Abstract :
[en] Internationally, foodborne diseases are receiving increasing attention. Salmonellosis is one of the most common foodborne diseases worldwide, accounting for around 93.8 million foodborne illnesses and 155,000 deaths per year. As a foodborne disease, Salmonella Enteritidis (SE) not only causes huge economic losses to the poultry industry, but also seriously threatens human public health. Poultry are considered to be important sources and carriers of the disease. SE also tends to be highly resistant to multiple antimicrobials, which has the potential to complicate treatment of animal and human disease. An improved understanding of host immunological resistance and response mechanisms in chickens should be a top priority. The aim of the research described in this thesis was divided into two parts: (1) To identify the splenic microRNAs and mRNAs that were differentially expressed following infection of chickens with Salmonella Enteritidis using RNA sequencing; (2) To investigate whether fructooligosaccharides (FOS) addition alters the expression of inflammatory genes involved in MyD88-dependent signaling in immune tissues during Salmonella infection. To achieve these objectives, the research strategy involved construction of an improved disease model, transcriptome screening, identification of resistance genes and pathways, and evaluating the effect of FOS addition, both by dietary supplementation and by exposure of chicken immune cells in vitro.
In the first part, differentially expressed microRNAs and gene transcripts (mRNAs), as well as signaling pathways were investigated in resistant (R, SE challenged-slight clinical symptoms and < 105 cfu SE / 10 μL blood), susceptible (S, SE challenged-severe clinical symptoms and > 107 cfu SE / 10 μL blood) and control birds (C, non-challenged, no SE in blood) using the splenic microRNAome and transcriptome. A total of 934 significant differentially expressed (DE) genes and 32 DE miRNAs were identified in comparisons among the C, R and S birds. First reported here, the DEG involved in the Forkhead box O (FoxO) signaling pathway, especially FoxO3, were identified as potential markers for host resistance to SE infection. There was evidence of cross-talk among these pathways, perhaps contributing to susceptibility to Salmonella infection, including the FoxO signaling pathway, cytokine-cytokine receptor interaction and Jak-STAT signaling pathway. Importantly, TLR4 signaling was also significantly enriched among C, R and S birds. In addition two DE miRNAs, gga-miR-101-3p and gga-miR-155, directly repressed luciferase reporter gene activity by binding to 3'-untranslated regions of the immune-related target genes IRF4 and LRRC59. When gga-miR-155 and interference gga-miR-101-3p were over-expressed in chicken HD11 macrophages, expression of their target genes was significantly altered. These two miRNAs were identified as candidates being potentially associated with SE infection.
The second part of the study investigated whether provision of FOS altered the expression of inflammatory genes involved in MyD88-dependent signaling during Salmonella infection. It was first necessary to determine the optimum level of adding dietary FOS for effective protection against Salmonella infection. One day-old specific-pathogen-free (SPF) chicks were fed throughout with a basal diet containing 0, 10, 20, and 30 g/kg supplemental FOS. Three days post-hatch, all chicks were orally challenged with SE. Lower concentrations of dietary FOS reduced the bacterial burden in liver and cecum, decreased the serum levels of interleukin-1β, and decreased relative abundance of TNF-α and IL-6 transcripts in spleen and cecal tonsils. These results suggest a beneficial effect of adding FOS, probably at 1% of the diet of young chicks, prophylactically against SE infection.
The effects of dietary FOS addition on the expression of inflammatory genes and TLR4 signaling genes were also investigated in young chickens and direct actions of FOS on chicken macrophages challenged with the bacterial toxin, LPS, were examined. Three days post-hatch, birds from two treatment groups (diets with or without 1% FOS) were also orally challenged with SE or vehicle PBS. Dietary FOS significantly reduced the gene expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as the transcript abundance of inflammation-related pathway genes TLR4, MyD88, TRAF6 and NF-κB in spleen and in cecal tonsils during S. Enteritidis infection in young chickens. Using HD11 chicken macrophages in vitro, exposure to FOS directly increased the expression of IL-6 and TNF-α and reduced the extent of increase in abundance of pro-inflammatory factors, otherwise provoked by added LPS. Taken together, these findings provide novel information that FOS may reduce production of the pro-inflammatory cytokines through TLR4-MyD88-dependent signaling during the early stages after Salmonella infection. It is emphasized that further research of this direct immunomodulatory role of FOS on TLR4 signaling is warranted.
In conclusion, this research with chicks has systematically exposed novel information on the immune mechanism of the host in providing some protection against Salmonella by use of a high-throughput sequencing combined with an improved experimental design strategy. Several important signaling pathways and miRNAs have been identified and will be the focus of future research. In addition, evidence for FOS having a direct regulatory influence on innate immunity in chickens was obtained. These mechanistic findings will help facilitate the understanding of resistance and susceptibility to Salmonella infection in the earliest phases of the host immune response, they will provide new approaches for developing strategies for Salmonella prevention and treatment, and they may aid in enhancing innate resistance using genetic selection.
