The heterophil/lymphocyte (H/L) ratio has been extensively studied to select poultry that are resistant to environmental stressors. Chickens with a low H/L ratio are superior to the chickens with a high H/L ratio in survival, immune response, and resistance to Salmonella infection. However, this disease resistance ability is likely to be associated with enhanced intestinal immunity. Therefore, to expand our understanding of these underlying resistance mechanisms, it is crucial to investigate the correlation between the H/L ratio as a blood immune indicator in live chickens and the intestinal barrier function and immunity. Jinxing yellow chickens H/L line one-day-old were divided into non-infected (NI) and Salmonella enteritidis infected (SI) at 7-days old. After dividing the birds into NI and SI, blood samples were taken for H/L ratios determination, and subsequently, birds from the SI group were infected with Salmonella enteritidis (SE). We assessed the effects of SE infection on the (i) goblet cells number from the ileum and caecum gut-segments, (ii) ileal mucosa morphology, and (iii) immune gene mRNA expressions from the ileum and caecum of NI and SI chickens at 7 and 21 days-post-infection (dpi). We found that the H/L ratio was negatively correlated with most intestinal immune indices, particularly with the goblet cells number and with IL-1β, IL-8, and IFN-γ ileal expressions. In conclusion, these results suggest that the H/L ratio is associated with the intestinal barrier and immune response for SE clearance and that the chickens with a low H/L ratio displayed enhanced intestinal immunity. This study expands the current knowledge that is related to using the H/L ratio to select and breed resistant broiler chickens.

Heat stress is one of the most prevalent issues in poultry production that reduces performance, robustness, and economic gains. Previous studies have demonstrated that native chickens are more tolerant of heat than commercial breeds. However, the underlying mechanisms of the heat tolerance observed in native chicken breeds remain unelucidated. Therefore, we performed a phenotypical, physiological, liver transcriptome comparative analysis and WGCNA in response to heat stress in one native (Beijing You, BY) and one commercial (Guang Ming, GM) chicken breed. The objective of this study was to evaluate the heat tolerance and identify the potential driver and hub genes related to heat stress in these two genetically distinct chicken breeds. In brief, 80 BY and 60 GM, 21 days old chickens were submitted to a heat stress experiment for 5 days (33 °C, 8 h/day). Each breed was divided into experimental groups of control (Ctl) and heat stress (HS). The results showed that BY chickens were less affected by heat stress and displayed reduced DEGs than GM chickens, 365 DEGs and 382 DEGs, respectively. The transcriptome analysis showed that BY chickens exhibited enriched pathways related to metabolism activity, meanwhile GM chickens’ pathways were related to inflammatory reactions. CPT1A and ANGPTL4 for BY chickens, and HSP90B1 and HSPA5 for GM chickens were identified as potential candidate genes associated with HS. The WGCNA revealed TLR7, AR, BAG3 genes as hub genes, which could play an important role in HS. The results generated in this study provide valuable resources for studying liver transcriptome in response to heat stress in native and commercial chicken lines.

Salmonella causes significant economic loss to the poultry industry and represents a real threat to human health. The region of difference 21 (ROD21) pathogenicity island removal is a genetic mechanism by which Salmonellaenteritidis (SE) invades the intestinal epithelium and induces systemic infection in mice. The heterophil/lymphocyte (H/L) ratio reflects the chicken’s robustness and immune system status. The H/L ratio is considered a disease resistance trait, and it could be used as a marker for selecting Salmonella resistance in live chickens. However, the association of the H/L ratio with Salmonella resistance and the inflammatory response remains to be elucidated. Moreover, the kinetics of ROD21 excision in the intestine and immune organs of chickens is unknown. Therefore, this study aimed to investigate the bacterial load, the ROD21 excision, the IL-1β, IL-8, and INF-γ blood serum concentration kinetics, and the association with the H/L ratio in chicken at 1, 3, 7, and 21 days post-SE infection. The results showed a significant correlation between the H/L ratio and the bacterial load in the ileum and caecum at 7 dpi. The ROD21 pathogenicity island absolute and relative excision in the caecum were positively correlated at 1 dpi but negatively correlated at 7 dpi with the H/L ratio. However, in the liver, we found the opposite tendency. The association of the H/L ratio with IL-1β, IL-8, and INF-γ blood serum concentrations showed that a low H/L ratio is correlated with increased IL-1β and INF-γ at 21 dpi. This study confirmed that the H/L ratio is associated with robustness and Salmonella-resistance in chicken. The methodology used in this study can separate individuals into susceptible and resistant and can help in the selection and breeding of Salmonella-resistant chickens.

Bacterial infections remain an important cause of morbidity in poultry production. The molecular characteristics and dynamic changes in immune cell populations after bacterial infection have yet to be fully understood. Beijing-You chicken and Cobb broiler, two broiler breeds with different disease resistance, were infected with Salmonella typhimurium, and inflammation models were constructed. Compared to Beijing-You, Cobb showed higher survival rates, lower liver load, and milder spleen damage after Salmonella infection. We characterized chicken spleen CD45+ immune cells by single-cell RNA sequencing and identified 9 distinct cell types among 54,487 cells. In Beijing-You, mono-macrophages expressed higher levels of pro-inflammatory factors, including IL1B, IL6, and M-CSF, after bacterial infection. In Cobb, Tregs exhibited intense inflammatory inhibition and highly expressed CTLA4, LAG3 and other immunosuppressive regulators. In addition, we found complex macrophage phenotypes during bacterial infection, with a tendency in macrophages from pro-inflammatory phenotypes (Mac-IL1B) to anti-inflammatory phenotypes (Mac-C1QC/Mac-MARCO). This study represents the first single-cell transcriptomic analysis of chicken spleen and compares the immune responses of Beijing-You and Cobb after bacterial infection. These findings provide insight into the mechanism of inflammation regulation in different broiler breeds.

Salmonella Typhimurium (ST) is a foodborne pathogen that adversely affects the health of both animals and humans. Since poultry is a common source and carrier of the disease, controlling ST infection in chickens will have a protective impact on human health. In the current study, Beijing-You (BY) and Cobb chicks (5-day-old specific-pathogen-free) were orally challenged by 2.4 × 10¹² CFU ST, spleen transcriptome was conducted 1 day post-infection (DPI) to identify gene markers and pathways related to the immune system. A total of 775 significant differentially expressed genes (DEGs) in comparisons between BY and Cobb were identified, including 498 upregulated and 277 downregulated genes (fold change ≥2.0, p < 0.05). Several immune response pathways against Salmonella were enriched, including natural killer-cell-mediated-cytotoxicity, cytokine–cytokine receptor interaction, antigen processing and presentation, phagosomes, and intestinal immune network for IgA production, for both BY and Cobb chickens. The BY chicks showed a robust response for clearance of bacterial load, immune response, and robust activation of phagosomes, resulting in ST resistance. These results confirmed that BY breed more resistance to ST challenge and will provide a better understanding of BY and Cobb chickens’ susceptibility and resistance to ST infection at the early stages of host immune response, which could expand the known intricacies of molecular mechanisms in chicken immunological responses against ST. Pathways induced by Salmonella infection may provide a novel approach to developing preventive and curative strategies for ST, and increase inherent resistance in animals through genetic selection.

Salmonella negatively impacts the poultry industry and threatens animals’ and humans’ health. The gastrointestinal microbiota and its metabolites can modulate the host’s physiology and immune system. Recent research demonstrated the role of commensal bacteria and short-chain fatty acids (SCFAs) in developing resistance to Salmonella infection and colonization. However, the complex interactions among chicken, Salmonella, host–microbiome, and microbial metabolites remain unelucidated. Therefore, this study aimed to explore these complex interactions by identifying the driver and hub genes highly correlated with factors that confer resistance to Salmonella. Differential gene expression (DEGs) and dynamic developmental genes (DDGs) analyses and weighted gene co-expression network analysis (WGCNA) were performed using transcriptome data from the cecum of Salmonella Enteritidis-infected chicken at 7 and 21 days after infection. Furthermore, we identified the driver and hub genes associated with important traits such as the heterophil/lymphocyte (H/L) ratio, body weight post-infection, bacterial load, propionate and valerate cecal contents, and Firmicutes, Bacteroidetes, and Proteobacteria cecal relative abundance. Among the multiple genes detected in this study, EXFABP, S100A9/12, CEMIP, FKBP5, MAVS, FAM168B, HESX1, EMC6, and others were found as potential candidate gene and transcript (co-) factors for resistance to Salmonella infection. In addition, we found that the PPAR and oxidative phosphorylation (OXPHOS) metabolic pathways were also involved in the host’s immune response/defense against Salmonella colonization at the earlier and later stage post-infection, respectively. This study provides a valuable resource of transcriptome profiles from chicken cecum at the earlier and later stage post-infection and mechanistic understanding of the complex interactions among chicken, Salmonella, host–microbiome, and associated metabolites.