Identification Of Avian Toll Like Receptors On Avian Pasteurella Multocida And The Regulation To Downstream Signaling Molecules

Fowl bacilli is a contact septic infection caused by Avian Pasteurella multocida.The infected birds are chickens,turkeys,ducks,geese and so on.Usually,acute infection is the main cause of infection,with high infection rate and high mortality rate.At present,the prevention of avian cholera mainly depends on antibiotics,inactivated vaccine,attenuated vaccine and subunit vaccine.Antibiotics are susceptible to drug resistance,while other vaccines are not immune.The delay in developing the ideal vaccine may be due to the neglect of congenital immunity,especially the lack of understanding of the innate immune mechanism of Pasteurella infection.In order to explore the innate immune mechanism of avian Pasteurella C48-1 infected poultry,this study first established a fluorescence quantitative PCR for detecting Ch TLRs,signal transduction molecules and inflammatory cytokines,and used to detect the change of ChTLRs expression in HD11 cells and chickens infected with C48-1.The RNA interference technique was used to interfere with ChTLR.The expression of downstream related signal molecules and inflammatory cytokines was detected by fluorescence quantitative PCR.The activation of MAPK family and NF-?B by C48-1 was detected by Western blotting.Finally,protein inhibitors were used to inhibit MAPK family and NF-?B signaling pathway and research P38 MAPK,JNK,ERK and NF-?B regulation role of inflammatory cytokines.The results of the study:(1)Established fluorescence quantitative methods for detecting Ch TLRs(ChTLR1.1,ChTLR1.2,Ch TLR2.1,ChTLR2.2,ChTLR4,ChTLR5 and ChTLR15)? signal transduction molecules MyD88,TRIF,TRAF6,NF-?B and inflammatory cytokine(IL-1??IL-6?IL-12?TNF-?)and ?-actin,with good sensitivity,good reproducibility and strong specificity.(2)HD11 cells and chickens were infected with Pasteurella C48-1 and the expression of ChTLR15 was significantly up-regulated at different times and different tissues,indicating that the Toll like receptor for the identification of C48-1 was ChTLR15.(3)After ChTLR15 gene silencing,it was found that ChTLR15 recognized the C48-1 infection and passed signal to TRAF6 and NF-?B via MyD88 dependent pathway,and also found that the inflammatory cytokine,IL-1 ?,IL-6,IL-12,TNF-?)was also regulated by ChTLR15,indicating that ChTLR15 mediates the inflammatory signaling pathway.(4)Western blotting found that C48-1 could rapidly activate ERK,P38 and JNK proteins in the MAPK signaling pathway and activate NF-?B to enter the nucleus,indicating that NF-?B and MAPK participate in the innate immune response.(5)After the cascade of NF-?B and MAPK signal was inhibited,it was found that the release of TNF-? was related to P38 MAPK pathway,the release of IL-1? was related to the ERK pathway,the release of IL-6 was related to the P38 MAPK,ERK and NF-?B pathway and the release of IL-12 was related to all of them.Therefore,this study further enriched the innate immune response mechanism of poultry to Pasteurella multocida,and provided the theoretical basis for the development of a safe and efficient new vaccine of Pasteurella multocida.