| ABSTRACT Shigellosis of chicken is a zoonotic infectious disease caused by shigella.spp. With widely popular and high incidence, this disease can cause chicken's dysentery on different species and different day-old, and can also cause the death of young chicks. The spread of this epidemic disease has caused great economic losses to the poultry industry in China, at the same time, it has given a serious influence on human security and public. So, making the diagnosis as early as possible to prevent and treat shigellosis of chicken better has become the key to control it. Therefore, to establish practical and rapid diagnostic method is receiving increasing attention of domestic poultry industry. In this study, the flat agglutination test and indirect ELISA methods of diagnosising shigellosis of chicken have been established and applied in clinical. The aim is to provide the ideal method to diagnosis shigellosis of chicken, which have great specificity, sensitivitily and practicality,and provide technical support for preventing and controlling the shigellosis of chicken.The research carried out two serodiagnosis methods of the flat agglutination test and indirect ELISA using Shigella from chicken which was isolated and identificated in the laboratory.1. Choose a variety of reaction conditions of plate agglutination test methods using the lectin antigen and its standard serum of Shigella from chicken. The results showed that:the best agglutinating antigen concentration was 4×109/ml, the best dilution of carbolic acid was 0.5% saline, and the optimum pH was 7.2, the optimum reaction temperature was 20℃～35℃, the best reaction time was between 3 and 5 minutes, the reaction conditions were established, the disease diagnosis of plate agglutination test was established. The results show that the method is specific,rapid,accurate and user-friendly. The establishment of serological diagnosis provides the important conditions and the reliable basis for rapid diagnosis and effective control of the shigellosis.2. The plate agglutination test was used to diagnosis 810 chicken serum samples which were collected from different parts of Henan Province, the positive rate of S.boydii and S.dysenteriae were 31.5% and 14.6%. The results show that the method had specificity, reproducible and reliable results for clinical epidemiology in the investigation, while got to know epidemiological characteristics of shigellosis.3. Successfully establish an indirect ELISA method of diagnosising chicken shigellosis by using formaldehyde inactivated whole bacteria of Shigella as a diagnostic antigen, horseradish peroxidase labeled rabbit anti-chicken IgG-HRP as the second antibody, TMB as color reagent. By a series of trial on indirect ELISA reaction conditions, determine the optimal composition of the working conditions:ELISA plates used in the coefficient of variation was 4.19% to achieve the requirements of ELISA; the optimum coating antigen concentration of chicken shigella bacteria was 108cfu/mL, the optimum coating conditions was 4℃overnight; to be found, the best dilution of serum was 1:160, with the coating antigen at 37℃role 1h; ELISA antibody concentration for optimal work was 1:4000, the reaction conditions for rabbit anti-chicken IgG-HRP was 37℃role 1.5h; the optimum conditions for the closure is 2% gelatin at room temperature role 1.5h; the best optimal reaction conditions for substrate are at room temperature role 15min; the critical value of negative and positive was 0.414. The specificity test and repeatability test show that the establishment of an indirect ELISA method had high specificity, sensitivitily and reproducibility, and can be used for diagnosis of chickens infected with Shigella and detection of antibodies.4. By application of established indirect ELISA method for diagnosis,589 chicken serum samples were tested which were collected from different parts of Henan Province, 309 were found positive serum, the positive rate was 52.46%, which was 11.71% higher than that detected by plate agglutination test. The results showed that this method can be used to clinical diagnosis of chicken shigellosis and epidemiological with high value of popularization and application. |
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