Flavanol-Rich Cocoa Powder Interacts with Lactobacillus rhamnossus LGG to Alter the Antibody Response to Infection with the Parasitic Nematode Ascaris suum

Abstract

Consumption of the probiotic bacteria LactobacillusrhamnosusLGG and flavanol-rich cocoa have purported immune modulating effects. This study compared the host response to infection with Ascaris suum in three-month-old pigs fed a standard growth diet supplemented with a vehicle control: LGG, cocoa powder (CP) or LGG + CP. Pigs were inoculated with infective A. suum eggs during Week 5 of dietary treatment and euthanized 17 days later. Lactobacillus abundance was increased in pigs fed LGG or LGG + CP. Specific anti-A. suum IgG2 antibodies were decreased (p < 0.05) in LGG + CP-fed pigs compared to pigs fed CP alone. Pigs fed LGG had significantly reduced expression (p < 0.05) of Eosinophil peroxidase (EPX), Interleukin 13 (IL-13), Eotaxin 3 (CCL26), Toll-like receptor 2 (TLR2), TLR4, and TLR9 and Interleukin-1Beta (IL1B) in the tracheal-bronchial lymph node (TBLN) independent of CP treatment. These results suggested that feeding LGG significantly reduced the localized prototypical Th2-related markers of infection with A. suum in the TBLN. Although feeding CP does not appear to affect the A. suum-induced Th2-associated cytokine response, feeding LGG + CP reduced anti-A. suum antibodies and delayed intestinal expulsion of parasitic larvae from the intestine.

Keywords: LGG; cocoa flavanols; gut microbiota; porcine model; toll-like receptors; type-2 immune response.

Figure 1

Ascaris suum fourth-stage larvae (L4) from intestinal luminal contents 17 days post-inoculation. Data represent mean larvae counts ± SEM (n = 8 per treatment). Different letters denote differences among treatments after ANOVA (p < 0.05).

Figure 2

Bacterial LGG abundance in feces of pigs fed different diets. Lactobacillus rhamnosus abundance was measured by real time PCR and converted to copy number per gram of feces (cpg). Values shown are means ± SEM (n = 8 per treatment). Different letters denote differences among treatments after ANOVA (p < 0.05).

Figure 3

Gene expression related to a type-2 immune response in Tracheobronchial lymph nodes (TBLNs) and Mesenteric Lymph nodes (MLNs). Gene expression in TBLNs and MLNs were measured by RT-PCR (n = 8/group): (A) EPX (Eosinophil peroxidase); (B) IL-13 (interleukin 13); and (C) CCL26 (C-C motif ligand 26). Bars represent a mean fold change. The control group was designated as one-fold change. Labeled means without a common superscript letter differ (p < 0.05).

Figure 4

Toll-like receptor gene expression in TBLNs and MLNs. Toll-like receptor (TLR) 2 (A); TLR4 (B); TLR9 (C); and IL-1β (D) gene expression in TBLNs and MLNs were measured by RT-PCR (n = 8/group). Bars represent a mean fold change. The control group was designated as one-fold change. Labeled means without a common superscript letter differ (p < 0.05).

Figure 5

Anti-Ascaris IgG2 antibody response. The anti-Ascaris IgG2 antibody response was measured using A. suum proteins extracted from isolated third and fourth stage larvae and serum obtained 10 days post-inoculation with infective A. suum eggs. Bound serum antibody at a 1/1000 dilution was detected using biotinylated anti-pig IgG2 followed by streptavidin-HRP complex. Values shown are means ± SEM (n = 8 per treatment). Labeled means without a common letter differ, p < 0.05.