L. interrogans proliferated in the intestine, destroyed the intestinal structure, and increased intestinal inflammation.

(A) Flow diagram of the experiment. Six-week-old female hamsters were injected intraperitoneally with 107 leptospires. Hamsters were euthanized at 0 h, 6 h, 48 h, 96 h, and AM p.i.. The ileums and colons were collected aseptically for further analysis. (B) Leptospiral burdens in the ileums and colons of Leptospira-infected hamsters were determined by qPCR (n = 6). (C) Left panel, histopathology of colons of D0 hamsters (scale bar, 50 μm; n = 4) and AM hamsters (scale bar, 50 μm; n = 4). Representative photographs are presented. Right panel, histopathology scores of colons. D0, uninfected hamster. AM, articulo mortis. (D-I) The gene expression of Lcn2 (D), TNF-α (E), IL-1β (F), IL-10 (G), TLR4 (H), and Nos2 (I) was analyzed by RT-qPCR. The mRNA levels of genes in the colons of the D0 group (n = 3) and the AM group (n = 3) were normalized to the expression of the housekeeping gene GAPDH. Each infection experiment was repeated three times. Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001.

L. interrogans infection changed the composition of the gut microbiota.

(A) Flow diagram of the experiment. Six-week-old female hamsters were injected intraperitoneally with 107 leptospires. Fecal pellets were collected aseptically for 16S rRNA gene sequencing at 0 days, 2 days, and AM p.i.. D0, uninfected hamster. AM, articulo mortis. (B-C) Observed species (B) and Ace (C) in the feces of hamsters. Observed species and Ace indicate species richness. (D-E) Simpson index (D) and Shannon index (E) in the feces of hamsters. Simpson and Shannon index indicate species diversity. (F) Principal coordinates analysis (PCoA) of fecal samples based on 16S rRNA gene sequencing using weighted UniFrac. (G) PCoA of fecal samples based on 16S rRNA gene sequencing using weighted UniFrac. (H) Relative abundance of the top 10 phyla in the feces of hamster. (I) Linear discriminant analysis (LDA) of effect size (LEfSe) between D0 and AM hamsters (LDA score > 3). Cambridge blue bars indicate taxa enrichment in D0 hamsters, and pink bars indicate taxa enrichment in AM hamsters. (J) The ratio of Firmicutes to Bacteroidetes in the D0 and AM. (K-M) The relative abundance of Proteobacteria (K), Lactobacillus (L), and Allobaculum (M) in the D0 and AM groups. (D0, n = 10; D2, n = 10; AM, n = 8). Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01.

L. interrogans infection disrupted intestinal tight junctions.

Six-week-old female hamsters were injected intraperitoneally with 107 leptospires. Blood was collected to determine intestinal permeability at 0 days, 2 days, and AM p.i.. Colons were collected aseptically to measure gene expression at 0, 2, and AM p.i.. (A) The intestinal permeability of Leptospira-infected hamsters was analyzed with a fluorescence spectrophotometer at indicated time (D0, n = 4; D2, n = 6; AM, n = 6). AM, articulo mortis. (B-G) The relative gene expression of ZO-1 (B), Claudin-3 (C), JAMA (D), Claudin-2 (E), and Mucin-2 (F) in Leptospira-infected hamsters (n = 6-10 per group) was determined by qPCR. D0, uninfected hamster. AM, articulo mortis. Each infection experiment was repeated three times. Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001.

Translocated microbiota in the intestinal epithelium following L. interrogans infection.

Six-week-old female hamsters were injected intraperitoneally with 107 leptospires. Hamsters were euthanized at 0 days, 2 days, and AM p.i.. AM, articulo mortis. Colons were collected aseptically. The intestinal contents were excluded from the intestine with PBS and a segment of colon was fixed in 4% paraformaldehyde solution overnight and analyzed by FISH with an EUB 338 probe (red). (A) Th results are representative photographs of three groups. (B) Number of positive bacteria per field in the three groups. n = 6 per group. D0, uninfected hamster. AM, articulo mortis. Each infection experiment was repeated three times. Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. ***p < 0.001.

Microbiota depletion exacerbated leptospirosis.

(A) Flow diagram of the experiment. Hamsters were administered with Abx or water intragastrically once daily for ten consecutive days. Then, fecal pellets were collected aseptically for 16S rRNA gene sequencing. (B-C) Observed species (B) and Chao1 (C) in the feces of hamsters. Observed species and Chao1 indicate species richness. (D-E) Simpson index (D) and Shannon index (E) in the feces of hamsters. Simpson and Shannon index indicate species diversity. (F) Relative abundance of the top 10 phyla in the feces of hamsters. (G) The relative abundance of Bacteroidetes, Firmicutes, and Proteobacteria in the Con, Abx, and FMT groups. n = 4 per group. (H) Hamsters were treated with Abx as described above. After a 2-day washout period, hamsters were intraperitoneally infected with 106 L. interrogans. Then, the survival rate of the hamsters was recorded. n = 6 per group. (J) Leptospiral load in the blood of the Con, Abx, and FMT groups. n = 6 per group. (K-M) Gene expression of TNF-α (K), IL-1β (L), and IL-10 (M) in the blood of the three groups. n = 5 per group. Each infection experiment was repeated three times. Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. Survival differences between the study groups were compared by using the Kaplan-Meier log-rank test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Leptopsira infection induced intestinal and systemic inflammation that was partially inhibited by LPS neutralization.

(A) Flow diagram of the experiment. Fecal filtration or serum of uninfected hamsters and AM hamsters was directly added into dishes or pretreated with PMB at 37 °C for 2 h, and then cocultured with macrophages for 4 h. RNA was extracted for gene expression analysis. (B-E) The gene expression of TNF-α (B), IL-1β (C), IL-10 (D), and TLR4 (E) of fecal filtration of uninfected hamsters (n = 3) and AM hamsters (n = 3) were analyzed by RT-qPCR. (F-I) The gene expression of TNF-α (F), IL-1β (G), IL-10 (H) and TLR4 (I) of serum of uninfected hamsters (n = 3) and AM hamsters (n = 3) were analyzed by RT-qPCR. (J-M) The effect of LPS neutralization on the gene expression of TNF-α (J), IL-1β (K), IL-10 (L) and TLR4 (M) in the fecal filtration of AM hamsters (n = 3) were analyzed by RT-qPCR. (N-Q) The effect of LPS neutralization on the gene expression of TNF-α (N), IL-1β (O), IL-10 (P) and TLR4 (Q) of in the serum of AM hamsters (n = 3) was analyzed by RT-qPCR. The mRNA levels in untreated controls were set as 1.0. Each infection experiment was repeated three times. Data are shown as the mean ± SEM and analyzed by the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001.

LPS neutralization combined with antibody therapy or antibiotic therapy improved the survival rate of female hamsters with severe leptospirosis.

(A) Flow diagram of the experiment. Six-week-old female hamsters were injected intraperitoneally with 107 leptospires. Group 1: saline control; Group 2: Polymyxin B (PMB) (1 mg/kg, i.p.); Group 3: Antibody against Leptospira (Ab) (16 mg/kg, subcutaneous injection); Group 4: Doxycycline (Dox) (5 mg/kg, i.p.); Group 5: PMB (1 mg/kg, i.p.) and Ab (16 mg/kg, subcutaneous injection); Group 6: PMB (1 mg/kg, i.p.) and Dox (5 mg/kg, i.p.); Hamsters were treated twice a day for 3 consecutive days. Hamsters were monitored daily for 21 days. (B) Survival rate of female hamsters of severe leptospirosis after different treatments. n = 6 per group. (C) Leptospiral load in the blood of different groups. n = 5 per group. (D-F) Gene expression of TNF-α (D, IL-1β (E), and IL-10 (F) in the blood of different groups. The mRNA levels of cytokines in uninfected controls were set as 1.0. n = 5 per group. Each infection experiment was repeated three times. Survival differences between the study groups were compared by using the Kaplan-Meier log-rank test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.