Preliminary Study On The Lesions Of Immune Organs In Ducklings By Goose Parvovirus

Goose parvovirus(GPV)is a single-stranded linear DNA virus that infects 4 to 21 days old goslings and young muscovy ducks.Novel goose parvovirus(NGPV)is the causative agent of duck scorpion-dwarf syndrome,NGPV is very similar to GPV in heredity and antigenicity.In recent years,epidemiological investigations have shown that GPV can co-exist or infect other pathogens in commercial duck populations.It is reported in the literature that artificial infection of ducklings by GPV can cause damage to different organs of ducklings,but there is no direct evidence to confirm the spread of GPV in ducks,and the damage of GPV to duck immune organs has not been reported.This study adopted the separation and identification of GPV in Anhui from 2015 to 2018,and the VP gene was amplified,and a genetic evolution tree was drawn.A classic GPV AH-1 strain with the closest relationship to NGPV was selected for duckling challenge test to observe GPV.In order to study the damage of immune organs in ducklings,the purpose was to study the proliferation of GPV in the immune organs of ducklings,and to provide a reference for explaining the pathogenesis of GPV.1.PCR detection was performed on 12 cases of suspected GPV cases with typical clinical symptoms and 2 cases of suspected NGPV cases in Anhui Province in 2015-2018.The test results showed positive results,GPV was named AH-1 to 12,NGPV was named for AH 13 and AH 14.Then,14 parts of the disease materials were inoculated into the 13-day-old non-immune goose embryos.As a result,the embryos and organs showed different degrees of bleeding.The dead embryonic allantoic fluid was collected,and 14 VP genes and ITR genes were amplified.And sequencing and genetic evolution analysis showed that 12 strains of GPV belong to a subset and have high affinity.The GPV AH-1 strain(GenBank accession number:MK333463)has high homology with NGPV.Therefore,the GPVAH-1 strain was selected for the median lethal dose determination of goose embryos,and the results showed that the ELD50 of the AH-1 strain was 10-4.17/0.2 ml,and the strain was used as a candidate for subsequent studies.2.A pair of specific primers were designed with reference to the VP3 fragment of GPV standard B strain(GenBank accession number:U25749),and the VP3 gene was amplified.After sequencing,the pMD18-T-VP3 plasmid was constructed and the plasmid was used as a positive standard.And SYBR Green I fluorescence quantitative PCR method was established.The linear regression equation of the VP3 standard curve is:y=-3.2568X+37.987(R2=0.9986),the amplification efficiency is between 90%-120%,and the lowest detection limit is 46.95copies/?L,which indicates the established SYBR Green I The validity of the PCR and the linear relationship between the logarithmic value and the Ct value of different concentration gradients showed a good linear relationship.In the specific test,DTMUV,GPMV,ALV,ILTV and CIAV were negative.The SYBR Green I real-time PCR method was used to detect the change of GPV in the gosling tissue.The results showed that it could be in the tissue within 3-5 days after infection.The virus was detected,indicating that the method has high sensitivity and specificity.3.The GPV AH-1 strain of 1000 times ELDso was injected intramuscularly with non-immune ducklings of 2 days old to study the pathological damage of the immune organs of ducklings by GPV.By artificially infecting ducklings,the tissues of Id,3d,5d,7d,10d,and 14d after the challenge were collected for gross examination,pathological section observation,antigen quantification and localization analysis.The results showed that after GPV infection of ducklings except for mild depression and anorexia on the 1st day after infection,there was no abnormal change.There was no death within 14 days.There was bleeding point in the thymus 3d after infection,5d spleen and intestinal swell in 5d,and a little bit of hemorrhage in 7d liver.The thymus and spleen were more severely observed 5-7 days after infection,and the 3-5d Had's gland was more severely congested.All organ damage is reduced or disappeared after 10-14d.The results of qPCR showed that the proliferation of virus reached the highest level in the bursa of Fabricius,thymus and Had's gland and showed a decreasing law.The proliferation of spleen,cecal almond and bone marrow cells increased first and then decreased.The results of antigen localization showed that the positive signal of GPV in the immune organs was strong within 3-5d,and the positive signal gradually weakened after 7d.These results indicated that GPV could replicate in duck immune organs and cause pathological damage of some immune organs.In summary,12 cases of GPV and 2 cases of NGPV in Anhui Province were isolated and identified,and VP gene amplification analysis was carried out.SYBR Green I fluorescence quantitative PCR method for detecting GPV was established.A strain of GPV virulent strain AH-1 was selected by intramuscular injection of ducklings to observe the pathological damage of ducklings by GPV,and the quantitative and localization of GPV were performed on the immune organs of 1-14d infected ducklings.It provides a reference for studying the pathogenesis of GPV.