Histopathological Observation Of Gout In Goslings And Molecular Identification Of Vertically Transmitted Pathogens

Since 2015,a gosling gout characterized by visceral and articular urate deposition has broken out in Anhui,Jiangsu,Shandong and other major goose-producing provinces in China.The mortality rate is as high as 20% ～ 70%,which has caused huge economic losses to goose raising industry in China.At present,The cause for gosling gout remains unclear.While Goose Astrovirus(GoAstV)is believed to be the main pathogen of gout,the full-blown disease of gout cannot be completely reproduced by infecting the goslings with GoAstV.And the mortality and morbility of the disease were far less serious than those of natural cases in the field,suggesting the possibility of other infectious factors may be related to the occurrence and development of gosling gout.To assess other possible infectious factors,13 cases of gosling gout in Anhui province from October 2018 to July 2019 were studied in this paper,and the clinical autopsy,histopathology,pathogen molecular identification and virus antigen distribution were explored.On the basis of this,according to the sources of goslings in the surrounding areas where gout occurred,this paper explored the pathogens carried by goose embryos from different sources in Anhui Province from the perspective of vertical transmission,in order to provide a reference for further determining the etiology of gout in goslings.The main research contents are as follows:1 Preparation of polyclonal antibody against GoAstV capsid protein ORF2To expression protein fragment from GoAstV capsid protein ORF2 gene,partial capsid gene fragments(1273-2115 bp)were cloned into expression vector pET-32a(+).The recombinate pET-32a-ORF2 plasmid was constructed and transformed in competent BL21(DE3)cells.After optimizing the experiment conditions,the highest expression of the protein was obtained by adding IPTG at a final concentration of 1.0 mmol/L at 30℃ for 5h.The obtained ORF2 recombinant protein was consistent with the expected size(49.1 kDa).The concentration of the ORF2 recombinant protein was 2.348 mg/mL after purification by His label protein purification method.The purified recombinant ORF2 protein was used to immunize Kunming mice and prepare specific polyclonal antibody against GoAstV-ORF2.The highest titer of antibody prepared by indirect ELISA was 1:51200 with p GEX-4T-1-ORF2 protein as coating antigen.Western-blot confirmed that the antibody could specifically bind to ORF2 recombinant protein.IFA assay determined that the antibody could produce a specific fluorescence signal with LMH cells infected with GoAstV for continuous passage.2 Histopathological observation of gouty disease in goslingsFrom October2018 to July2019,we collected tissues from gout-affected goslings in 13 goose farms in Anhui province.The clinical necropsy,histopathology,pathogen molecular identification and virus antigen distribution were investigated from 13 cases.Postmortem examinations showed that the joints of geese were swollen and the diffuse urate distribution was observed in internal organs,severe bleeding in kidneys of some goslings,swelling and hardness in duodenum and ileum,and appearance of "sausage-like embolism".Histopathology showed typical kidney injury,including tubular necrosis,epithelial cell shedding and the presence of eosinophilic protein-like substances in the lumen.In addition,satellite phenomenon and neurophagocytic phenomenon were observed in brain tissue.A large number of basophilic intranuclear inclusion bodies were found in liver,kidney,spleen and other tissues,and local needle-shaped radial urate deposits were observed,accompanied by infiltration of macrophages and lymphocytes.There was a large number of heterophilous myeloid cells and heterophilous granulocytes were found to be focal hyperplasia.Our data showed that all gout-affected goslings carried both GoAstV and GPV determined by RT-PCR/PCR.Sequence analysis showed that GoAstV ORF2 amino acid sequence of 13 cases(AH-1 ～ AH-13)had high homology with the GoAstV SD01 strain sequence,reaching 98.2% ～ 98.8%.The sequence of VP3 in part of GPV had a high homology with that of standard strain B,up to 96.2% ～ 98.7%.mIHC detection showed that GoAstV was mainly distributed in liver and kidney,followed by lung,brain and bursa of fabricius.Notably,GoAstV and GPV are co-located in renal tubular epithelial cells,hepatocytes,ileal mucosal muscle cells and follicular epithelial cells of bursa of fabricius.3 Molecular Identification of main vertically transmitted pathogens in geese13,20,28-day-old goose embryos and 1-day-old goslings were collected in four batches from October to December in 2020.A total of 143 samples(all from the goose incubation companies in surrounding areas of the gout)were collected for molecular identification of10 main vertical transmission pathogens in geese.The detection pathogens such as Escherichia coli(E.coli),Salmonella spp,GoAstV,GPV,Tembusu virus(TMUV),Goose circovirus(GoCV),Avian nephritis virus(ANV),Goose reovirus(GRV),Fowl adenovirus serotype 4(FAdV-4)and Duck enteritis virus(DEV).Thirty 28-day-old goose embryos were detected by histomathological observation and viral antigen distribution.The results showed that all 143 samples were single and mixed infection,12 were single infection and131 were mixed infections,the infection positive rates were 8.39% and 91.61%respectively.Among the detected viruses,the detection rate of GPV was the highest,up to67.13%,and all of them were mixed infections.Fluorescence mIHC staining revealed co-expression of GPV,TMUV,FAdV-4 and GoAstV in the same kidney tissues from28-day-old goose embryos infected with four viruses.In summary,polyclonal antibodies against GoAstV-ORF2 protein were prepared in this study.13 cases of gosling gout in Anhui were co-infected with GPV and GoAstV.143 samples of main vertical transmission pathogens in goose embryos in Anhui area were identified as GPV,TMUV,FAdV-4,GoAstV,E.coli and Salmonella spp,and most of them were mixed infections.