Figure 3.

Body weight change, bacterial load, and pro-inflammatory cytokine response of mice challenged with L. interrogans serovar Canicola. (A) Mouse body weight (% change) was recorded from 0 day to 13 days upon infection; concurrent assessment of clinical status (grooming, eating, drinking, energy level) was also observed. G-I and G-II mice were sacrificed at 6^th and 5^th day (‡ and †,). Statistical analysis was performed to determine statistical significance in body weight between the PBS control and vaccinated groups using two-tailed unpaired, Mann-Whitney T-Test. p values: VM mix vs PBS, p - 0.0152 *: VM unlabeled vs PBS, p – 0.0005*: VM unlabeled vs VM mix, p < 0.0001 ****: t3490 vs PBS, p - 0.3869, ns. Error bars indicate the standard error. Total genomic DNA was extracted from the kidney (B) and liver (C) and analyzed by qPCR performed in duplicates with lipL32 primers and SYBER Green probes to quantify leptospiral tissue load. Statistical analysis was performed using the Kruskal–Wallis test and Dunn’s multiple comparisons test. p < 0.0001 was considered significant. (D) Pro-inflammatory cytokine response in pooled serum samples from each group: G-I (PBS control), G-II (t3490), G-III (mix of 5 VM proteins), and G-IV mice (mix of 2 VM proteins) pre-challenge and post-challenge were used to measure the levels of TNF-α, IL-6, IL10, KC/GRO, IFN-γ and IL-1β by V-PLEX Proinflammatory Panel 1 Mouse Kit (Meso Scale Discovery, MD, USA), an immunoassay based on electrochemiluminescence. PIB denotes pre-immunized bleed. *p < 0.05, **p < 0.001, ****p < 0.0001.