Enterotoxigenic Escherichia coli K88 (ETEC-K88) is a primary causative agent of post-weaning diarrhea in piglets, yet most in vitro infection models evaluate epithelial injury at a limited number of endpoint time points, failing to capture the temporal dynamics of host–pathogen interactions. Here, we established a time-resolved ETEC-K88 challenge model in IPEC-J2 cells from 0 h to 48 h. The data showed that ETEC-K88 adhesion significantly increased after 4 h and peaked after 24 h, with a critical response transition at 4–8 h characterized by coordinated changes in mRNA expression of tryptophan metabolism, tight junction, aquaporins, and Solute Carrier Transporters (SLC). Additionally, we also evaluated the protective effects of L-tryptophan supplementation in IPEC-J2 with ETEC-K88 infection and found that its addition significantly restored the disrupted gene expression related to tryptophan metabolism, transporter channels, aquaporins, and cell cycle. Finally, two different mouse strains, C57BL/6J and BALB/c mice, were challenged with ETEC-K88 to assess strain- and segment-specific intestinal responses. Although overt diarrhea was not clearly induced in mice, the ETEC-K88 fimbria receptor genes were induced in both mice strains. Additionally, both mice strains exhibited obvious intestinal histomorphological abnormalities and showed the strain- and segment-specific expression of intestinal stem cell marker genes (Lgr5, SOX9), goblet cell marker gene TFF3, and aquaporin genes. In conclusion, we have defined a temporal epithelial response framework for ETEC-K88 infection in IPEC-J2 cells and mice, providing a theoretical basis for developing nutritional strategies against ETEC-associated intestinal dysfunction in pig production.

NingXiang (NX) pig has been recognized as one of the most famous Chinese indigenous breeds due to its characteristics in high fertility and stress resistance. However, the global intestinal gene expression profiling and the feature of gut microorganisms in NX piglets have not been studied. Here, we compared the intestinal microbiome and transcriptome between NX and Duroc × Landrace × Large white (DLY) piglets and found the high enrichment of several colonic Bacteroides, Prevotella and Clostridium species in NX piglets than DLY piglets. Further functional analyses of these microbes revealed their predominant function in methane, glycolysis and gluconeogenesis metabolism. Our mRNA-sequencing data unraveled the distinct colonic gene expression between these two pig breeds. In particular, NX piglets exhibited improved intestinal barrier function and cellulose digestibility, as well as varied immune features than DLY piglets. Finally, by integrating metagenomic and transcriptomic analyses, we further showed that the improved intestinal function in NX piglets may be determined by intestinal genes through modulating the composition of the gut microbes. Together, our study revealed the intestinal characteristics of NX piglets, providing their potential application in improving breeding strategies and developing dietary interventions.