Formula milk alters microbial diversity in porcine colon and impacts immune response
Authors: Saraf Manish Kumar , Bowlin Anne K , Chintapalli Sree V. , Shankar Kartik , LeRoith Tanya , Ronis Martin J. , Badger Thomas M , Yeruva Laxmi 
Published: June 2021

Formula feeding in infants is associated with increased risk of upper respiratory tract infections, allergies and gut dysfunction possibly by compromising gut immune system. We hypothesized that formula feeding alters the host microbiome, resulting in morphology changes and modulation of large intestine immune response. This hypothesis was tested in piglets fed formula diet from postnatal day (PND) 2 to PND 21(n=12, either sex, age‐matched) and compared to sow fed control group (n=12) by measuring the distal colon microbiome, cytokine mRNA and histomorphometry of proximal (PC) and distal colon (DC) on day 21. Microbial richness and diversity was measured via 16sRNA amplicon sequencing using V4 variable primers. Data analyses were carried out by using QIIME 1.9.1. Principal component analyses (PCoA) indicated clear separation of the formula fed group from the control sow group. Formula feeding showed significantly more microbial diversity (Bonferroni corrected, p=0.01) in comparison to the sow fed group. The order of microbial abundance was Bacteroidietes>Firmicutes> Proteobacteria>Verrucomicrobiae at phylum level in both formula and sow‐fed groups. Formula feeding showed 2‐ to 8‐fold abundance of Bacteroidaceae, Porphyromondadaceae, Rokenellaceae, Odoribacteraceae within Bacteroides phylum at family level in comparison to sow group. However, in the sow fed group 5‐fold higher Paraprevotellaceae was observed (FDR corrected, p<0.01). In Firmicutes only Streptococcus spp was 5‐fold higher in formula fed piglets (FDR corrected, p<0.05). Furthermore, 11‐fold higher Gammaproteobacteria and 20 folder higher Verrucomicrabae was observed in formula fed pigelts (FDR corrected, p<0.01) in comparison to sow‐fed, indicating that formula‐fed piglets show more microbial diversity than sow fed piglets in distal colon. Cytokine analyses showed 1.5‐ to 2.0‐fold increases (p<0.05) in gene expression of BMP4 (gut mucosal barrier dysfunction), CCL21 (critical for dendric cell migration), CCL25 (helps in gut‐specific migration of leukocytes), CSF3 (granulocyte development), VEGF‐A (growth factor), and 1.5‐ to 4‐fold (p<0.05) decreases in CXCL‐11 (Th1‐associated chemokines), IL‐27 (B and T lymphocytes regulation) in PC and DC in formula group, suggesting the participation of specific cytokines in alteration of mucosal barrier and activation of gut‐associated immune response. No significant changes were found in length and perimeter of the large intestine; however, a significant increase in crypt density (p<0.05) was noticed in PC of formula‐fed group, suggesting alterations in colon morphology. In summary, formula diet‐driven microbiome changes accompanied alterations in colon crypt density and cytokine response. Studies are being carried out to determine if the formula diet impacts on mucosa membrane are a consequence of microbial interactions with the developing gut‐associated lymphoid tissue.Support or Funding InformationUSDA‐ARS Project 6026‐51000‐010‐05S.