Effects Of H/L Ratio Level On The Immunomodulatory Mechanisms Of Intestinal Microbiota Against Salmonella Infection In Chicken

The heterophil/lymphocyte(H/L)ratio reflects the chicken’s robustness and immune system status.The H/L ratio is considered as a disease resistance trait,and it could be used as a marker for selecting Salmonella resistance in live chickens.Previous studies have shown that the H/L ratio is heritable and individuals with low H/L ratio are superior in terms of resistance to Salmonella,immune response,survival,performances and others beneficial traits.However,the association of the H/L ratio with Salmonella resistance and the inflammatory response,the characterization of the gut-microbiota composition of individuals resistant to Salmonella,the determination of the bacterial taxa involved in the resistance to Salmonella and the host-genes associated with the traits which influence significantly the resistance to Salmonella infection remains to be elucidated.Therefore,this study aimed to establish the links between the H/L ratio and the resistance to Salmonella mediated by the gut-microbiota and identify the host-genes associated with the immune traits providing this resistance.To achieve these objectives,3 major experiments were conducted using the Jinxing yellow chicken H/L line: The first experiment used 360 Jinxing yellow chicks of one day old involved in an infection trial of 21 days.The chicks were divided into non-infected and Salmonella Enteritidis(SE)infected groups.In this experiment,the effects of SE infection on some immune traits and inflammatory responses during the infective cycle(at 1,3,7 and 21 dpi)have been evaluated.Then the correlations with the H/L ratio has been established.The second experiment involved chickens from the SE group(at 7 and 21 dpi),which were divided into low and high H/L ratio subgroups.The 16 S rRNA and metagenomic analysis were conducted to examine microbiome and functional capacity between the 2 groups,and Short Chain Fatty Acids(SCFAs)and histopathology were conducted to explore the potential difference between susceptible and resistant groups at 7 and 21 days post-infection(dpi).This experiment led us to identify the potential bacterial taxa involved in the resistance to Salmonella infection in chicken.The third and last experiment used the same experimental design as the second,with the only exception that transcriptome and weighted gene co-expression network analysis(WGCNA)were performed.In the third experiment,the candidate genes(including driver and hub genes)promising for Salmonella resistance mediated by the gut-microbiota and its metabolites were identified.From these experiments meaningful results were obtained.In the first experiment,it has been found that the H/L ratio is associated with Salmonella resistance mediated by increased inflammatory response(IL-1β and INF-γ blood serum concentrations)and improved intestinal immunity.It was remarkable that chickens with low H/L ratio were more resistant than chickens with high H/L ratio.The exploration of the cecal microbiota composition of individuals with low and high H/L ratio SE-infected revealed that low H/L ratio chickens,compared to high H/L ratio chickens,displayed a significantly higher abundance of Proteobacteria(Escherichia coli)and Bacteroidetes(Bacteroides plebeius)at 7and 21 dpi,respectively.Anaerostipes(r = 0.63)and Lachnoclostridium(r = 0.63)were identified as bacterial genus significantly correlated with H/L(P < 0.001).Interestingly,Bacteroides was significantly and positively correlated with bodyweight post-infection(r = 0.72),propionate(r = 0.78)and valerate(r = 0.82)contents,while Salmonella was significantly and negatively correlated with bodyweight post-infection(r =-0.67),propionate(r =-0.61)and valerate(r =-0.65)contents(P <0.001).Furthermore,the comparative analysis of the functional capacity of cecal microbiota of the chickens with high and low H/L ratio revealed that the chickens with low H/L ratio possess more enriched immune pathways,lower antibiotic resistance genes,and virulence factors compared to the chickens with high H/L ratio.In the third experiment,Differential gene expression(DEGs)and developmental dynamic genes(DDGs)analyses,and weighted gene co-expression network analysis(WGCNA)were performed using transcriptome data from caecum of Salmonella Enteritidis infected chicken at 7 and 21 days after infection.DEGs analysis identified,FBXO32,FKBP5,NDUFAF8,IL8,TIMD4,SOCS3,IGF2BP3,PER2,EXFABP,MST1,S100A12,and others genes.Besides,based on the DDGs analysis,HESX1,SMAD5,IRF1,RAF1,NFATC3,NFKBIB and JAK2 showed significant changes in their expression from 7 to 21 dpi.Furthermore,the driver and hub genes associated with important traits such as the heterophil/lymphocyte ratio,body weight post-infection,bacterial load,propionate and valerate cecal contents,and Firmicutes,Bacteroidetes and Proteobacteria cecal relative abundance were identified.Among the multiple genes detected as highly correlated with each of the traits used in this study,FAM168 B,HESX1,USP8,C2CD5,PIGC,EMC6,NFU1 and others were found as potential candidate gene and transcript(co-)factors for resistance to Salmonella infection.Taken together,these results suggest that the chickens with low H/L ratio are more resistant to Salmonella,and it is possible that the commensal Proteobacteria and Bacteroidetes are involved in this resistance against Salmonella infection.Moreover,these results lay the foundation of the complex interactions among chicken Salmonella host-microbiome and associated metabolites.Our study suggests that genes with significant temporal change during Salmonella infection could be potential candidate genes.This thesis provided valuable resource of microbiome and transcriptome profiles from chicken caecum at the earlier and later stage post-infection.The bacterial taxa and candidate genes identified in this thesis can be used in further molecular studies and selection for improvement of resistance to Salmonella infection in chicken.