Fig. 3. LmCen^−/− immunized hamster protects and induces pro-inflammatory type of immune response upon challenge with L. donovani.

a Schematic representation of experimental plan to determine the efficacy and immune response of LmCen^−/− parasites hamsters against L. donovani via needle challenge. b, c Parasite load were determined by limiting dilution after various periods post challenge and expressed as number of parasites per Spleen (b) and per gram of Liver (c). LmCen^−/− immunized (Imm Chal) (n = 6 for 1.5 and 3 months and n = 10 for 9 and n = 11 for 12 months) and age-matched nonimmunized (Non-Imm Chal) hamsters (n = 6 for 1.5 and 3 months and n = 10 for 9 and 12 months) after various periods of post-needle challenge (1.5, 3, 9, and 12 month) with L. donovani. Results (Mean ± SD) are representative of cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test). d Heat map showing the differential gene expression in the splenocytes (with or without 24 h of L. donovani freeze–thaw antigen restimulation; ±FTAg) of age-matched nonimmunized (Non-Imm Chal) and LmCen^−/− immunized (Imm Chal) hamsters after 1.5 months of L. donovani challenge by needle injection (n = 6/group). According to the general function, transcripts are annotated in the left side. Downregulation and upregulation of the transcripts are shown in pink, yellow and blue, respectively. For heat map, results were shown as log₁₀ fold change over naive hamster. e Expression profile of IFN-γ, TNF-α, IL-4, IL-21, IL-10, and IFN-γ/IL-10 Ratio in the spleen, which was evaluated by RT-PCR. The data were normalized to γ-Actin expression and shown as the fold-change relative to age-matched naive hamster. Results (Mean ± SD) represent cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test).