Effects Of Reticuloendotheliosis Virus Infection On Interleukin-18 Production In The Primary Immune Organs Of Broilers

IL-18(Interleukin-18, IL-18), also known as IFN-γinducible factor (interferon-gamma-inducing factor, IGIF), is a kind of multi-directional and multi-level immunomodulatory cytokine which is closely associated with the occurrence and development of carcinoma, allergy, auto-immune diseases, and graft-versus-host disease. Therefore, IL-18 will play an important role in basic research and clinical applications of many diseases. Now intensive immunosuppressive disease has brought great harm to the poultry industry in China. There have been many reports that REV(Reticuloendotheliosis virus, REV) infection can cause serious immunosupression of humoral and cell-mediated immune responses. Understanding the mechanism of immunosuppression and developing strategies to improve the immune responses of chickens have become the current research hotspots. In this study, immunosuppression caused by REV was used as a model and the role of chicken IL-18 in the immune suppression was discussed. And this study was divided into two parts:1. A real-time reverse-transcription PCR(RRT-PCR) based on SYBR Green I for detecting chicken IL-18 was established successfully. According to the chicken IL-18 gene sequence available in GenBank, a pair of primers was designed for developing a SYBR GreenⅠquantitative real-time PCR method to detect chicken IL-18 gene while the chicken ribosomal protein subunit 4(RPL4)gene was used as an internal control. To establish the standard curve, the positive plasmid of each gene served as a standard. The melting curve was also analyzed. The results showed that the amplification efficiency of this assay was 107.16%, and the linear range was 10-2~10-6 with good correlation coefficient of 0.998, as well as the detection limit reached 100 copies/μL. The melting curve presented a single peak at(80.4±0.6)℃. It only took 4 hours from extracting RNA to the end of real-time fluorescent quantitative RT-PCR. The developed real-time PCR assay could quickly detect IL-18 gene in expansion range with high efficiency, thus providing the basis for quantitative analysis of IL-18 gene expression.2. Study on IL-18 gene expression in the primary immune organs of broilers infected with REV by real-time quantitative RT-PCR. In order to study expression levels of IL-18 gene in broilers infected with reticuloendotheliosis virus(REV), 120 one-day-old broilers were randomly divided into 2 groups. The treatment group chickens was infected with REV while the control group was inoculated with saline. Six chickens of each group were randomly chosen and killed at day 7,14,21,28,35 and 42 post infection. Spleen, thymus and bursa were sampled for RNA extraction. SYBR GreenⅠq uantitative real-time PCR method was applied to compare the relative expression quantity of broiler IL-18 gene. The following results were obtained: IL-18 gene expression levels were significantly higher at day 7,14(P<0.05) and were significant at day 21,28,35 and 42(P<0.05) in the spleen, thymus and bursa in broiler chickens from treatment group than those in the control group. Our study laid a certain foundation for investigating the mechanism of immune suppression of REV infection.