| The bursal lesions is the key point of the immunosuppression in chikens infected with infectious bursal diaease virus(IBDV), which belongs to double-stranded ribonucleic acid(dsRNA)virus. Melanoma differentiation associated gene 5(MDA5) is the pattern recognition receptor detecting for ds RNA virus. In the context, 14 days old specific pathogen free chicks infected with IBDV as the research object, using histopathology and molecular biology techniques to detect the expression of chMDA5 signaling pathway associated factors, IBDV RNA load, pathology and morphology changes of chicks bursa. The research is to reveal function of chMDA5 signaling pathway in pathomorphism changes of chicks bursa infected IBDV.Firstly, Total RNA was extracted from the IBDV-infected chick bursa and reversed into c DNA.Then, the IBDV VP2 was amplified by reverse transcription PCR(RT-PCR), at the same time, the prokaryotic expression plasmid pET30a-VP2 was constructed successfully and recombinant protein was expressed with E. coli Rosetta system. Rabbits were immunized with recombination protein to generate rabbit-anti-IBDV VP2 polyclonal antibody. The polyclonal antibody had good reactivity and specificity by western blot, ELISA and IFA.Chick were infected with IBDV standard strain(I group) and immunized moderate-virulent vaccine(V group), which were detected IBDV load and pathomorphism changes of bursa in IBDV infected chicks. The results showed that the relative expression of IBDV mRNA increased continually and rise to a peak at post infection days(PIDs) 4(P<0.01), and then reduced gradually,the expression of IBDV in I and V group close to the control level at PIDs 35 and 21, respectively.The IBDV VP2 protein had the same results. After IBDV standard strain infected in chicks, eye view pathology injury of bursa mainly displayed as atrophied to a degree, serosal surface had gelatin sample exudate and sporadic hemorrhage spots, but only observed as atrophied later. After IBD moderate-virulent vaccine immunized in chicks, only observed as mildly atrophied and then restored gradually. After IBDV standard strain infected in chicks, the histopathology injury major performance showed that inflammatory responses, massive lymphocyte were necrosis or even disappeared, heterophil, macrophages cells infiltrationand and reticular cells proliferated,hyperplasia of connective tissue, the pathological changes recovered gradually at PIDs 35, but there were still apparent atrophy of bursal lymphoid follicles. Chick with the IBD moderate-virulent vaccine, the histopathology injury major performance showed that mild inflammatory responses, and then returned to normal gradually at PIDs 21.The expression changes of chMDA5, ch IPS-1, ch IRF3, chNF-κB, chIFN-β, chIL-6, chTNF-αand chIL-1β were assessed. The results showed that the expression of chMDA5 mRNA and protein,all the cytokines mRNA increased continually and rise to a peak at post infection days(PIDs) 4(P<0.01), and then returned to normal gradually. The above factors of V group had the same results,but the expression lower than the I group.The results indicated that IBDV could activate the chMDA5 signaling pathways in chicks,chMDA5 its signal transduction pathways involved in the process of IBDV infected chicks, in addition, the degree of ativation which is in direct proportion to the virus virulence. IBDV activate the chMDA5 signaling pathways in chicks, before inducing release of inflammatory cytokines,resulting in bursa injury, and the degree of injury in connection with the degree of chMDA5 signal transduction pathway activation.The study from the gene and protein expression levels confirmed ch MDA5 had closely relationship with injury in the IBDV infection chick bursa. The test make further illustrates the process of IBDV infected chicks of the molecular mechanisms, and provide ascientific basis for IBD prevention. |
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