| Gallibacterium anatis (G.anatis) is the type species of Gallibacterium which is grouped as a new genus within the family Pasteurellaceae. As a important pathogen to affect the egg production, G.anatis could infect layers to cause a series of chronic and progressive diseases of reproductive tracts, such as salpingitis and oviduct cysts. The researches mainly studied on the pathogenicity and etiopathogenesis of G.anatis in recent years, but there is a little study on serology of G. anatis.A indirect enzyme-linked immunosorbent assay (ELISA) was developed in this study for detection of all serotypes antibodies against G.anatis in chickens. The coating antigen was made by ultrasonie method with the strain YU-PDS-RZ-1-SLG (1-S) of G.anatis. The optimum G.anatis antigen concentration was found to be 10μg·mL-1, coating time was 2 hours at 37℃and then overnight at 4℃. The blocking material was 1% gelatin and blocking time was 1 hour at 37℃. The dilution of the positive or negative sera was 1:100, and the optimum dilution of rabbit-anti-chicken IgG labelled with horseradish peroxidase was 1:1000. The optimum reaction time of the enzyme-substrate was determined as 15 min. The indirect ELISA was confirmed to be reproducible and specific by cross-assay, blocking-assay and reproductive assay. The coefficient of variation within plate was 2.01%5.75%, and the coefficient of variation with between plates was 2.43%6.20%. The sensitivity of indirect ELISA was 25 to 100 times than micro-agglutination test. Three groups of 20 three-day-old SPF chinkens were used and divided into 3 groups, one group was inoculated with 1.4×106 colony-forming units of G.anatis strain 1-S, the other two groups were used as cohabitating group and negative control group. The indirect ELISA technique was used for testing the antibodies against G.anatis from these chickens in different time after infection. The response curve was established when the OD450nm values were varies along with the time, the changes of the antibodies were observed that the antibody level reached the peak in two months post-infection,but the lasting duration of the antibodies was short,and then the antibody level dropped gradually. The results showed that the indirect ELISA could be used for detecting the sera of chickens from clinical cases and to predict the efficiency of vaccination. Lipopolysaccharide(LPS) obtained from G.anatis strain 1-S was used as coating antigen for detection of antibody against G.anatis, and the optimum work conditions for indirect ELISA were developed as follows: the concentration of coating antigen was 2.77μg·mL-1 ;coating time at 37℃was 1.5 hours and then at 4℃overnight; the blocking material was 1% gelatin and blocking time at 37℃was 1 hour; the dilution of the positive or negative sera was 1:1600; the optimum dilution of goat-anti-mouse IgG labelled horseradish peroxidase was 1:3000, and the optimum reaction time of the enzyme-substrate was determined as 15 min.Three hybridoma cell strains secreting monoclonal antibodies (McAbs) against G. anatis LPS were developed by cell fusion after immunizing the Balb/c mice with G. anatis LPS antigen and named as 1H11, 1B12 and 4D4. These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing-thawing three times within a three-month storage at -196℃.The ELISA titers of antibodies against G. anatis LPS in culture supernatant were 1:3200, 1:6400 and 1:3200 ,and in ascites were 1:80000, 1:100000, 1:80000 for strains 1H11, 1B12 and 4D4, respectively. Pathogen detection of indirect ELISA showed the McAb had no cross-reaction with Pasteurella multocida, Brucella melitensis, Chicken Sh. flexneri and E.coli.The subtypes of three McAbs were IgM, IgG2a and IgG2a, respectively. The development of the three hybridoma cell strains secreting McAbs against G.anatis LPS laid the foundation for rapid and accurate monitoring of pathogen diagnosis and relevant scientific research of G.anatis in chickens. |
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