Isolation And Identification Of Goose Astrovirus And Application Of Its Yolk Antibodies

Astrovirus is a non-enveloped,single-stranded positive-strand RNA virus with a wide range of hosts.It mainly causes enteritis,nephritis,and viral hepatitis in birds.In recent years,a disease with gout as the main symptom has broken out in many places.The sick goose has pale and swollen kidneys,urate deposits in the liver and heart,and other tissues and organs.In severe cases,urate deposits also appear in joints and serosa.Therefore,the purpose of this study is to isolate and identify the pathogens that cause the disease,and then establish a detection method,and use the detection method to screen a suitable laboratory breeding system,prepare yolk antibodies to achieve treatment of the disease,and reduce losses in the goose breeding industry.The virus isolation and characteristics were studied in this paper by using of RT-PCR detection,electron microscope observation,animal regression experiments and gene sequence analysis.After the isolated virus was inoculated into a 10-day-old goose embryo through the chorioallantoic membrane,the goose embryo appeared dead and showed reddening and swelling of the embryo body,growth inhibition,thickening of the chorioallantoic membrane,and white deposits at the inoculation site.The specific bands appeared obviously while the specific PCR amplification primers were used to detect the goose embryos from 1?10 generations.Virus particles with a diameter of 28?30 nm were observed in the collected allantoic fluid by electron microscopy.In the animal regression test,the goose showed a mortality rate of 40%.The dead goose displayed urate deposits on the surface of the heart and liver,and the kidneys were obviously pale and swollen.Urine particles were observed in bile and joint.The distribution of the virus in the infected goslings showed that the virus detection rate was the highest between 2?8 days after infection.Further investigation of the incidence of goslings under the condition of high protein feed showed the more severe symptoms under the condition of high protein feeding.The ORF1b,ORF2 and whole gene sequences of the isolated virus were to be in the same branch as the reported SDPY,SD01,and GD strains,and the homology was above 98.2%.According to the results of virus isolation and identification,the pathogen causing the disease was finally identified as goose astrovirus and named as GAstV-AH.In order to conveniently detect the astrovirus and screen the virus propagation system,a real-time quantitative PCR(SYBR Green I)detection method was established according to the designed primers of ORF2 sequence of the isolated strain in this study.The results showed that the established method has a good specificity,sensitivity and reproducibility.Under the optimized conditions,a standard curve is constructed.The curve R2=0.999,the amplification efficiency is 102.8%and the linear equation output by the real-time quantitative PCR system is Y=-3.257X+35.317.The results of viral load in the main tissues of the infected goose at different time point showed that the viral load in all tested tissues was much higher from day 2 to 8 and then gradually decreased.The viral load was no longer changed significantly after virus challenged from day 20 to 30.Meanwhile,three goose astrovirus propagation systems such as goose embryos,primary goose embryo kidney cells,and LMH cells were studied.The results showed that goose embryo was a more suitable virus propagation system for subsequent inactivated vaccine preparation.After immunized three times with the inactivated vaccine,the neutralizing titer of the antibodies of egg yolk was 1:512.After injected with 0.5 mL,1 mL,and 2 mL of antibody subcutaneously into the neck of 1-day-old goslings,the goslings did not show pathological manifestations and the injection site was well absorbed.The experiment using 0.5 mL and 1 mL of antibodies to treat virus-infected goslings showed that 1 mL of antibody injection can protect 100%protection rate from virus challenge.The results of goose body weight also showed that there is no difference in weight between the treatment group and the healthy control group,however there is a significant difference between the treatment group and the virus infection control group.It indicated that the prepared antibody is safety and has therapeutic effect,and provided reference for the subsequent antibody development.