Study On Pathogenicity Of EDSV To Breeding Ducks

Egg drop syndrome virus belongs to adenovirus type Ⅲ, which can cause the dropping of egg production accompanied by the production of soft-shelled or shell-less eggs in laying hens and make serious economic loss. The disease made egg production rate of chicken decreased by 20% to 30%, breakage rate of egg reached 30% to 40%, which was listed as one of the most serious four viral diseases in chicken industry of the world. Van Eck first reported the occurrence of this disease in the Netherlands in 1976 and EDS virus was isolated in 1977.Since 1991, EDS spread rapidly from north to south, from east to west and widely popular in China. EDSV was isolated from a chicken farm for the first time in Nanjing that named NE4 strain in 1991 and duck-original EDS virus was isolated from which appeared reduction in egg production in 1992 named JE1 strain. Egg drop syndrome-76(EDS-76) virus is a pathogen which is usually considered apathogenic in ducks and as one of the natural reservoirs. Since the virus was firstly isolated from ducks with laying drop and severe diarrhoea in 1984, more and more researches on duck egg drop syndrome were studied in China and abroad. In recent years, researches in China and abroad showed that: the ducks infected with EDS-76 virus could also be pathogenic to ducks and cause the lessening of egg production under certain conditions. So strengthen the research on the disease can help to protect and promote the development of poultry industry of China.In this study, sixty-two healthy 62-week-old ducks were randomly divided into two groups.Every group had 31 ducks which included 10 ducks used to observe changes of egg production, 21 ducks used to collect the necessary materials. The infected group was artificially inoculated with the challenge virus SD01 strain in 1.5 ml 10-3.6EID50/0.2 mL undiluted amnio-allantoic fluids by oral route. Incidence and clinical symptoms of experiental ducks were observed, birds were euthanized at different times to observe changes in the tissues and organs. Tissues, cloacal and oropharyngeal swabs were gathered to detected the viral copys and viral shedding of experimental ducks. Clotted blood samples were gathered from random birds to detect the change of EDSV antibody titers, TL-4 and IFN- γ. The organs were fixed in 10% neutral buffered formalin, prepared by conventional paraffin sections and HE staining to examine changes of histopathology. The results showed that, mild respiratory symptoms and the decline of egg production were observed in infected ducks.The levels of IL-4 and IFN-γ in challenged group were significantly higher than controls; The most distinct microscopic lesions were hemorrhage of spleen and glomerulus, edema of oviduct mucosal, swelling and degeneration of liver cells, hemorrhage and inflammatory cellsinfiltration of ovary and follicle.Highly conserved hexon protein gene were chose to design a couple of pecific primers and one probe. PCR products were cloned to the pMD18-T vector plasmid, the screened plasmid was 10 tiems dilution as the template for building a standard curve of Taqman-MGB fluorescence quantitative PCR. The results showed that, established Taqman-MGB quantitative PCR method could detect minimum concentration template of about 10Copies/μL and standard curve had a good linear relationship, R2 value of 0.99. The results of specificity test indicated that, in addition to external amplification curve of EDSV other virus had no amplification curves which indicated the method could specifically detect EDSV;reproducible test results showed coefficient of variation of intra and inter were both less than1.5%. The above study indicated that the Taqman-MGB fluorescence quantitative PCR method established in this test is stable, specificity, high sensitivity, and can be used for early detection of EDSV.Using the Taqman-MGB fluorescence quantitative PCR method to detect the viral loads in various tissues of breeding ducks at 3, 7, 14, 21, 28, 35 dpi and viral shedding of cloacal and oropharyngeal swabs. The results showed, viral loads were various in different tissue and times. EDSV could detect form the different organizations during experimental period after inoculation. Where the viral load in liver, fallopiantube, was higher than other organs, trachea and intestine were the lowest.