Weight loss, kidney colonization, shedding in urine and survival to challenge with Leptospira interrogans following a single exposure to L. biflexa.

Male C3H/HeJ mice were inoculated once with 108 L. biflexa (LB) at 6 weeks and they were challenged with 108 L. interrogans serovar Copenhageni FioCruz (LIC) at 8 weeks. A) experimental layout; B) body weight measurements (%) acquired for 15 days post challenge with LIC; C) mouse survival within the 15 days post challenge with LIC; D) 16s rRNA qPCR quantification of live LIC in urine; E) 16s rRNA qPCR quantification of Leptospira burden in kidney tissue harvested on d15 post challenge with LIC and F) 16s rRNA qPCR from kidney EMJH cultures containing live Leptospira previously observed by dark field microscopy (DFM). DFM positive culture from the total data is represented in numbers under the graph. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups and their respective controls, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, N = 6-8 mice per group. Data represents two independent experiments.

Weight loss, kidney colonization, shedding in urine and survival to challenge with Leptospira interrogans following a double exposure to L. biflexa.

Male C3H/HeJ mice were inoculated twice with 108 L. biflexa at 6 and 8 weeks, and at 10 weeks they were challenged with 108 L. interrogans ser Copenhageni FioCruz (LIC). A) experimental layout; B) body weight measurements (%) acquired for 15 days post challenge with LIC; C) mouse survival within the 15 days post challenge with LIC; D) 16s rRNA qPCR quantification of live LIC in urine; E) 16s rRNA qPCR quantification of Leptospira burden in kidney tissue harvested on d15 post challenge with LIC and F) 16s rRNA qPCR from kidney EMJH cultures containing live Leptospira previously observed by dark field microscopy (DFM). DFM Positive culture from the total data is represented in numbers under the graph. Statistical analysis was performed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups and their respective controls,*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. N = 6-8 mice per group. Data represents two independent experiments.

Kidney histopathology and quantification of renal fibrosis.

Representative H & E-stained kidney tissue sections from both single and double exposure studies are included in A and C, respectively. The images were captured at 40X magnification. B and D represent the mRNA expression of kidney fibrosis marker ColA1 by qPCR normalized to endogenous b-actin expression. Data was analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison correction between challenged groups with their respective controls; * p values are included in the graphs. Data represents one of two independent experiments.

Detection of IgG1, IgG2a and IgG3 specific to L. interrogans in serum from experimental mice.

A) represents IgG isotypes specific to L. interrogans in 10-week serum of mice exposed once to L. biflexa before L. interrogans challenge. B) represents IgG isotypes specific to L. interrogans in 12-week serum of mice exposed twice to L. biflexa before L. interrogans challenge. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups with their respective controls; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns= not significant; N= 6-8 mice per group. Data represents two independent experiments.

Frequency of lymphocytes in spleen of mice subjected to a double exposure of L. biflexa before challenge with L. interrogans.

A to E) show B cell (CD19+), T cell (CD3+), NK cell (CD49b+), Helper T cell (CD4+) and Cytotoxic T cell (CD8+) frequencies in groups of experimental mice. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups and their respective controls; **p<0.01, ***p<0.001, ****p<0.0001 and ns= not significant; N= 3-4 mice per group. Data represents one of two independent experiments.

Frequency of T cell subsets (CD62L/CD44) in spleen of mice subjected to a double exposure of L. biflexa before challenge with L. interrogans.

A to D) represent naïve, early effector, effector and memory subsets of CD4+ helper T lymphocytes, respectively. E to H) represent naive, early effector, effector and memory subsets of CD8+ cytotoxic T lymphocytes, respectively. Ordinary one-way ANOVA followed by Tukey’s multiple comparison correction test was used to compare between challenged groups and their respective controls; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns= not significant; N = 3-4 mice per group. Data represents one of two independent experiments.