Isolation, Identification And Tissue Distribution Of Goose Parvovirus Anhui Strain

The Gosling Plague is a highly fatal disease mainly occurring on geese and young muscovy ducks with the necrotizing enterocolitis as the main pathological symptom.It is one of the most serious infectious diseases threatens the development of China's goose industry.10-day-old goslings and occasionally 60-day-old geese were infected,the morbidity due to GPV reach up to 80% of birds in a flock while the mortality is frequently about 60%.Therefore,it is important to study the epidemic regularity of the gosling plague,the virus replication and distribution in the tissues,which benefit the understanding of the pathogenesis of the gosling plague.Two strains of gosling plague virus(GPV),named GPV strain YZ and WX,were successfully isolated from the samples of died geese which collected from two farms in Anhui Province.Two GPV strains were identified by PCR method and by electron microscope and sequence analysis of genome.The results of the pathogenicity test showed that ELD50 of GPV strain YZ was 10-4.840.2ml and GPV strain WX was 10-5.160.2ml.Five pairs of specific primers were designed and used to amplify the complete genome of GPV strain YZ and WX by PCR,the result indicated that the complete genome sequence of GPV strain YZ and WX were 5049 bp in length with a length of 1605 bp on VP3 fragment.The sequence similarities of the complete genome and VP3 fragments of the two strains were 99.7% and 99.4%.Analysis of phylogenetic tree showed that two strains had a higher homology with the strains isolated from surrounding cities,such as GPV strains YZ99-6and LH from Jiangsu,GPV strain SHFX-1201 from Shanghai,GPV strain Y from Anhui.Compared with the standard virulent GPV strain B,both strains YZ and WX lack 14-bp fragments in each of the regions 67-80nt(CACATGCTTCCGGT),334-347nt(GCATGTGACCGGAA),4767-4780nt(CACATGCTTCCGGT)and 5033nt-5046nt(GCATGTGACCGGAA).All missing sections were located in the inverted terminal repeats(ITR),but they had no affects on virus hairpin structure formation.In order to study the proliferation regularity of GPV in goling bodys,one animal model was established with GPV strain YZ.Quantitative PCR combining with fluorochrome of SYBR Green I were used and a pair of specific primers was designed based on the sequence of VP3 fragment.The flurescence standard curve was established by using the plasmid as standard template.The application of SYBR Green I fluorescent quantitative PCR technique was used to evaluate the virus concentrations in each organ at different times.The results shown that the content of the virus was relative low before 48 hours and then the amount amplified rapidly in 48-96 h.The virus concentration in each organ reached the highest level at 96 h,and it decreased at 144 h after infection.Hence,the mathematical model of proliferation of GPV in golings shown a normal distribution curve,which regarded the 96 h as the axis.At 96 h,the virus concentrations in organs were:kidney>duodenum>ileum>spleen>jejunum>liver>heart>brain.In order to reveal the localization of GPV antigen and the histological changes,the experiment studied the organ changes at 96 h by using immunohistochemistry and HE staining techniques.The fell down of the intestinal villi,swelled renal tubular epithelial cells,and hemorrhaged liver were observed by the HE staining.The positive reactions of GPV antigen were detected in the cytoplasm of intestinal villi epithelial cells,renal tubular epithelial cells and nerve cell in brain by the immunohistochemistry staining.The positive signal detected in the intestine and kidney were stronger than that of in other organs such as liver,the result was consistent with the results of fluorescence quantitative PCR.It is found that GPV could cause various degrees of organ damage and hemorrhage,so it confirmed that GPV resprented pantropic after infected golings.Based on the isolation and identification,the messages of the complete genome and tissue distribution of two GPV strains,this study revealed the phylogenetic relationships of the GPV strain YZ and WX from Anhui Province,and the rule of proliferation of GPV in goslings,which provided theoretical knowledge for pathogenesis.