| A total of 14 strains of FAdV,sampled from sick broiler,layer,sheldrake in Shandong and Jiangsu provinces,were isolated through inoculation in embryonated SPF chicken.The study of biological characteristics demonstrated that the viruses could not agglutinate chicken erythrocytes,and the viruses were fully inactivated with56°C or 65°C treatment for 30 min and partial inactivated with pH3 or pH9 treatment for 60 min,respectively,in addition,the viruses were resistant to 4.4%volume chloroform treatment for 10 min at 4°C.The partial genes of hexon protein were amplified by PCR for sequences analysis and RELP,from the sequencing and RELP data,11 strains,including derived from the sheldrake were FAdV-4 serotype but the other 3 strains were FAdV-8a,8b,and 11 respectively.Subsequently,a PCR detection method based on viral DNA polymerase gene was established,which was capable of detecting FAdV covered 5 species and 12 serotypes in theory.With the advantages of sensitivity,specificity and accuracy,it could detect templates of the order of 2.8×102copies viral DNA and the above.Three DNA viruses,EDSV,MDV and FPV,which were susceptible to chicken,were tested,and totally found to be negative.The viruses were successfully isolated from the fresh samples with positive PCR results.Moreover,the samples of infected SPF chicken including heart,liver and spleen,lung,kidney,glandular stomach and muscle were entirely detected to be positive.The hexon protein genes of 11 FAdV-4 isolates were cloned and the sequence analysis indicated that the ORF was 2814 bp encoding 937 amino acids.It was highly homologous to the nucleotide and amino acid sequences of other type 4 viruses and the homology even reached from 97.1%to 99.7%,the amino acid sequence shared99.8%homology with PK-01,and the prevalent FAdV-4 strains in China belong to the independent genetic group via the phylogenetic tree analysis.The data of chicken regression test infected with isolated 4 serotypes showed that the symptom and necropsy were consistent with clinical cases with the characterization of ruffled fur,sluggish,pale cocks,except the infected died chickens,some of the survivors recovered after 2 to 4 days with symptoms.The lesions,typical pericardial effusion,inclusion body hepatitis,and kidney swelling were clearly visible.There existed partial glandular stomach bleeding,gizzard erosion in the autopsy.11 strains of FAdV-4 viruses were used to challenge SPF chickens,and the ELD50 of viruses was also determined.It was shown that the mortality rate was quite different,ranging from40%to 100%.From the results of ELD50,some were as high as 10-7.84ELD50/0.2 mL,but the lowest were only 10-5.0ELD50/0.2 mL.In combination with the challenge assay and ELD50 test,FAdV-4-sdbc-1 was finally selected as an inactivated candidate strain for vaccine development.In order to maintain the purity and stability of the virus seeds,the FAdV-4-sdbc-1seed batches including primary seed and working seed were prepared and a series of tests were performed.The titer of primary seed was ELD50?107.84/0.2 mL and the LD500 of 30-day-old SPF chicken was 10-4.84/1.0 mL,as for working seed,the titer of viruses was ELD50?10-8.0/0.2mL,and it was supposed to passage no more than 3generations for primary seed and working seed.In accordance with the rules for the inspection of biological products,foreign viruses,bacteria,and mycoplasma were tested,the results showed that the foreign ALV,REV,H5/H9 AIV,NDV,EDSV,IBV,ILT,IBDV,FPV,ARV and MDV were totally negative,and there was no bacterial or mycoplasma contamination.The virus passaging in embryos from generation 5 to 13did not affect the virus proliferation and immunogenicity.The water-in-oil?W/O?emulsion inactivated vaccine was prepared with qualified virus working seeds,and the production process was conducted.The titer of working seed more than 108.0ELD50/0.2mL was diluted 10-22 to inoculate yolk sac of 6-day-old embryos,then,the yolk fluid was harvest from died embryos between 96 h and 144 h.At this time,the virus titers should be more than 108.0ELD50/0.2 mL.The semi-finished product was prepared by adding an equal volume of physiological saline and inactivated by 1‰formaldehyde solution at 37°C for 32 h.According to the regulations,the inactivation test and the sterility test were conducted.After passing the detection,the vaccine was prepared with the ratio of oil phase to aqueous phase of3:2?V:V?.The prepared vaccine successfully passed various inspection indicators in the aspects of appearance,dosage form,stability,viscosity,asepsis,and formaldehyde content.To determine the safety,effectiveness,and convenience of the vaccine in clinical use,the safety and efficacy evaluation and storage period of the three batches vaccine was carried out.The 18-day-old SPF chickens were used to intramuscularly injected with three batches of vaccines respectively,and then the injected included a dose of 2mL per animal only and every 3 days,0.5 mL per animal for 3 times in total to perform the biosafety assay.The animal were observed for 14 days with the safety reached up to100%,from which revealed that all the SPF chickens were healthy and normal.Efficacy tests were conducted using two methods:challenge route and AGP antibody detection.Three batches of vaccines were immunized against 18-day-old SPF chickens,after 21 days,100LD50/mL FAdV-4-sdbc-1 strain was injected with 1.0mL of each and observed for 14 days.All of the immunized groups survived with a protection rate of 100%compared to the all died control group.The sera of chickens detected at 14d,21d,and 42d were 1:2,1:8,and 1:16,respectively.The vaccines could be stored at 4°C for 12 months and 20°C for 6 months,however the efficacy was unchanged.The successful development of this vaccine has provided biological agents for prevention and control of the disease. |
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