Isolation, Identification And Study On Pathogenicity Of Duck Plague Virus SDWF Strain

Duck plague(DP) is an acute septic infectious disease that caused by duck plague virus (DPV), and characterized by its high prevalence, morbidity and mortality. It is going to serious threat to the development of duck industry.Since the first report of DPV in the Netherlands by Baudet in1923, it was spread rapidly and widely in many countries. In1957, Yinxian Huang reported duck plague in Guangzhou, China. So far, it had been reported in all the country areas raising ducks. In order to efficiently prevent and control DPV, it’s essential to establish simple, rapid and sensitive methods for diagnosis and detection of DPV, and further study of its pathogenesis. Four parts are contained in this present research:Part1:Isolation and identification of duck plague virus SDWF strainA strain virus (SDWF) isolated from a suspected case of duck plague through duck embryo allantoic membrane was identified as duck plague virus (DPV) by serum neutralization test, animal experimental infection and PCR identification. The isolated virus has the same serum type as the standard virulent virus. SPF ducks infected artificially showed the same or similar clinical symptoms and pathological changes as the natural infections in the pathogenicity test. The virus does not agglutinate red blood cells of other animals and it is sensitive to chloroform and ether. It also can not tolerate acid,alkali and heat. Toxicity tests showed that ELD50of SDWF-strain is10-433/0.2mL. It was identified by PCR, and the amplification products were sequenced. It revealed that the amplification result is right, compared with the standard virulent strain, the gene of DPV-SDWF has the homology99.9%in nucleic acids level.Part2:Development and application of a loop-mediated isothermal amplification assay for the rapid detection of duck plague virusTo develop a rapid and sensitive method for detecting duck plague virus(DPV), a loop-mediated isothermal amplification(LAMP) assay was established using three pairs of specific primers. The assay was optimized to amplify DPV DNA by incubation at63℃for1 hour. The results visualized directly or under the UV light with added SYBR Green I dye. The diagnostic method was sensitive and specific, for the amplification results of duck hepatitis virus, H9N2avian influenza virus, duck paramyxovirus and duck avian metapneumovirus were negative, and the lowest detection limit of0.245pg/μL, which was10-fold higher than the conventional PCR. The positive rate of forty duck samples suspected duck plague was30%. Therefore the LAMP assay developed in this study provided a rapid and practical method for DPV detection.Part3:The dynamic study of histopathology of SPF duck infected with duck plague virus virulent strainTwo-month-old SPF ducks were inoculated with duck plague virus(DPV), slaughtered in different period and observed the histopathology changes of tissues and organs. At the same time the blood were collected to be carried out the routine blood test and the detection of blood parameters. Results revealed that24hours later, the number of lymphocyte of experimental ducks’central immune organs, including thymus and bursa, decreased and tissue space enlarged. While the histopathology of liver and spleen was serious, other organs was slight.48-96hours later, the number of lymphocyte decreased severely in central immune organs, hyperplasia of lymphocytic netted cell, blurry configuration, serious hyperemia and hemorrhage, some irreversible pathological changes, cell degeneration and hemorrhage, were observed.120hours later, cell degeneration and focal necrosis of tissue appeared. Histological changes of ducks infected by conjunctival and intraocular-nasal vaccination were similar to those subcutaneous injection appeared with a delay of24-48hours. Histopathology of control group was not observed. WBC,HGB,AST and ALT showed obvious changes. These results suggested tissues and organs, especially the immune organs, of SPF ducks inoculated DPV severely damaged, which even resulted in immunosuppression.Part4:Application of indirect immunofluorescence assay(IFA) for detection and antigen location of duck plague virus virulent strain in paraffin sectionsTo obtain the primary antibody, two clean New Zealand rabbits were vaccinated with oil-emulsion vaccine of DPV. The rabbit anti-DPV specific IgG was extracted by caprylic-ammonium sulfate law method, purified by Sephadex G200chromatography after dialyzing. An IFA test was developed to the rapid diagnosis of DPV by using the anti-DPV IgG as the primary antibody and the fluorescein isothiocyanate(FITC) conjugate goat anti-rabbit IgG as the second antibody. The research suggested that the dilution of primary antibody was1:100and sections were incubated overnight at4℃. Then sections incubated at37℃for1h with diluted FITC-labeled-secondary antibody (1:200). The IFA was applied to detect the DPV antigen in different organs of the artificially infected SPF ducks. The IFA was applied in detecting the virulent DPV antigen in different organs of the artificially infected adult ducks, and the viral antigen was detected in the spleen, thymus, bursa, liver, esophagus, duodenal, renal, lung and trachea of dead infected ducks. The fluorescence intensity of liver and spleen is the strongest, which suggested that the target organs that DPV mainly attacked are liver and spleen.