Pathogen Detection Of Infectious Gosling Gout And Preparation Of Polyclonal Antibody Against The Spike Protein Of Goose Astrovirus

Infectious gosling gout broke out in goslings in various provinces in China since2015.The disease is mainly characterized by urate deposition in the serosal layer of the organs and kidney enlargement.A large number of pathogenic testing studies have confirmed that infectious gosling gout is associated with GoAstV-2.There are mixed infections of GoAstV-2,Goose Astrovirus(GoAstV-1),Goose parvovirus(GPV),etc.in natural infections.The occurrence of mixed infection often leads to an increase in morbidity and mortality,which seriously threatens the development of goose industry.To understand the pathogen distribution of infectious gosling gout in the gosling flocks in Guangdong Province.272 gout gosling tissue samples were collected from192 goose farms with infectious gosling gout in Foshan,Huizhou,Jiangmen,Shantou,Yangjiang,Zhaoqing.272 samples were detected by 16S r DNA sequencing and specific PCR.The results showed that the positive detection rate of GoAstV-2 was 100%(272/272),of which 43.38%(118/272)were GoAstV-2 single positive,56.62%(154/272)were GoAstV-2 and GoAstV-1,GPV,Salmonellae,RA,NDV,Escherichia mixed positive.Among the 256 tissue samples of gout goslings collected in 2019,the co-infection of GoAstV-2 and GoAstV-1 was the highest,followed by the co-infection of GoAstV-2 and Salmonellae.It shows that infectious gosling gout is related to GoAstV-2 infection,and the mixed infection of GoAstV-2 and other pathogenic factors in natural infection cases is high and diverse,suggested that the prevention and control of infectious gosling gout should focus on the prevention of GoAstV-2 and GoAstV-1,and GoAstV-2 and Salmonellae co-infection.To establish a sensitive and rapid detection method for Goose Astrovirus,SYBR Green I real-time fluorescent quantitative PCR methods for detecting GoAstV-1 and GoAstV-2 was established.The correlation coefficient(R~2)of the GoAstV-1 standard curve is 0.9987.The minimum detection limit of GoAstV-1 is 10 copies/μL.The coefficient of variation of inter-assay and intra-assay repetition was less than 2.5%.The RT-PCR reaction of GoAstV-1 did not cross-react with DHV-1,H9N2,NDV,GPV,and GoAstV-2.The correlation coefficient(R~2)of the GoAstV-2 standard curve is 0.9987,the minimum detection limit is 10 copies/μL,and the inter-assay and intra-assay coefficient of variation are all less than 2.5%.The RT-PCR reaction of GoAstV-2 did not cross-react with DHV-1,H9N2,NDV,GPV,and GoAstV-1.Use the established detection methods of GoAstV to quantitatively detect the virus content in the various organs of gout goslings.It’s showed that the content of GoAstV-1 in the intestine was the highest,and the spleen,liver,and kidney contained high levels of GoAstV-2.The content of GoAstV-2 in each organ was much higher than the content of GoAstV-1.It shows that GoAstV-1 and GoAstV-2 in mixed gout goslings have different tissue tropism,suggesting that GoAstV-2 may occupy a dominant position,and GoAstV-1may be a secondary infection factor after GoAstV-2 infection.To understand the molecular evolution of GoAstV-1 and GoAstV-2.Virus isolation,capsid protein sequence determination,amino acid sequence similarity analysis,genetic distance calculation,and phylogenetic analysis were performed on tissue samples that were identified as GoAstV-2 positive and GoAstV-2 and GoAstV-1co-infected in 2018 and 2019.The results showed that the amino acid sequence of the capsid protein between the two GoAstV-1 strains was 99.9%similar,which was 99.3%similar to the 2017 GoAstV-1 reference strain AHDY(AYJ00429),but it was similar to the 2014 GoAstV-1 reference strain FLX(NC＿034567)The similarity between the two is only 86.7%,indicating that GoAstV-1 has undergone significant mutation.The similarity of the capsid proteins between the four GoAstV-2 strains reached 98.5%-99.4%,and the similarity between the GoAstV-2 reference strains from 2014 to 2018was also 97.7%-99.5%,indicating that the GoAstV-2 No significant mutation has occurred,which is in line with the evolution rate of positive-strand RNA viruses.With the goose Astrovirus isolated and identified in 2018 as the research object,bioinformatics methods were applied to analyze the molecular evolution of the isolates and the mutation of the capsid and spike protein.In order for the expression of spike protein and capsid,the prokaryotic expression plasmid of the spike and capsid was constructed.New Zealand white rabbits were immunized for the preparation of rabbit anti-spike antibodies,while the reactivity of capsid protein was verified against the level of prokaryotic expression.According to the results,the amino acid residues 425-665 are categorized into the spike of the capsid protein.In addition,it was found out that the spike of GoAstV-2 strains isolated from different regions from 2014 to 2018was highly similar in both sequence and structure,suggesting that there is still no significant mutation occurring to the GoAstV-2 strains across different parts of China.The rabbit anti-spike serum is capable of precise binding to the spike and the capsid protein,indicating that the high immunogenicity of recombinant spike and the strong reactivity of rabbit anti-spike polyclonal antibody.In summary,this study carried out pathogenic testing on 272 gout gosling tissue samples,and confirmed that infectious gosling gout is related to GoAstV-2 infection.In natural infection cases,the co-infection of GoAstV-2 and other pathogenic factors is high and has Diversity.An efficient,sensitive and specific fluorescent quantitative PCR detection method for GoAstV-1 and GoAstV-2 has been established,which provides technical support for the rapid diagnosis of GoAstV-1 and GoAstV-2 co-infection.Based on the genetics of GoAstV-2,successfully expressed the capsid protein and spike protein of GoAstV-2,and successfully prepared a reactive polyclonal antibody against the GoAstV-2 spike protein,in order to further develop GoAstV-2 Immunological testing and immune prevention provide technical support.