Fig. 4.

LIC12143 mutant fails to induce acute leptospirosis in hamsters but retains renal colonization capacity. (A) Hamsters (n = 5) were intraperitoneally infected with 10⁷ cells of either wild-type or LIC12143 mutant L. interrogans serovar Canicola LAD-1. Animals were monitored daily for clinical signs of leptospirosis and changes in body weight. Negative control animals received medium only. (B) On day 3 post-infection, blood samples were collected via retroorbital plexus for bacterial quantification, expressed as genome equivalents (Geq) per mL. (C, D) On day 7, all animals were humanely euthanized. Liver (C) and kidney (D) tissues were harvested for bacterial burden analysis by qPCR. (E) Kidney macerates were inoculated into HAN medium supplemented with 5-fluorouracil (5-FU) for bacterial isolation. Isolates were phenotypically confirmed by immunoblotting using anti-Canicola polyclonal antiserum. (F) Mutants isolated from the initial experiment were used to re-infect a second cohort of hamsters (n = 5), which were monitored daily and euthanized upon reaching humane endpoint criteria. (G–I) Bacterial burden was assessed by qPCR in blood on day 3 (G), and in liver (H) and kidney (I) at endpoint or at the conclusion of the experiment. (J) Kidney-derived isolates were re-validated by immunoblotting. Endpoints of animals were compared using the Log-rank (Mantel–Cox) test. For bacterial burden analysis, comparisons between control and mutant-infected groups were performed using non-parametric Mann-Whitney test (*P ≤ 0.05).