Exploring the modulatory role of bovine lactoferrin on the microbiome and the immune response in healthy and Shiga toxin-producing E. coli challenged weaned piglets

Abstract

Background: Post-weaned piglets suffer from F18⁺ Escherichia coli (E. coli) infections resulting in post-weaning diarrhoea or oedema disease. Frequently used management strategies, including colistin and zinc oxide, have contributed to the emergence and spread of antimicrobial resistance. Novel antimicrobials capable of directly interacting with pathogens and modulating the host immune responses are being investigated. Lactoferrin has shown promising results against porcine enterotoxigenic E. coli strains, both in vitro and in vivo.

Results: We investigated the influence of bovine lactoferrin (bLF) on the microbiome of healthy and infected weaned piglets. Additionally, we assessed whether bLF influenced the immune responses upon Shiga toxin-producing E. coli (STEC) infection. Therefore, 2 in vivo trials were conducted: a microbiome trial and a challenge infection trial, using an F18⁺ STEC strain. BLF did not affect the α- and β-diversity. However, bLF groups showed a higher relative abundance (RA) for the Actinobacteria phylum and the Bifidobacterium genus in the ileal mucosa. When analysing the immune response upon infection, the STEC group exhibited a significant increase in F18-specific IgG serum levels, whereas this response was absent in the bLF group.

Conclusion: Taken together, the oral administration of bLF did not have a notable impact on the α- and β-diversity of the gut microbiome in weaned piglets. Nevertheless, it did increase the RA of the Actinobacteria phylum and Bifidobacterium genus, which have previously been shown to play an important role in maintaining gut homeostasis. Furthermore, bLF administration during STEC infection resulted in the absence of F18-specific serum IgG responses.

Keywords: E. coli; Immune modulation; Lactoferrin; Microbiome.

Fig. 1

Timeline of the bLF microbiome trial and F18⁺ STEC challenge in vivo. In both trials, bovine lactoferrin (bLF) was administered orally twice a day (for a total of 500 mg/d) for 10 consecutive days. To study the gut microbiome different samples were taken: faecal swabs were collected during the experiments, as indicated in the timeline, and mucosal scrapings and intestinal content of both ileum and colon were sampled upon euthanasia, indicated by †. The challenge infection with an F18⁺ STEC strain (F107/86) was carried out on D0 and D1. Blood and faeces were collected on different timepoint to determine effect of bLF on F18-specific immune responses and faecal shedding. bLF: bovine lactoferrin, STEC: Shiga toxin-producing E. coli

Fig. 2

Effect of bLF administration on the microbiome richness and diversity in post-weaned piglets. A Chao1, ACE, Shannon and Simpson of all samples, B–D ACE and Simpson of faecal content (B), mucosal scraping (C), and faecal swabs (D). Values represented as mean ± SD; n = 7 (PBS and bLF) and n = 5 (STEC and STEC + bLF). bLF: bovine lactoferrin, LP: lamina propria, PP: Peyer’s patches

Fig. 3

Principal coordinate analysis (PCoA) of bLF administration on the microbiome of post-weaned piglets. A and B PCoA of the microbiome trial experiment (n = 7), C and D PCoA of the STEC challenge trial (n = 5). A and C Faecal content ileum (left) and mucosal scraping ileum LP (right), B and D Faecal swabs. Data was analysed by PCoA analysis using the Bray-Curtis distance. bLF: bovine lactoferrin, LP: lamina propria, STEC: Shiga toxin-producing E. coli

Fig. 4

Effect of LF administration on the relative abundance of bacterial phyla. A and C Stacked bar chart of the 4 most abundant bacterial phyla in mucosal scrapings and faecal content. A Sample from the non-infected groups (bLF and PBS), C Samples from pigs challenged with STEC (STEC and STEC + bLF). B Relative abundance (RA) microbiota mucosal scrapings Ileum from the non-infected group, D RA microbiota mucosal scrapings ileum from the STEC challenge group. bLF: bovine lactoferrin, LP: lamina propria, PP: Peyer’s patches, STEC: Shiga toxin-producing E. coli. Data was shown as mean ± SD; **P < 0.01

Fig. 5

Effects of LF administration on the relative abundance of bacterial genera. A and B Stacked bar charts of the 30 most abundant bacterial genera identified in the faecal content and mucosal scrapings in unchallenged piglets (A) and in F18⁺ STEC challenged piglets (B). C and D Relative abundance of bacterial genera in the mucosal scrapings of colon and ileum with PP and without PP (=ileal LP) in unchallenged piglets (C) and in F18⁺ STEC challenged piglets (D). Al: Alistipes, Bf: Bifidobacterium, Bif: Bifidobacteriaceae (f), Cg: Candidatus Glomeribacter, Ch: Christensenella, Cl: Clostridium, Cm: Campylobacter, Cp: Coprococcus, Ent: Enterobacteriaceae (f), Fc: Faecalibacterium, Gm: Gemmiger, Hl: Helicobacter, Int: Intestinimonas, Lc: Lactobacillus, Ol: Olsenella, Pr: Prevotella, Prp: Propionibacterium, Ps: Pseudobutyrivibrio, Rm: Ruminococcus, Rs: Roseburia, St: Streptococcus, Ws: Weissella, bLF: bovine lactoferrin, IL: ileum, LP: Lamina propria, PP: Peyer’s patches, STEC: Shiga toxin-producing E. coli. Data was shown as mean ± SD; **P < 0.01

Fig. 6

Effect of bovine lactoferrin on the F18⁺ STEC excretion and immune modulatory effect upon F18⁺ STEC challenge infection. A Mean faecal excretion of F18⁺ STEC (log10)/g faeces (± SD). The mean faecal excretion of F18⁺ STEC was calculated by plating faecal dilutions and confirmed by dot blot analysis. The detection limit is represented by a dotted line at 2 log₁₀ STEC/g faeces. B and C: Effect of bovine lactoferrin on the F18-specific serum antibody levels upon F18⁺ STEC challenge, B F18-specific IgA and C IgG serum responses. D F18-specific IgA⁺ antibody secreting cells after oral administration of bovine lactoferrin. ELISpot of F18-specific IgA⁺ ASCs from PBMCs (left) isolated on −1, 9 and 21 days post infection (dpi) and mononuclear cells isolated from mesenteric lymph nodes (middle) and intestinal tissues (right) at 21 dpi. ASC: antibody secreting cells, bLF: bovine lactoferrin, ILP: ileal lamina propria, IPP: ileal Peyer’s patches, JLP: jejunal lamina propria, JPP: jejunal Peyer’s patches, MLN: mesenteric lymph nodes, PBMC: peripheral blood mononuclear cell. Data was shown as mean ± SD; ^*Q < 0.05; ∆ Q < 0.05 (n = 3/group)