Studies On Attenuation Of Novel Goose Parvovirus Strain NMG21 In Duck Embryos

The goose parvovirus disease is a infections disease in goose farming.This disease had been well controlled by using attenuated vaccines in breeding geese and goslings and positive serum.Since 2015,the disease had emerged in duck flocks which exhibited short beak,large tongue,dwarfism,and loss of back feathers.Because of its high morbidity and low mortality rate,it caused serious economic losses to the duck farming industry in China.Novel goose parvovirus（NGPV）were firstly isolated and identified by our team.The strain used in this study on attenuation was from a duck farm in Inner Mongolia,which was named NMG21.Amplification of NMG21 to 35 generations by using 10-day old duck embryos.First dilute the allantoin of NMG21,NMG21-5,NMG21-15,NMG21-25,and NMG21-35 with saline to 10^-7.Then,each dilution gradient of virus was inoculated into five10-day-old duck embryos through the allantoic cavity.Finally,calculated the EID₅₀of the virus according to the Reed-Muench method.The results indicated that the ability of NGPV NMG21 to kill duck embryos became weaker and its virulence decreased with the increasing of amplification.A pair of specific primers and a probe were designed based on the gene sequence of NGPV VP3 and the target fragment was connected to the vector p MD18-T to construct a recombinant plasmid.After successful construction,the standard plasmid was diluted 10-fold and used as templates to construct a fluorescent quantification standard curve to establish a TaqMan probe fluorescent PCR method for NGPV detection.The test results show that the method is specific,sensitive,reproducible and it has a high detection rate for clinical samples.Therefore,it can be used in animal experiments for the qualitative and quantitative detection of NGPV.One-day-old ducklings were infected by intra-muscular injection with 0.2 m L(EID₅₀of each strain was 10^-7,10^-5,10^-5.2,10^-4.2,10^-3.7/0.2m L)of virus solution per duckling for NMG21,NMG21-5,NMG21-15,NMG21-25,and NMG21-35.Control ducklings were injected with the same dose of saline.Then we observed and recorded the clinical behaviors and mortality of different groups of ducklings daily.Six randomly selected individuals from each group were weighed and dissected on the 7th,14th,21st,and 28th after infecting NGPV.Organs such as liver,spleen,intestinal,lung,and proventriculus were fixed for pathological study.Collection of cotton swabs from cloacal cavities and various tissues for viral load determination.Blood was collected and serum was prepared for determination of viral load and antibody levels.The beak length and tongue length of ducklings in the control group were measured to establish a standard curve for the incidence of short beak,and then the incidence of short beak in infected ducklings was counted.The length of the femur and tibia were measured,and the bone density were determined to investigate the lesions.The results of animal experiments showed that the clinical behaviors of the ducklings in the different generations of NMG21 varied considerably.The NMG21 group had the most severe clinical symptoms,with mortality rates of 40%and severely stunted duckling development.Some ducklings were paralyzed,with severe hemorrhaging of the lungs,liver,and pancreas,and hemorrhaging of the spleen,kidneys,and thymus.Mortality in the NMG21-5 and NMG21-15 groups was 30%and 26.7%respectively,and duckling development was impaired.The thymus,lungs and liver were bleeding.The mortality rate in the NMG21-25 and NMG21-35 infected groups was low,at 6.7%and 2.8%respectively,and the development of the ducklings was slightly impaired.The organs were slightly hemorrhagic or normal,except for NMG21-35,which showed"dorsal spur"symptoms,but no other symptoms.The pathological histology was similar,with congestion in the lung compartment,emphysema of the lung cells,swelling and hyperplasia of the hepatocytes and splenocytes,and varying degrees of loss of intestinal villi with inflammatory cell exudation in the infected groups.The results of the viral load tests in different organs showed that the infected ducklings had higher viral loads in the heart,liver,proventriculus and intestinal.The tissue viral load of infected ducklings decreased gradually as the virulence of the different generations of strains decreased compared to the different infection groups.The viraemia results show that the more virulent the infecting strain,the higher the viral level in the blood.The regularity of cloacal detoxification shows that the more virulent the infecting strain,the greater the cloacal detoxification.ELISA results show that the weaker the strain of infection,the stronger the antibody response of the ducklings.In conclusion,this study successfully passed through the duck embryo to the strong strain NMG21 of NGPV,and with the increase in the number of amplifications,the virulence showed a decreasing trend,and the pathogenicity to ducklings was also decreasing.This experiment provides reliable experimental data and theoretical basis for the development of NGPV attenuated vaccine.