishmaniasis (VL) and malaria under controlled experimental conditions. BALB/c mice, which are susceptible to both Leishmania donovani and Plasmodium berghei infection, were used to generate single- and co-infection models. A bioluminescent L. donovani strain (LdLuc) enabled longitudinal, non-invasive quantification of parasite burden in target organs, facilitating accurate timing of secondary malaria infection. In parallel, a GFP-expressing P. berghei strain (PbGFP) allowed rapid and quantitative assessment of blood-stage parasitemia by flow cytometry, eliminating the need for routine microscopic examination of Giemsa-stained blood smears. This experimental platform provided a reproducible system for investigating the impact of pre-existing VL on malaria infection.
2. Determine how acute and chronic L. donovani infection influence malaria progression following sporozoite infection - The impact of acute and chronic VL on malaria progression was investigated using sporozoite challenge. Pre-existing L. donovani infection significantly reduced P. berghei liver-stage infection and delayed the appearance of blood-stage parasitemia. This protective effect was evident during both acute and chronic VL, with chronic infection producing the strongest suppression of hepatic parasite burden. Histological and quantitative analyses demonstrated that protection was associated with reduced hepatocyte infection and impaired exoerythrocytic parasite development. In contrast, when the hepatic stage of malaria was bypassed through direct inoculation of infected erythrocytes, chronic—but not acute—L. donovani infection exacerbated malaria disease severity. These findings demonstrate that pre-existing VL exerts stage-specific effects on malaria, limiting liver-stage parasite development while promoting increased susceptibility to blood-stage pathology during chronic infection.
3. Evaluate the reciprocal effects of Plasmodium infection on visceral leishmaniasis - Analysis of hepatic parasite burden following secondary P. berghei infection demonstrated that malaria challenge did not significantly alter L. donovani parasite burden during either acute or chronic VL. Based on these findings, further investigation of the reciprocal effects of malaria on VL progression was not pursued. Experimental efforts were instead focused on defining the influence of pre-existing L. donovani infection on malaria pathogenesis, where clear biological effects were observed.
4. Characterization of immune responses during mono-infection and co-infection - Comprehensive hepatic immunophenotyping was performed by multiparametric spectral flow cytometry to characterize the cellular responses associated with mono-infection and co-infection. Acute L. donovani infection, both alone and during co-infection, was characterized by increased frequencies of PD-1⁺ CD4⁺ and CD8⁺ T cells, activated γδ T-cell populations, activated NK-like cells, and inflammatory monocyte and dendritic cell subsets, consistent with the establishment of granulomatous inflammation. In contrast, chronic L. donovani infection exhibited a distinct hepatic immune landscape enriched in differentiated γδ T cells, CD69⁺ γδ and CD8⁺ T cells, activated B-cell populations, and dendritic cell-like subsets. These immunological changes are consistent with the transition from developing granulomas during acute infection to mature and organized granulomas during chronic VL, providing mechanistic insight into the altered hepatic environment associated with suppression of malaria liver-stage development.
