| Shigellosis of chicken is a new infectious disease, which found in recent years. The disease is an acute infectious disease caused by Shigella, which can give rise to different species, different age of chicken diarrhea and death of chickling. The current study results show that Shigellosis of chicken was widespread and harmful. Because of the spread of Shigellosis, the poultry industry of China has tremendous economic losses. Shigella may cause cross-infection between human and poultry, which led to serious security risks for human public health. Therefore, the study of Shigellosis of chicken not only has important veterinary health significance, but also has great medical public health significance. The topic has established two methods for the detecting of Shigella boydii from chicken using Shigella boydii as researching materials.The finding as follow:1 Establishment of the Indirect ELISA method for the Detecting of Shigella boydii Antibody from chicken by using LPS as a coating antigenSuccessfully establish an indirect ELISA method of detecting Shigella boydii by using extracted LPS from S.boydii in chicken as a coating 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, the optimal working conditions are defined: the optimum coating antigen concentration of LPS from chicken S.boydii is 5μg/ml, the optimum coating conditions is 4℃overnight; the best dilution of serum to be found is 1:160, with the coating antigen at 37℃for 1h; Rabbit anti-chicken IgG-HRP concentration for optimal work is 1:2 000, and the reaction conditions is 37℃for 1h; the optimum condition for the closure is 37℃for 1h; the best reaction conditions for substrate is at room temperature in dark for 20 min; the critical value of negative and positive was 0.385. The specificity test,sensitivity and repeatability test show that the establishment of an indirect ELISA method had high specificity, sensitivitily, repeatability.By application of established indirect ELISA method for diagnosis, 268 chicken serum samples were tested which were collected from different parts of Henan Province, 93 were found positive serum, the positive rate was 34.70%. The results showed that this method can be used to serology diagnosis of chicken shigellosis and epidemiological investigation with significance value of popularization and application.2 Establishment of immunoperoxidase staining assay to detect Shigella boydii from chicken in the paraffin wax tissue splice and its application for antigen localization of infection chicken tissue.2.1 Estabilishment of the immunoperoxidase staining assay to detect Shigella boydii from chicken in the paraffin wax tissue spliceThe mouse were immunized with purified bacillus to obtain S. boydii-specific antibody,which were then extracted by caprylic-ammonium sulphate method,purified through DEAE columns.An immunoperoxidase staining method was established to detect the S.boydii antigen in paraffin slices.2.2 The application for S. boydii localization of infection chicken tissueDetected and located S. boydii in each organ tissues of dead chicken experimentally infected with S. boydii.The result showed that the immunoreactions can be observed in the heart,liver,spleen ,lung ,kidney, duodenum, ileum, appendix and rectum after intraperitoneal infection,and the positive signal mostly located in cytoplasm and cytolemma of tissue cells,phagocyte,monocytes,lymphocyte and so on .The result could be used to provide foundation study for the pathopoiesis mechanism of S. boydii from chicken. |
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