Raw potato starch changes the butyrate-producing microbiota and host immune responses in

pigsJulian Trachsel, Cassidy Briggs, Crystal Loving, Heather Allen

• Food Safety and Enteric Pathogens Research Unit1) Prevent foodborne illness such as Salmonella and E. coli (pre-harvest)

2) Find alternatives to antibiotics

• How can we maintain a safe (and profitable) food supply while reducing medically important antibiotic use in our farming systems?

Motivations

Motivations

• Recent focus on young piglets, post weaning• The most common time for antibiotic intervention, often prophylactic.

• Weaning is very stressful • Massive dietary change

• Transport stress

• First contact with non-littermate pigs

• Transitional microbiota• Less effective pathogen exclusion

• Critical window for training and modulation of the immune response

BUTYRATE

Transcriptional changes in host epithelia

Mucus SecretionAntimicrobial

PeptidesTight Junction

proteins

Preferred colonocyte energy source

pH reduction and pH dependent

antimicrobial activity

Immunomodulatory Activity

Induction of Treg cellsCD4+ T-cell anergy

Reduced epithelial sensitivity to IFN-γ

Reduced macrophage pro-inflammatory mediators

Tolerance of the microbiota and reduced inflammation

Maintenance of Colonic Epithelial Barrier

Butyrate reduces oxygenation of the intestinal epithelia

Pimonidazole staining (red) indicates hypoxia

Chavez et. al., Cell Host Microbe. 2016 Apr 13;19(4):443-54.

Mucosal pathogens benefit from community disturbance• Many pathogens do better with a little oxygen

• Salmonella

• E. coli

• Citrobacter

• Brachyspira

• Campylobacter

• Reduce epithelial oxygen potential to limit colonization?

• Some pathogens thrive during inflammation• Salmonella

Modulating butyrate producing community as a preventative intervention

• Young piglets do not have a well established butyrate-producing community, or microbiota in general

• Maybe partially responsible to susceptibility to enteric pathogens, (oxygenated epithelia, lack of competitive exclusion etc.)

• Can we speed the establishment of butyrate producers to help improve resistance to proteobacteria or other pathogens?• Selectively feed beneficial microbes

• Raw Potato Starch (RPS) prebiotic• Resistant starch, escapes host digestion

• Highly fermentable in the hindgut

RPS alters cecal VFA profiles

p = 0.2 p = 0.05 p = 0.01

RPS changes the abundance of several genera in the fecal microbiota

but gene amplicon: specific profiling of the butyrate-producing community• Developed a degenerate primer set to target the but gene

• Centrally important for butyrate production in the gut

• Trachsel et. al, AEM, 2016 Sep 9. pii: AEM.02307-16

• Very similar to 16S analysis, just specific to butyrate producers

• Allows us to see specifically which butyrate producing bacteria are reacting to a particular treatment

RPS significantly changes the butyrate-producing community

Lymphocytes from RPS treated pigs decrease secretion of pro-inflammatory cytokines in response to LPS stimulation

p = 0.004 p = 6.0e-4 p = 0.003

RPS fed pigs have altered T-cell populations in their cecal tissues

p=5.8e-4 p=0.017 p=5.8e-4

RPS fed pigs have indications of a more robust epithelial barrier

p = 0.1 p = 0.03 p = 0.03

Conclusions

• Inclusion of 5% raw potato starch in piglet diets causes significant changes in the intestinal microbiota as well as the host immune response to commensal microbes

• Increased butyrate production

• Improved tolerance of commensal microbes

• Improved epithelial barrier function

• Reduced niche for facultative anaerobes, Proteobacteria etc.

• Raw potato starch may help ease the stress of weaning and reduce prophylactic antibiotic use