**Background:** Salmonella infections threaten poultry production and public health. The gut microbiota and its metabolites (e.g., short-chain fatty acids) play critical roles in pathogen exclusion and immune modulation. Chickens with a low heterophil/lymphocyte (H/L) ratio have been shown to be more resistant to environmental stressors and infections. This study aimed to identify candidate genes and signaling pathways associated with resistance to Salmonella Enteritidis (SE) colonization mediated by the intestinal microbiota and its metabolites.
**Methods:** A total of 200 one-day-old Jinxing yellow chicks were housed in sterilized isolation cages and fed SPF feed. At 7 days old, all birds were orally challenged with 1 mL PBS containing 1×10^10 CFU of SE 50335. Cecal samples and blood were collected at 7 and 21 days post-infection (dpi). H/L ratios were determined from blood smears. Cecal propionate and valerate concentrations were measured by GC-MS. Cecal microbiota composition was assessed by 16S rRNA sequencing. Transcriptome profiling of cecum tissues was performed on 22 samples (4–7 per group) using RNA-seq (Illumina). Differentially expressed genes (DEGs) were identified using DESeq2 (|log2FC| ≥ 1, padj < 0.05). Developmental dynamics genes (DDGs) were detected using maSigPro. Weighted gene co-expression network analysis (WGCNA) was performed on 19 samples using 19,501 genes with a soft threshold of 8, yielding 27 co-expression modules. Hub genes were identified using |GS| > 0.5 and |MM| > 0.5 (p < 0.01). qRT-PCR validation was performed on cecum tissues from Dagu chickens infected with Salmonella Typhimurium (1.5×10^13 CFU/mL) at 24 h post-infection.
**Key Results:** Chickens with low H/L ratio showed significantly reduced body weight loss at 21 dpi, lower cecal bacterial load at 7 dpi (p = 0.0053), and increased propionate and valerate cecal contents. Cecal microbiota analysis revealed significantly higher relative abundance of Firmicutes (p = 0.030), Bacteroidetes (p = 0.0014), and Proteobacteria (p = 0.0090) in low H/L chickens. DEG analysis identified 276 DEGs (H7 vs. L7), 155 DEGs (H21 vs. L21), 855 DEGs (H7 vs. H21), and 737 DEGs (L7 vs. L21). Key DEGs included FKBP5 (upregulated in low H/L at 7 dpi), CEMIP (upregulated in low H/L at 21 dpi), EXFABP (log2FC = 4.23, padj = 1.21E−11, L7 vs. L21), and S100A9 (log2FC = 5.69, padj = 5.71E−07, L7 vs. L21). PPAR signaling pathway was significantly enriched in H7 vs. L7; oxidative phosphorylation was enriched in H21 vs. L21. WGCNA identified 10 modules highly correlated with traits. The blue module (r = 0.85, p = 5e−06) and brown module (r = 0.61, p = 0.005) were positively correlated with dpi; the yellow module (r = −0.75, p = 2e−04) was negatively correlated. The blue module was positively correlated with body weight (r = 0.83, p = 1e−05) and negatively with bacterial load (r = −0.77, p = 1e−04). The yellow module was negatively correlated with propionate (r = −0.64, p = 0.003) and valerate (r = −0.65, p = 0.003). The green module showed significant enrichment in immune-related GO terms (inflammatory response, lymphocyte activation, T cell activation) and KEGG pathways (cytokine-cytokine receptor interaction, Toll-like receptor signaling, intestinal immune network for IgA production). Hub genes included NDUFAF8 (blue module), MAVS (brown module), TADA2A (green module), and C2CD5 (yellow module). qRT-PCR validation confirmed significant upregulation of FKBP5 and S100A12, and significant downregulation of EMC6, FAM168B, and HESX1 in ST-infected vs. non-infected Dagu chickens.
**Clinical Implications:** This study provides a comprehensive gene co-expression network linking host genetics, gut microbiota, and microbial metabolites to Salmonella resistance in chickens. The PPAR signaling and oxidative phosphorylation pathways, along with candidate genes FKBP5, S100A9/12, CEMIP, EXFABP, and MAVS, represent potential targets for breeding Salmonella-resistant poultry or developing interventions. The findings suggest that low H/L ratio chickens mount an early strong immune response (via FKBP5, S100A9/12) followed by anti-inflammatory and antimicrobial activity (via CEMIP, OXPHOS) to clear infection and restore intestinal homeostasis. The positive correlation of Proteobacteria with immune-related gene modules (green module) suggests this phylum may play a role in adaptive immune maturation. These insights could help reduce economic losses in poultry production and improve food safety by controlling Salmonella colonization.