The Probiotic Bacillus subtilis MB40 Improves Immunity in a Porcine Model of Listeriosis

Abstract

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 10⁸ CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Keywords: Bacillus subtilis; Listeria; direct-fed microbials; foodborne illness; immunity; listeriosis; probiotics; swine.

Figure 3

Effects of B. subtilis MB40 supplementation and Listeria challenge on blood monocyte concentrations. The line connecting data from individual animals (circles) shows the change from d8 to d15. The dotted line shows the upper reference limit (2.3 cells/µL) for pigs up to 6 weeks of age [76]. Basal, basal diet; Control, no LM challenge; d, day; LM, Listeria monocytogenes challenge at d8; MB40, MB40-supplemented diet.

Figure 1

Experimental design.

Figure 2

(a) Liver and (b) spleen bacterial counts 7 days following intraperitoneal Listeria challenge (LM) in animals fed basal diet (Basal) or diet supplemented with B. subtilis MB40 spores (MB40). The box and whisker plots show the median, interquartile range, and minimum and maximum values (n = 8 per group). Statistical differences were determined by unpaired t-tests.

Figure 4

Relationship between organ bacterial counts and blood monocyte concentrations in (a) liver, (b) liver × diet, (c) spleen, and (d) spleen × diet 7 days following Listeria challenge in animals fed basal diet (Basal) or diet supplemented with B. subtilis MB40 spores (MB40). Statistical differences were determined by Pearson correlation. The areas shaded in gray indicate the 95% confidence interval.