Infection Dynamics of a Leishmania tropica Isolate in the BALB/c Mouse Model: Effects of Pre-infection Culture Conditions on Experimental Infectivity

Objective: To evaluate the effects of distinct pre-infection culture conditions [RPMI-1640 and Schneider's insect medium (SIM)] on the experimental infectivity of a clinical Leishmania tropica isolate and to determine how culture-associated differences are reflected in infection dynamics in the BALB/c mouse model. Methods:A clinical Leishmania isolate, obtained from a patient with cutaneous leishmaniasis and cryopreserved in liquid nitrogen, was recovered in NNN medium.Species identification was performed using an internal transcribed spacer 1-targeted probebased real-time polymerase chain reaction followed by melting-curve analysis.Following post-thaw recovery, logarithmic-phase promastigotes were transferred to RPMI-1640 or SIM and maintained for five days without serial subpassaging before experimental infection.BALB/c mice (n=5 per group) were subcutaneously inoculated in the right hind footpad with 1×10 7 promastigotes.Infection dynamics were evaluated over six weeks by monitoring footpad diameter, amastigote burden, and promastigote recovery.Results: Molecular analysis identified the clinical isolate as L. tropica (MHOM/TR/2025/CBU152).Mice infected with SIM-derived promastigotes developed visible lesions from week 2. At the same time, longitudinal footpad measurements differed significantly between the SIM and RPMI groups from week 1 onward (Sidak-adjusted p<0.0001 at each time point).By week 6, the mean absolute footpad diameter reached 4.22 mm in the SIM group, compared with 3.52 mm in the RPMI group.Amastigote burden was significantly higher in the SIM group than in the RPMI group [median (interquartile range [IQR]): 6 (5-6) vs. 2 (2-2); p=0.0079].Promastigote recovery on day 7 was also significantly higher in the SIM group [median (IQR): 1.15 (1.12-1.18) vs. 0.15 (0.14-0.16)×10 6 /mL; p=0.0079].No evidence of infection was observed in the control group. Conclusion:Pre-infection culture conditions significantly influence the experimental infectivity of an L. tropica isolate in BALB/c mice.SIM-derived promastigotes exhibited a more infectious phenotype than RPMI-derived promastigotes, supporting SIM preconditioning as a useful approach for establishing experimental L. tropica infection in BALB/c mice.