Monoassociation of Preterm Germ-Free Piglets with Bifidobacterium animalis Subsp. lactis BB-12 and Its Impact on Infection with Salmonella Typhimurium

Abstract

Preterm germ-free piglets were monoassociated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to verify its safety and to investigate possible protection against subsequent infection with Salmonella Typhimurium strain LT2 (LT2). Clinical signs of salmonellosis, bacterial colonization in the intestine, bacterial translocation to mesenteric lymph nodes (MLN), blood, liver, spleen, and lungs, histopathological changes in the ileum, claudin-1 and occludin mRNA expression in the ileum and colon, intestinal and plasma concentrations of IL-8, TNF-α, and IL-10 were evaluated. Both BB12 and LT2 colonized the intestine of the monoassociated piglets. BB12 did not translocate in the BB12-monoassociated piglets. BB12 was detected in some cases in the MLN of piglets, consequently infected with LT2, but reduced LT2 counts in the ileum and liver of these piglets. LT2 damaged the luminal structure of the ileum, but a previous association with BB12 mildly alleviated these changes. LT2 infection upregulated claudin-1 mRNA in the ileum and colon and downregulated occludin mRNA in the colon. Infection with LT2 increased levels of IL-8, TNF-α, and IL-10 in the intestine and plasma, and BB12 mildly downregulated them compared to LT2 alone. Despite reductions in bacterial translocation and inflammatory cytokines, clinical signs of LT2 infection were not significantly affected by the probiotic BB12. Thus, we hypothesize that multistrain bacterial colonization of preterm gnotobiotic piglets may be needed to enhance the protective effect against the infection with S. Typhimurium LT2.

Keywords: Bifidobacterium animalis subsp. lactis BB-12; Salmonella Typhimurium; immunocompromised; inflammatory cytokines; intestinal barrier; preterm host.

Figure 1

Experiment design. Gnotobiotic piglets (n = 24) were assigned into four groups with six piglets per group: (i) germ-free (GF); (ii) infected with Salmonella Typhimurium strain LT2 (LT2); (iii) associated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12); (iv) associated with BB12 and infected with LT2 (BB12 + LT2).

Figure 2

Colony-forming units (CFU) of B. animalis subsp. lactis BB-12 (BB12) in the gnotobiotic piglets. BB12 CFU was counted in the jejunum, ileum, colon, and blood (all CFU/mL) and mesenteric lymph nodes (MLN), liver, spleen, and lungs (all CFU/g) in the piglets monoassociated with BB12 (BB12; blue column) and the piglets monoassociated with BB12 and one week later infected with S. Typhimurium LT2 for 24 h (BB12 + LT2; violet column). Interferences between BB12 and LT2 were evaluated by unpaired t-test, and statistical differences were marked with asterisks at p < 0.05. Log CFU are depicted as mean + SEM and n = 6 piglets for all groups.

Figure 3

Colony-forming units (CFU) of S. Typhimurium LT2 in the gnotobiotic piglets. S. Typhimurium LT2 CFU was counted in the jejunum, ileum, colon, and blood (all CFU/mL) and mesenteric lymph nodes (MLN), liver, spleen, and lungs (all CFU/g) in the piglets infected with S. Typhimurium LT2 for 24 h (LT2; red column) and the piglets monoassociated with B. animalis subsp. lactis and one week later infected with S. Typhimurium LT2 for 24 h (BB12 + LT2; violet column). Interferences between B. animalis subsp. lactis BB-12 and S. Typhimurium were evaluated by unpaired t-test. Statistical differences were marked with asterisks at p < 0.05. Log CFU are depicted as mean + SEM and n = 6 piglets for all groups.

Figure 4

Histopathological evaluation of the ileum in the gnotobiotic piglets: germ-free (GF; A,B), infected with S. Typhimurium LT2 for 24 h (LT2; C,D), associated with B. animalis subsp. lactis BB-12 (BB12; E,F), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2; G,H). Bars represent 500 μm (G) and 100 μm (H), respectively. Histopathological changes affected the LT2 and BB12 + LT2 groups. Histopathological scores from the ileum of six piglets per group are depicted (I).

Figure 5

Relative expression (fold change) of claudin-1 (A,B) and occludin (C,D) mRNA in the ileum (A,C) and colon (B,D) of the gnotobiotic piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). The values are presented as mean + SEM. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple comparison post hoc test, and p-values < 0.05 are denoted with different letters above the columns. Six samples in each group were analyzed.

Figure 6

Intestinal levels of the inflammatory cytokines IL-8 (A,D,G), TNF-α (B,E,H) and IL-10 (C,F,I) in the jejunum (A–C), ileum (D–F), and colon (G–I) of the gnotobiotic piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). The values are presented as boxes indicating the lower and upper quartiles, the central line is the median, and the ends of the whiskers depict the minimal and maximal values. Statistical differences were calculated by the Kruskal–Wallis test with Dunn’s multiple comparison post hoc test, and p < 0.05 are denoted with different letters above the columns. Six samples in each group were analyzed.

Figure 7

Plasmatic levels of the inflammatory cytokines IL-8 (A), TNF-α (B) and IL-10 (C) in the gnotobiotic piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). The values are presented as boxes indicating the lower and upper quartiles, the central line is the median, and the ends of the whiskers depict the minimal and maximal values. Statistical differences were calculated by the Kruskal–Wallis test with Dunn’s multiple comparison post hoc test, and p < 0.05 are denoted with different letters above the columns. Six samples in each group were analyzed.