Preliminary Study On The Agent Of Porcine Proliferative Enteropathy In Guangxi

Porcine Proliferative enteropathy(PPE) is a contagious infectious disease,caused by Lawsonia Intracellularis(L.Intracellularis),which lead worldwide disease of possessing important economic meaning. PPE is global pandemic and has an increasing tendency of the clinical susceptible cases in domestic. It also has high infection rate in herds. L. Intracellularis ,seemed as one of key agents of PEDC, has wide host range and causes tremendous economic losses to the swine industry throughout the world. Therefore, the study of its epidemic and the agent will provide useful information for prediction of the disease and healthy development of the livestock.In the study, we optimize a PCR diagnostic method which is capable of detecting PPE with feces,and amplificate the L.Intracellularis gene from suspectable pathological changes ileum of pig using Nest-PCR. It shows that the isolate from this study had 98.9% to 100% nucleotide sequence homology with the isolates of L.Intracellularis from GenBank. This is confirmed that the pig herds in Guangxi province were infected with L.intracellularis. The infection rate of the slaughtered pigs from slaughterhouse and the weaned pigs from pig farm were 15.6% and 13.5% , respectively. Attention should be paid to this infection rate.We successfully inoculate the GXNN strain L.intracellularis to rabbit using ileal mucous membrane of infected pig. Animal infected test shows L.intracellularis from pigs can be reserved in rabbits and artificially infect mice. Since similar pathogenicity between rabbits and mice has been observated,we establish a preliminary model for L.intracellularis infection. We reproduce PPE by artificially infection, and confirm L.intracellularis is the agent of the desease. Organization of artificially infection pigs was made in sections,stained with HE, and proliferating crypt epithelial cells of ileum and colon were observated.we design specific primers to clone 16SrRNA gene of GXNN strain L.intracellularis.Comparison of nucleotide sequence shows that GXNN strain has 98.4% to 100% nucleotide sequence homology with the isolates of L.intracellularis from other species of anmals,71.8% to 88.6% homology with the other species of prokaryotic microorganisms.We clone three outer membrane protein genes (LI1022, LI1024,LI0902) and one of surface lipoprotein gene (LI0235 )of GXNN strain L.intracellularis. It shows 99.8%, 99.5%, 99.8%, 99.8% nucleotide sequence homology beteen GXNN strain and AM180252 of L.intracellularis, 99.4%, 98.8%, 99.3%, 98.9% amino acids sequence homology beteen two strains,respectively. We inserted four genes into the prokaryotic expression vector PET-32a+,respectively. The constructed recombinant expression plasmids,identified by sequencing,were transformed into the receptive cells of E.coli BL21,and induced 5 hours by IPTG with a finial concentration 1mmol/L at 20℃.It shows the recombinant protein PET32a+-LI1022, PET32a+-LI0902 were expressed by Western blotting using 6×His MAb. It also shows recombinant protein PET32a+-LI1022 probably have antigenicity to react with serum of infected rabbit by Western blotting.