| The genus Edwardsiella is within the family Enterobacteriaceae and is composedof three species: Edwardsiella hoshinae, Edwardsiella ictaluri, and Edwardsiella tarda.Edwardsiella tarda is one of the gram-negative facultative aerobic pathogens found inmany animals including fish, reptiles, amphibians, and humans. Most commonly, it isassociated with edwardsiellosis of fish and resulted in important loss of aquacultureworldwide. Now many researchers are focusing on the molecular pathogenesis of E.tarda infection and the design of efficacious vaccines against edwardsiellosis. Thedetection of specific antibody is an efficiency way to prevent the breakout of diseaseby finding out the specific disease in early stage and validating the efficiency of thevaccine. However, the normally methods of antibody detection need special skills inthe lab with the help of costly equipments which would not be used by the peoplewithout special skill, especially in the field diagnosis. Therefore, an easy and fast wayto detect the antibody is need to be developed. In this study, animmunochromatographic strip was developed for the serological detection ofantibodies against Edwardsiella tarda in turbot, Scophthalmus maximus.1. The preparation and application of inactivated vaccine and its antiserum fromeight common pathogens of Flatfish.In this study, formal dehyde was used to inactivate Edwardsiella tarda, Vibrioanguillarum, Vibrio harveyi, Vibrio vulnificus, Vibrio alginolyticus, Aeromonas caviae,Vibrio fluvialis, Aeromonas hydrophila, in order to preparethe inactivated vaccine.Then vaccine was used to immune turbot by intramuscular injection. In this way,wegot the antiserum of all bacterium above. Enzyme-linked immunosorbent assay wasused to determine the content of antibody in serum. The antibody can be detect afterfour week in the immune, and the level of antibody is research highest in the tenth week which is inoculated inactivated vaccine of Edwardsiella tarda, and the highesttiter of other vaccines are1:102400,1:6400,1:6400,1:12800,1:12800,1:6400,1:6400,1:12800. The results of cross reaction of the bacteria and their anti-serumshows that the effect is most intense when the bacteria react with its own antiserum,and it is very weak when the bacteria react with other antiserum.2. Characterization of monoclonal antibodies against turbot serum immunoglobulinM.Using monoclonal antibody of turbot serum immunoglobulin M, which wasprepared in our laboratory, as experimental material, we used Western blot and ELISAto detected the titers, sensitivity and specificity of monoclonal antibody. The resultsshowed that monoclonal antibody can recognize the heavy chain of purified IgM fromturbot. The monoclonal antibody do not react with Ctenopharyngodon idellus,Cyprinus carpio, Aristichthys nobilis and Carassius cuvieri. It have weak positivereaction with Lateolabrax japonicas, Hexagrammos otakii, Sebastodes fuscescens,Takifugu rubripes and Paralichthys olivaceus. The monoclonal antibody have strongpositive reaction with Scophthalmus maximus, the titer is1:1.024×106. Themeasuring sensitivity of the monoclonal to purified turbot IgM was as long as32ng/ml. These result shows that the monoclonal antibody have high sensitivity andspecificity.3. Development and application of a colloidal gold immunochromatographic stripfor rapid detection of antibory to Edwardsiella tarda in turbot.Thirty nanometer colloidal gold was produced by using trisodium citratereducing anrum chloride, and the monodispersed colloidal gold was synthesized byelectric heating of microwave and sodium citrate reduction. It was proved thatcolloidal gold was homogeneous, without fragments and agglutination characterizedby visual observation and ultraviolet-visible absorption spectroscopy. The optimizedprotein amount of conjugation between colloidal gold and monoclonal antibody wasconfirmed as10μg/ml. The colloidal gold-antibody was checked by using Dot-Elisato certify whether it could react with the antibody against Edwardsiella tarda or not.The result shows that the colloidal gold–antibody can react with the antibody against Edwardsiella tarda. In the strip, the colloidal gold-antibody conjugation wasdispensed on a porous glass fiber to form the gold conjugated released pad,Edwardsiella tarda and staphylococcal protein A were blotted on the nitrocellulosemembrane for the test and control lines, respectively.The positive serum samples from turbot vaccinated with Edwardsiella tarda andnegative serum samples from normal turbot were collected to evaluate thecharacteristics of the strip, and the results will be compared with the method ofenzyme linked immunosorbent assay (ELISA) established in this study. The resultsshowed that coincidence rate between positive and negative were100%, with goodstability and high specificity. The sensitivity for Edwardsiella tarda anti-serum is1:102400, and consistent with the ELISA results. The strip did not react withanti-serum against Vibrio anguillarum, Vibrio harveyi, Vibrio vulnificus, Vibrioalginolyticus, Aeromonas caviae, Vibrio fluvialis and Aeromonas hydrophila.The gold immunochromatographic test strip in this research is proved to besimple, accurate and specific for rapid detection of antibody against Edwardsiellatarda with no requirement of specialized equipments, reagent or professional skills.Therefore, the kit is a practical tool for rapid detection of antibody againstEdwardsiella tarda in turbot. |
PDF Full Text Request