| Goose Astrovirus(GAst V)is an emerging causative agent of gosling gout.Typical signs for the disease include visceral gout is an animal infectious disease.GAst V is mainly transmitted horizontally through the digestive and respiratory tracts.But vertical transmission also occurs.Gosling gout has widely broken out in China since 2016.Clinical symptoms include the presence of arthrogryposis and growth retardation.Dissection revealed large deposits of urate attached to the viscera and joint cavities.According to clinical survey statistics,goose gout mainly infects goslings under 20 days of age.With a morbidity rate of 80%in geese and a mortality rate of up to 73.2%.Rapid spread in geese because of significantly infectious.And no significant effect of antibiotic treatment.No drugs available for the clinical treatment of gout caused by GAst V in goslings,and there is no commercially available vaccine to prevent.Therefore the ability to prevent and control this disease outbreak is limited.In order to effectively prevent and control the disease,further research on the biological characteristics of goose astrovirus is needed to provide a theoretical basis for the prevention and control of goose astrovirus.The experiment conducted an investigation on the incidence of gout in each goose farm by epidemiological,and collected samples of the goose gout disease from18 goose farms across all over China.Samples of typical symptoms were collected and submitted to next-generation DNA sequencing.We confirmed the sequencing results by reverse transcription polymerase chain reaction method.The disease samples of sick goose were sampled from a goose farm in Weifang City,Shandong Province.Identification of goose astrovirus by inoculation of goose embryos with isolated disease material,pathogen sequencing analysis and artificial infection experiment.The whole genome was obtained by assembly and splicing and analyzed using biological software.Real-time quantitative PCR methods based on Taq Man probe and SYBR Green I dye were established.After optimizing the reaction conditions and system,specificity,sensitivity and reproducibility of these two methods were validated and compared.The results are as follows:1.Exploration of the causative agent of gout disease in geeseThe dead or close to death geese(totally 138 samples)from 18 investigated farms.At autopsy,uratosis was the main pathological change.Their age of clinical signs onset ranged from 8 to 37 days.Mortality of the different farms ranged from8.90%to 73.20%.Typical signs for the disease include diarrhea,anorexia,depression,dehydration,emaciation and paralysis.At autopsy,uratosis was the main pathological change which could be found at kidney,pericardium,air sac,muscle and leg joint.Epidemiological indicate that the disease was significantly infectious.Antibiotic treatment for the sick goose was ineffective A preliminary analysis of the disease was made in relation to the virus.First,a sample of diseased geese with typical symptoms of gout disease was selected.Samples of kidney,Fabricius bursa,spleen and jejunum were collected and submitted to next-generation DNA sequencing.Sequencing results and clinical analysis suggest that the disease may be associated with infection by the goose astrovirus.Based on the results of metagenomic sequencing,a pair of primers was designed to detect partial genome of GAst V.We confirmed the sequencing results by reverse transcription polymerase chain reaction method got consistent results.Expanded sample size and All the collected samples were conducted with detection,including GAst V AIV,NDV,DRV,DHAV,DTMUV,Du CV,MDRV and REV.Results showed that 127 samples were astrovirus-positive.But other viruses showed negative reaction.In summary,goose avastrovirus was highly related with this disease.2.Isolation and identification of GAst VTo further verify that the causative agent of goose gout is goose stellate virus.Isolation and identification of viruses in samples from a goose farm in Weifang,Shandong Province.The disease homogenate was inoculated with 11-day-old goose embryos.The results showed that goose embryos died within 24 h-96 h.The main lesion was systemic hemorrhage from the dead embryo,enlarged kidneys,mottled and white urate in the allantoic membrane.Bright bands were obtained by RT-PCR amplification with goose astrovirus-specific primers and sent for sequencing comparative analysis.The results showed positive for goose astrovirus and were named as goose astrovirus strain SD.Hemagglutination test showed no agglutination of SD.The results of animal regression experiments were consistent with those of naturally infected animals.No significant symptoms in the control group.The mean body weight and mean uric acid content of the surviving geese in both groups at the end of the observation period were Differences and significant.Slices showed that the liver tissue,kidney tissue,spleen tissue and intestinal tissue of the control group were normal.Dilated sinusoidal gaps and hepatocyte necrosis in the liver tissue,renal tissue with glomerular atrophy and tubular lumen filled with blue-stained urate particles,hemorrhage of splenic tissue and massive necrosis of parenchymal cells as well as dilated and congested intestinal capillaries both appeared in the experimental group.In summary,the isolated and identified virus strain SD was goose astrovirus.3.Whole genome sequence amplification and genetic evolution analysis of SD strainsIn order to deepen the understanding of goose astrovirus,this experiment performed whole genome sequencing and analysis of the isolated and identified SD strain of goose astrovirus.Refer to the published whole genome sequence of goose astrovirus with high similarity to the partial sequence of SD strain.The software Primer5.0 was used to design a total of 6 pairs of fragmented amplification primers,and the sequencing results showed that the 6 fragments were all goose astroviruses by NCBI online comparison;the whole genome sequence of SD strain was obtained by assembling and splicing the Seq Man program of DNAstar Lasergene v7.1 software.,the results show that the whole genome length of SD strain is 7128 nt,including5’-UTR and 3’-UTR and three open reading frames of ORF1a,ORF1b,ORF2,NCBI accession number OM937013;The whole genome sequence of the astroviridae virus was compared,and the phylogenetic tree was constructed by using the Mega software NJ program to construct the phylogenetic tree of the whole gene sequence of SD strain and the amino acid sequence of ORF2 and the aligned strains.The homology of the reference strain was 61.2%-98.9%,the homology of avian astrovirus was52.9%-98.9%,and the homology of mammalian astrovirus was 46.0%-50.7%;the phylogenetic tree analysis results show that SD The strains were on the same branch as HN03,AHAU3 and AHAU5 on the evolutionary tree,and FLX was a separate branch.In conclusion,SD strain has the typical structure of goose astrovirus,belong to the genus of avian astrovirus in the family Astroviridae,and was similar to duck astrovirus and turkey astrovirus in evolutionary relationship.4.Establishment of real-time quantitative PCR detection methodIn order to better applied in clinical detection,two q PCR methods based on Taq Man probe and SYBR Green I dye were established.Specific primers and probes were designed according to the conserved sequence of ORF1b gene of Go Ast V.After optimizing the reaction conditions and system,specificity,sensitivity and reproducibility of these two methods were validated and compared.Also,utilizing these two methods,113 samples which collected during 2020 to 2021 were detected.Results showed that both methods had good linear relationships(R~2=0.999)at concentration 2.5×10~8 copies/μL to 2.5×10~2 copies/μL of standard plasmids.The minimum detection limits of probe method and SYBR Green I dye method were2.5×10~1 copies/μL and 2.5×10~2 copies/μL,respectively.The coefficients of variation within and between the two methods were less than 2.50%.Positive detection rates for the clinical samples of the two methods were 69.91%and 60.18%,respectively.All these results indicated that both q PCR methods were specific,sensitive and duplicable for the Go Ast V detection and can be applied in the diagnosis for clinical gosling gout. |
PDF Full Text Request