Preliminary Development Of A Bivalent Oil-emulsion Inactivated Vaccine Against Duck Parvovirus Disease And Duck Viral Hepatitis

Duck viral hepatitis can cause large-scale deaths of ducklings and is highly infectious.In recent years,duck parvovirus disease,which causes slow growth and development of ducks,has occurred in many areas of China,and mixed infection with duck viral hepatitis has brought great economic losses to duck breeding industry.In this study,the epidemiological investigation and pathogen isolation of duck viral hepatitis were carried out.The inactivated oil emulsion vaccine was prepared by using duck viral hepatitis isolates and duck parvovirus isolates,and the safety evaluation of the vaccine was carried out,which provided technical support for the prevention and control of the two diseases.In order to understand the prevalence of duck viral hepatitis in China,20 suspected materials collected from the whole country from October 2017 to November 2018 were tested by clinical diagnosis,virus isolation,PCR,etc.The results were 20 among the suspected materials,there were 11 duck hepatitis virus type 1?DHV-1?positive disease materials and 2 duck hepatitis virus type 3?DHV-3?positive disease materials,mainly occurring in ducks at 7 days old,and also occurred at 10 to 30 days old.After the positive disease material was treated,9 days old duck embryos were inoculated for 5 generations,and 3 DHV-1 isolates YN strain,LY strain and SG strain were obtained,and the ELD50 of the YN strain was determined to be 10^-3.75/0.1 mL.The three isolates were sequenced,and the nucleotide sequences of the three isolates were analyzed by DNAstar software with 20other DHV-1 reference strains.The results show that the nucleotide sequence homology between YN strain with other reference strains is 92.2%⁹7.7%,the nucleotide homology between SG strain and reference strains is 92.1%⁹7.6%,the nucleotide homology between LY strain and reference strains is 92.7%⁹8%.and all three strains had close genetic evolution relationship with DHV-1.The nucleotide homology between YN strain and LY strain is 99.3%,the nucleotide homology between YN strain and SG strain is 99%,the nucleotide homology between SG strain and LY strain is 99.5%.Genetic evolution analysis show that YN strain has the closest relationship with SG strain and belong to a kind of branch.The YN strain was used to infect 1 day old ducks for animal regression experiments,which replicated cases of duck viral hepatitis.The diseased dead duck was in a retrograde position of the horn,and the liver was congested,yellowing and swelling.The kidney was congested.The results show that DHV-1 isolated from natural cases of duck viral hepatitis was associated with duck viral hepatitis.The duck parvovirus SD strain preserved in the laboratory was expanded,and the EID₅₀ of the strain was determined to be 10^-3.5/0.1 mL.The duck hepatitis virus YN strain and the duck parvovirus SD strain were filtered and sterilized,and the same amount of virus liquid was mixed uniformly,and inactivated with formaldehyde.The inactivated oil emulsion vaccine of duck parvovirus disease and duck viral hepatitis was developmed preliminarily with white oil and Span-80 as adjuvants.And the dosage form,stability,viscosity and safety of the vaccine were tested.The test results show that the dosage form of the vaccine is water-in-oil type,has good stability,the viscosity meets the standard,and the sterility test and safety test are all qualified,and there is no adverse reaction in the injected animal.This study provides new resources for the epidemiological investigation of duck viral hepatitis.At the same time,it provides a theoretical basis for how to prevent and control duck parvovirus disease and duck viral hepatitis.