| Hydropericardium hepatitis syndrome(HHS)and Inclusion body hepatitis(IBH)are two acute infectious diseases infected by group I fowl adenovirus(FAdV).In recent years,these two diseases have caused serious economic losses to the poultry industry in China.Group I FAdV has five genotypes(A-E)and these are further subdivided into 12 serotypes(FAdV-1 to 8a and-8b to 11).These serotypes belong to the five genotypes as follows:(a)FAdV-A includes FAdV-1;(b)FAdV-B,FAdV-5;(c)FAdV-C,FAdV-4 and-10;(d)FAdV-D,FAdV-2,-3,-9,and-11;(e)FAdV-E,FAdV-6,-7,-8a,and-8b.The study confirmed fowl adenovirus serotype 4(FAdV-4)as agent of HHS in current China,whereas fowl adenovirus serotype 8b(FAdV-8b)is pathogen of IBH.Therefore,the development of a bivalent oil-emulsion inactivated vaccine for FAdV-4 and FAdV-8b has important application value.1 Isolation and identification of FAdV-8b QD2016 strainIn this study,the FAdV QD2016 strain was isolated from chickens suspected with IBH in Shandong Province.33 pairs of primers were designed and the whole genome sequence of QD2016 strain were determined and analyzed,which showed that the full length genome of the strain is 43632 bp.It belongs to an evolutionary branch with the CR119,YR36,TR59,764,HG,UPM04217 and HLJ/151129 strains of FAdV-E.The nucleotide homology ranged from 93.4%to 98.4%.QD2016 strain and UPM04217 strain have the closest relationship,which belong to FAdV-8b.2 Development of a bivalent oil-emulsion inactivated vaccine for FAdV-4 and FAdV-8bA bivalent oil-emulsion inactivated vaccine was developed using FAdV-8b QD2016 strain and the FAdV-4 GX2013 strain previously isolated by our laboratory.According to the requirements of "Veterinary Pharmacopoeia of the People’s Republic of China",the seed banks of two strains were established.No exogenous virus(ALV and REV)were detected by chick embryo and cell test.LMH cells were cultured with cell factory to amplify FAdV-4 GX2013 strain and FAdV-8b QD2016 strain.The amplified GX2013 strain and QD2016 strain were quantified using the real-time fluorescence quantitative PCR,and their optimal multiplicity of infection(MOI)on LMH cells was 1.2 and 3.2,respectively,and the optimum time for harvest was 72 hours after inoculation.The viruses were inactivated by formaldehyde with a final concentration of 1 ‰,and then oil adjuvant was added to make the bivalent oil-emulsion inactivated vaccine.7-day-old SPF chickens were divided into 2 groups(n=10).The first group was an immunization group and the chickens were inoculated with the inactivated vaccine subcutaneously,0.5mL per chicken.The second group was a control group and the chickens were inoculated subcutaneously with an equal dose of sterile oil.The mental status and feed intake of chickens in immunization and control groups were normal within 21 days after the inoculation.No injury,inflammation and vaccine residue were found at the inoculation site through necropsy.Above of all,the results indicated the good safety of the vaccine.7-day-old SPF chickens were divided into 6 groups(n=10)and immunized with the vaccine at the dosage of 25,50,100,250 and 500 μL per chicken in vaccinated groups via subcutaneous inoculation in the neck,with an unvaccinated control group.At 21 days after immunization,5 chickens were taken from each group and challenged with FAdV-4 GX2013 strain(105.5 TCID50/mL)and FAdV-8b QD2016 strain(105.5 TCIDso/mL)via intramuscular injection respectively,0.2 mL per chicken.The virus content in throat swabs and cloacal swabs(collected on days 3,5,7 and 10)were detected after challenge by using the real-time fluorescence quantitative PCR.The results showed that the amount of virus shedding in the throat and cloacal reached a peak within 7 days after challenge.The amount of virus shedding in 25 μL,50 μL and 100 μL immunization groups was not significantly different from the control group(P>0.5).The amount of virus shedding in 250 μL and 500 μL immunization groups was significantly different from the control group(P<0,01).However,the difference in the amount of virus shedding between the two immunization groups was not significant(P>0.5),It was confirmed that the immunization dose of the vaccine was 250 μL and 500 μL per chicken,which could effectively prevent virus shedding.The minimum immunization dose of the vaccine was 250 μL per chicken.7-day-old SPF chickens were divided into 3 groups(n=10).Chicken in group 1 and 2 were immunized subcutaneously in the neck with 250 μL inactivated vaccine.Chicken in group 3 was inoculated subcutaneously in the neck with an equal dose of sterile oil and treated as control.Serum samples of group 1 were collected at 7,14,21,28 and 35 days after immunization.The immunoprotective efficacy of the inactivated vaccine was detected by serum neutralization test(SNT)and agar gel diffusion precipitation test(AGPT).At 21 days after immunization,5 chickens were taken from group 2 and group 3 and challenged with FAdV-4 GX2013 strain(106.5 TCID50/mL)and FAdV-8b QD2016 strain(106.5 TCID50/mL)via intramuscular injection respectively,0.2 mL per chicken.The virus content in throat swabs and cloacal swabs(collected on days 3,5,7 and 10)were detected after challenge by using the real-time fluorescence quantitative PCR.SNT showed that the FAdV-4 and FAdV-8b serum neutralizing antibodies could be detected at 7 days after immunization and reached a peak at 21 days(210 7 and 210 6).Afterwards,they gradually declined and remained a higher level at 35 days(210 2 and 29.9).AGPT showed that the FAdV-4 and FAdV-8b antibodies could be detected at 14 days after immunization,and the positive rates of antibodies were the highest at 21 days after immunization,reaching 100%and 90%,respectively,then they began to decline slowly.The chickens in the control group all died within 3 days after challenge with FAdV-4 GX2013 strain,while the chickens in the immunization group were normal,the clinical protection rate of the vaccine reaching 100%.The chickens in the control group,reduced feed intake,depressed spirit,excreted water-like dilute feces appeared after challenge with FAdV-8b QD2016 strain,while the chickens in the immunization group were normal.In addition,the amount of virus shedding in throat swabs and cloacal swabs was extremely significant difference between immunization group and control group(P<0.01).All of the results proved that the bivalent oil-emulsion inactivated vaccine has a good immune efficacy. |
