| Clostridium perfringens can cause a variety of Clostridium perfringens disease in animal,and easy to cause human food poisoning,gas gangrene,Which are seriously affect human health,so the diagnosis of Clostridium perfringens disease is particularly important.Each type of Clostridium perfringens can produce alpha toxin,so the detection of alpha toxin can provide strong evidence for the diagnosis of Clostridium perfringens disease.The research used the following methods to diagnose 30 suspected sick rabbits which were collected from three large-scale rabbit farms during from October 2015 to December 2016 in Shandong Province.A preliminary diagnosis was made on the suspected sick rabbits with clinical symptoms,autopsy results and histopathological examination.Bacterial separation and identification,DAS-ELISA and immunohistochemistry were used as the main methods for diagnosis of rabbits which are suspected of being infected with Clostridium Perfringens.Clinical symptoms observation and the results of necropsy showed that the clinical features and the pathological changes of suspected sick rabbits were consistent with that of rabbits which are infected with Clostridium perfringens.The rabbits which are suspected of being infected with Clostridium Perfringens were characterized by the following pathological changes: granular and vacuolar degeneration of liver cells,partial liver cell necrosis,hepatic sinusoidal dilatation,granular and vacuolar degeneration in cardiac muscles,necrosis and exfoliation of gastric mucosa,congestion of submucosal blood vessels,necrosis and exfoliation of mucosal epithelial cells in the small intestine,necrosis and exfoliation of caecum mucosal epithelial cells,neutrophil infiltration of lamina propria,congestion of submucosal blood vessels,granular and vacuolar degeneration of renal tubular epithelial cells,and renal interstitial hyperemia and hemorrhage.A total of 25 Clostridium perfringens were isolated by bacterial separation,the separation rate was 83.3%.Using multiple PCR identification,all of the isolated strains were Clostridium perfringens type A.The monoclonal antibody against alpha toxin was used as capture antibody,a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)was established by optimizing reaction conditions and establishing a standard curve.And the concentration of alpha toxin(expressed as a multiple of mouse LD50)was used as the abscissa of the standard curve,OD450 nm was used as the ordinate of the standard curve.Then,the performance of DAS-ELISA was evaluated and the DAS-ELISA was also applied in clinical detection.For DAS-ELISA,the variable coefficients of intra batch wre less than 10%,and the variable coefficients of inter batch were less than 12%.The results showed that the method had good repeatability and stability,Clostridium perfringens alpha toxin could be detected at a minimal concentration of 0.353 times mouse LD50,and the results showed that the quantitation range was 0.353-90.368 LD50.Healthy rabbits were used as negative control,the results showed that the concentration of alpha toxin in diseased rabbits’ intestinal contents were significantly higher than that of healthy rabbits(P <0.05).It was determined to be positive when the OD450 nm value of the suspected rabbits’ intestinal contents was greater than 0.3578,there were 26 cases of positive rabbits,the positive detection rate was 86.7%.Furthermore,the results of the DAS-ELISA were verified by immunohistochemistry testing results of ileum,stomach,kidney,and liver in diseased rabbits.It was determined to be positive when alpha toxin was detected in one organ of rabbit.There were 27 cases of positive rabbits,the positive rate was 90%.Based on the anti Clostridium perfringens alpha toxin monoclonal antibody(monoclonal antibody MAb),the distributions of alpha toxin on 30 diseased rabbits’ organs were detected by immunohistochemistry.The distributions of alpha toxin were detected in liver,kidney,heart,and so on of naturally infected rabbits.Among them,the signal of alpha toxin is strong in the stomach,liver,kidney.The detection results of several methods showed that the 3 outbreaks of scale farms’ rabbit disease during from October 2015 to December 2016 were related to Clostridium perfringens infection.This study provided an important basis for evaluating the incidence of Clostridium perfringens infection and improved the accuracy of diagnosis.What’s more,it provided a theoretical basis,technical support for the diagnosis and prevention of the disease in this region. |
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