Isolation And Identification Of Novel Duck Reovirus And Preliminary Study On Its Immunogenicity

Novel duck reovirus disease is an infectious disease caused by novel duck reovirus(NDRV),the typical symptoms of this disease in ducks include liver enlargement and hemorrhage,spleen necrosis and weight loss of ducks.In November 2017,an infectious disease characterized by liver enlargement and hemorrhage,spleen necrosis was occurred within 10 day old Cherry Valley Duck in a commercial duck farm in Heze city,which caused large economic loss in the local duck breeding industry.This study provided technical support for the effective prevention and control of NDRV infectious diseases.The main contents include:1.Isolation and identification of NDRVThe liver and spleen of 10 day old commercial Cherry Valley Duck in Heze area were collected and grind.After filtrating the diseased materials,inoculated respectively SPF chicken embryos,SPF duck embryos,Duck embryo fibroblasts(DEF cells)and African green monkey kidney cells(Vero cells).After culturing for 3 to 5 days,the allantoic fluid of the dead embryo and the supernatant of the diseased cells were collected,and the NDRV?C gene specific primers were used for RT-PCR identification and sequencing analysis.The results showed that one strain of NDRV was isolated and named as NDRV017.The similarity between the isolation and 9 reference strains of NDRV on NCBI ranged from95.3%to 99%.2.Preliminary development of inactivated vaccinesThe virus liquid was inoculated into Vero cells,and when the degree of cytopathic changes reached 80%,3‰formaldehyde was used to inactivate for 36 h to prepare NDRV oil emulsion inactivated vaccine.3.Studying on the immunogenicity of inactivated vaccinesOne day old ducklings hatched from the eggs 28 days after the second immunization were used and the offspring of the same batch of unimmunized breeding ducks were set as negative controls.NDRV017E3 strain was used for artificial infection.Negative control group was inoculated with 0.8 ml sterilized PBS with leg muscles,and the other three groups were respectively inoculated with 8 ELD₅₀strains of NDRV017E3(virus content4.50×10⁵/feather)in the leg muscles.After 10 days of infection,the animals were sacrificed and spleen tissues were collected.We took the spleen necrosis as a judgment index,and found that the protection rate of maternal antibodies to ducklings was 53.3%,and the protection rate of yolk antibodies to ducklings was 40%,compared with the positive control group,maternal antibodies could effectively reduce the risk of NDRV infection in ducklings(P<0.05)and had better protective effect on ducklings.The statistical results of the weight gaining change of each group showed that compared with the control group,the weight of the ducklings in the positive control group was reduced by 16%(P<0.05),and the maternal antibody group and the yolk antibody group were reduced by 4.57%(P>0.05)and9.43%(P>0.05),respectively.The above results showed that more than 50%of offspring ducklings could be protected by carrying maternal antibodies when Cherry Valley breeding ducks were immunized with the vaccine after 28 days,which was better than the immune protection effect of commercial vaccines on the market.