The Study On Immunoprotection And Virulence Of Haemophilus Parasuis NanH And LsgB Gene Deletion Strains

Haemophilus parasuis is a Gram-negative bacterium,which is a species of opportunistic pathogen in swine upper respiratory tract.This bacterium can cause Glaser's disease when the pig is under low immunity or in stress.Although the virulence factors and pathogenic mechanism of Haemophilus parasuis are still unclear,the surface sialylation of pathogens is an important mechanism for evading and resisting the host immune system.In addition,vitro experiments have shown that the sialidase of Haemophilus parasuis and Sialyltransferase is closely related to bacterial virulence.so this thesis researched about piglets of immunoprotective effect and pathogenic ability of Haemophilus parasuis sialic acid gene deletion strain JS0135?nanH and sialyltransferase gene deletion strain SH0165?lsgB.The main results of this study were as follows.1.Immune protection of piglets by JS0135?nanH and SH0165?lsgBJS0135?nanH and SH0165?lsgB were immunized piglets by muscle route and intraperitoneal route,commercial inactivated vaccine and PBS immunization as positive control and negative control,and then challenged with virulent strain SH0165.The results showed that the antibody level of H.parasuis in the serum of piglets increased,which show that the antibody level of piglets in JS0135?nanH group increased faster than that of SH0165?lsgB group,and the piglets in SH0165?lsgB group were faster than commercial inactivated piglets;JS0135?nanH piglets immunized by the intraperitoneal route were significantly faster than the piglets immunized by the muscle route;There was no significant difference between the two immunization routes in the SH0165?lsgB group.After the piglets were challenged,the immune protection effects of different immunized groups were comprehensively evaluated,which show that JS0135?nanH was superior to SH0165?lsgB,and SH0165?lsgB was superior to commercial inactivated vaccine.2.Wild and mutant strains infected cells stimulate cell inflammatory factorsJS0135 and JS0135?nanH,SH0165 and SH0165?lsgB were infected into 3D4/2 cells respectively;After 4 hours,the mRNA expression levels of inflammatory cells were detected,and IL-1?,IL-8 and TNF-? were differentially expressed.Then,at 2h,4h and 8h,the expression of IL-1?,IL-8 and TNF-? was detected,and the results showed that the deletion strain produced higher inflammatory factors than the wild strain stimulator cells(P?0.01).3.Wild and mutant strains challenge pigletsJS0135 and JS0135?nanH,SH0165 and SH0165?lsgB were challenged respectively on piglets;By recording the clinical symptoms,gross pathological changes and tissue and organ division of piglets,which results show that the pathogenicity of SH0165?lsgB relative to wild strain SH0165 was significantly decreased,JS0135?nanH had no significant change in the pathogenicity of piglets relative to wild strain JS0135;and JS0135?nanH was cleared completely in the survived piglets.