Establishment And Study On The Protoplast Fusion Of Esherichia Coli And Riemerella Anatipestifer

Ducks are often mixed infectious by Esherichia coli and Riemerella anatipestifer, which leads to a high mortality, high culled rate and very serious economic losses. Inactivated vaccines are used to prevent these two bacterial diseases at this stage.. In this study, ZYD2 and YL1 as parent bacterial strains for protoplast fusion were selected from drug sensitive test and serological test. Erythromycin and Gentamycin as drug resistance tag induced Riemerella anatipestifer YL1 (Erys, Gen1) and duck Esherichia coli ZYD2(Eryr, Gens). The best time of protoplast formation was obtained from bacterial growth curve:7h for YL1 and 5h for ZYD2. The optimized concentration of antibiotics in hyperosmotic selected culture medium is 20μg/mL for Ery,20μg/mL for Gen.Protoplast formation was got by Lysozyme-EDTA method, and protoplasts fusion can be successfully obtained through PEG method. Finally, based on the drug resistance tag, the fusion strains were got from the hyperosmotic selected medium. In this study,64 strains were regarded as fusions by observation on colonial morphology, characteritic of colouration with Gram-Solution, Serum type test, cultural characteristics and biochemical identify.4 of all the fusions had the typical characteristics between YL1 and ZYD2. In the process of protoplast fusion, the concentration and action time of lysozyme were systematic optimized. Riemerella anatipestifer YL1 was added with lysozyme 0.1 mg/mL with 20 min. Esherichia coli ZYD2 was added with 0.2mg/mL with 20 min.The 4 selected fusions were hereditary stability which has the characteristics of two parent strains after continuous culture without any change. Median lethal dose of the 4 fusions are higher than that of any parent strain (LD50 of DL4 is 2.8 x 108 CFU/mL, LD50 of DL5 is 1.7X 108 CFU/mL, LD50 of DL8 is 1.Ox 109 CFU/mL, LD50 of DL29 is 4.4×109 CFU/mL, LD50 of YL1 is 5.0 x 107 CFU/mL, LD50 of ZYD2 is 1.5 x 107 CFU/mL), and the toxicity are lower than the two parent strains. The protection rate of YLi were 40%after initial immunization, and 60% after secondary immunization; The protection rate of ZYD2 were 50% after initial immunization, and 70%after secondary immunization. The antibody which can be resisted antigen YL1 and ZYD2 were induced by means of the fusion inactive accine inoculation. The antibody level reached the peak after 27 days, and began to fall aftoul lays. The highest antibody titer of YL1 was 4.8 afrer the initial immunization, and 5.2 after the secondary immunization. The highest antibody titer of ZYD2 was 4.6 after the :''iii!i Innination, and 4.9 after the secondary immunization.The duck Eshcrichia coli. and Riemerella anatipestifer fusion strains were successfully obtained. The study show that the fusion strains have the characteristics of the two parent strains. The bivalent inactive vaccine of the fusion can induce antibody to resist the parent strain and have protective effect to some degree. This study lay solid foundations for protcv,ius'i.,1< ii duck Esherichia coli. and Rieru iclla anatipestifer and provide information and data for bivalent multiple inactive vaccine of fusion strains.