Isolation And Identification Of A New Type Of Astrovirus Causing Gout In Goose And Its Pathogenicity To Goose

A severe infectious disease characterized with gout has affected goslings in Shandong,Jiangsu,Anhui,Henan,Liaoning and Guangzhou provinces in China since February 2017.The disease mainly affects goslings within 2 weeks of age.The goslings have severe urate deposition in the body cavity and joints with a mortality up to 50%,which caused serious economic losses to the goose industry of our country.In order to determine the cause of gosling as soon as possible,this study conducted an epidemiological survey in the affected areas.The epidemiological survey results showed that the feed varieties and drug types used in different regions and different breeds of goslings were different,but the age of onset was concentrated at 5 to 20 days of age,and the symptoms of necropsy were basically the same.Bacterial isolation and PCR detection were performed on the collected materials.The results of bacterial isolation were all negative,while the PCR results were positive for astrovirus,and the positive rate was as high as 93%?316/340?.Based on this,the virus was isolated and identified.The kidney tissue of diseased goslings was collected aseptically and inoculated with 11-day-old goose embryos and goose kidney cells for virus isolation,the virus began to stabilize after three generations of inoculation of goose embryos,which could lead to the death of goose embryos,the time of death was mostly concentrated in 48⁷2 hours,and the mortality rate of goose embryo could reach 90%within 2⁵ days.The embryo showed thickening of allantoic membrane,punctate bloodstains on the skin and peritoneal surface of the whole body,and bleeding spots in the kidney,liver,lung and other organs.GKC began to shrink,round,and fall off after 72 hours of infection.After the allantoic fluid and cell fluid were positive by PCR and free of miscellaneous,a geese-origin astrovirus was isolated and named as AstV/SDPY/Goose/1116/17.The whole gene sequence of SDPY strain was analyzed with 26 strains of AstV from other generations.The results of homology analysis showed that the homology of AStV between SDPY strain and other turkey,duck and goose sources was low,and the nucleotide homology was only 51.8%⁶3.2%,but with the TAstV isolates homology is relatively high,the homology of the nucleotide is 63.2%⁶3.3%.The results of genetic evolution analysis showed that SDPY strain was located in an independent branch and was a new type of AstV.Animal regression test showed that 1 day old healthy goslings infected with SDPY strain after sixth days began to die,dissect the incidence of death has a lot of urate deposition of goose organs visible surface,part of the death of white urate deposition in Goslings joint cavity,and natural infection symptoms consistent.The above results show that this study successfully isolated astrovirus from gosling,and prove that the virus is the main pathogens of gosling gout.A pair of primers and a specific probe were designed based on the GAstV ORF2 gene sequence,and the amplification length was 89 bp.Connect the amplified fragment into pMD18-T vector to construct recombinant plasmid and through screening,sequencing,dilution to construct a standard curve,the establishment of detection method for TaqMan fluorescence quantitative PCR of GAstV after the optimal reaction conditions.There is a good linear relationship between the standard curve method,the linear regression equation is y=-2.9779x+37.196,correlation coefficient R²=0.9977.The sensitivity is good.The minimum concentration of detected template is 10 copies/?L,which is 1000 times higher than that of ordinary PCR.It has good specificity and has no cross reaction with other viruses.The coefficient of variation between batches and between batches was less than 0.7%,and the reproducibility was good.The above experiments show that the TaqMan probe fluorescence quantitative PCR method established in this study has high sensitivity,specificity and reproducibility,and can be used for the clinical detection of GAstV.150 one-day-old healthy goslings were randomly divided into three groups:50subcutaneous challenge group,50 oral administration group and control group.Each subcutaneous and oral challenge group was inoculated with 0.2 mL of virus solution(ELD₅₀was 10^-5.25/0.2mL),the control group was inoculated with 0.2mL of normal saline,and the clinical symptoms and necropsy changes of each group were observed.Every 2 days weighing was used to monitor the change of body weight.Three cloacal swabs,three non-anticoagulant swabs and three anticoagulant swabs were collected to monitor the rule of detoxification,the change of viral content in blood and the biochemical indexes in serum.Three goslings were randomly dissected at 2,4,6,8,10,12,14 and 16 days after challenge,and the viral load of each group was measured.The results showed that gout symptoms occurred in both the oral group and the subcutaneous group,and the subcutaneous group died earlier.The weight of goslings in the attacking group was slower than that in the normal control group,and the most significant in the subcutaneous group.The results of detoxification rule of cotton swab showed that detoxification occurred in the early stage of infection in the attack group.The change of virus content in blood showed that the virus was detected in the early stage of infection in the attack group,and the change of virus content was basically consistent with the rule of detoxification of cotton swab.The results of blood biochemical indexes showed that the contents of UN,UA,AST and ALT in serum were significantly higher than those in control group.The results of tissue and organ viral load showed that after artificial infection with SDPY strain,virus could be detected in heart,liver,spleen,lung,kidney,bursa,thymus,pancreas,brain,stomach and duodenum.The organs with the highest virus content in the challenge group and the oral challenge group were kidneys.Combining with the detoxification rules of cloacal swab,blood and tissues and organs,it can be concluded that after artificial infection with SDPY strain virus in goslings,the viral content in Goslings reached the peak on day 6,decreased on day 8,and began to rise on day 10,reaching the second peak,but the second peak load was generally lower than the first peak.The dynamic distribution of GAstV in goslings can provide a reliable theoretical basis for the prevention and control of goose gout.