Development Of Inactivated Gosling Plague Vaccine (TZ10 Strain)

Gosling plague is one of the common diseases resulting in massive death in goslings,causing huge economic losses and posing a serious threat to goose industry.The widespread use of existing attenuated live vaccines and gosling plague antibodies has played an important role in the prevention and control of this disease in China.However,the attenuated live vaccine has the potential of virulence reversion,which limits its extensive application in the field.Therefore,itis particularly important to develop the inactivated vaccines against gosling plaque.In this study,we systematically analyzed the biological characteristics of five gosling plague virus isolates,selected one of the field strains with good immunogenicity and high reproductive titer as an inactivated vaccine candidate,and performed research work concerning the vaccine production technique,emulsification process and protective efficacy.We expected to prepare a highly efficient inactivated gosling plague vaccine to provide a powerful tool for the prevention and control of the gosling plague in China.1.Selection of the vaccine candidate strain of gosling plague virusIn order to develop a vaccine that is compatible with the current circulating gosling plague strains,5 goose parvoviruses(GPVs)were isolated from clinical samples of suspected gosling plague cases in goose raising areas of Jiangsu province from 2009 to 2010.We then sequenced all the VP3 genes and constructed the corresponding phylogenetic tree,and the results showed that the 5 GPV isolates belonged to the clade that involving most of the domestic isolates,but obviously diverged from the vaccine strain SY61 and the strains from Taiwan.Subsequently,we determined the reproductive ability,virulence,specificity and immunogenicity of the 5 GPV strains after viral purification by using limited dilution method.It was found that the 5 GPVs were all virulent strains,in which the strain virus/Taizhou/10(TZ10)possessed the highest virus the titer(over 105 0 ELD50/0.2 ml of generation F10)and its inactivated antigens induced the highest antibody level in immunized geese 4 weeks post vaccination.Therefore,the TZ10 strain was selected as a candidate strain for the gosling plague vaccine development.According to the requirements of the "Guiding Principles of Experimental Techniques for the Establishment of seed batches of Veterinary Biological Products(Toxins,Insects)" issued by the Ministry of Agriculture,the purified TZ10 strain of generation F0 was subjected to continuous passage in goose embryos until generation F30.We then conducted the agar gel precipitation(AGP),immunogenicity specificity and purity tests of each different generation of seed viruses,as well as the determination of the median lethal dose in goose embryo(ELD50).Subsequently,we established the master seed batches(F16-F20)that complied with the GPV TZ10 strain and determined the highest generation limit(F25)for seed production.2.Study on the production technology of the inactivated vaccine against gosling plagueIn the process of vaccine production,the production technique of antigen determines the quality and cost of vaccine,especially the complete inactivation of antigen and the emulsification technology of vaccine are the key steps related to vaccine safety,efficacy and vaccine quality,respectively.In this study,we chose the previously established GPV seed batch F25,and tested the allantoic fluid of optimal dosage and optimal day-age for inoculation in goose embryos.According to the statistics of embryo death,antigen harvest volume and virus titer,the optimal seed dilution ratio was 1:100 with sterile saline while the optimal inoculation condition was 0.2 ml per embryo dose through allantoic cavity route on 11-day old non-immunized goose embryo.And the optimal virus culture time was 48?120 hours post inoculation according to the data from embryo death,antigen harvest and virus titer at different time after injection.To further determine the optimal method for antigen inactivation,we investigated the concentration and inactivation time of formaldehyde solution.When choosing formaldehyde solution as the inactivator,the GPV strain TZ10 could be completely killed at a final formaldehyde concentration of 0.2%with the inactivation time of 48 hours at 37?.In addition,studies on vaccine emulsification process confirmed that the optimal emulsification effect of the inactivated GPV strain TZ10 antigen was acquired with the following elements:fully dissolved and mixed aqueous phase prepared from every 96 antigen with 4 sterile Tween-80,optimal matched ratio of water phase and oil phase with 1:3,shear rate of 3000?4000r per minute for secondary emulsification,and an temperature below 25?during the whole emulsification process.3.Safety evaluation of the inactivated vaccine against gosling plagueAccording to the determined production technology for the inactivated vaccine(TZ10 strain)against gosling plague,3 batches of inactivated vaccines were test in the laboratory.The safety evaluation of the 3 batches of inactivated vaccine(TZ10 strain)was conducted by injection with one single dose of vaccine,repeated injection with one single dose and with double dose at 3?5 day-old and 1-month-old geese,and 7-month-old and 9-month-old breeeding geese,respectively.All the immunized geese showed normal mental state,appetite and drinking,without systemic adverse reactions.No swelling was present in the inoculation sites,and a small amount of vaccine particles were not absorbed only in the experimental group with boosting one-dose or double-dose injection on day 21 post-immunization(p.i).Except for the slight red flush of the muscle in the injection site from the geese received double-dose,no significant change was observed in the other immunized groups.On day 21 p.i,there was no significant difference in the weight growth between the immunized and control groups.The egg production in one week after injection were slightly less than those in the control group,and returned to normal level soon,also with no difference in hatching rate.These data indicated that the inactivated vaccine(TZ10 strain)against gosling plague was safe for breeding geese,and no clinical symptoms were observed in the gosling after injection.4.Immune efficacy of the inactivated vaccine against gosling plagueBased on the immune efficacy from the 3-batch inactivated vaccines,the GPV agar dififusion antibody level in serum from breeding geese injected with the vaccine was positively correlated with the passive GPV immunization antibody in serum from 3-day-old goslings and the protection rate.The relation of the he GPV agar diffusion antibody level between the breeding geese and the corresponding offsrpings showed that the protection rate of goslings was 92.3%when the titer of the serum from the breeding geese was 1:2,and the protection rate reached 100%when titer from the breeding geese was no less than 1:4.Considering the uncertainty in the breeding environment and the goose health status,the standard of the immune efficacy of the inactivated vaccine is that the geometric mean titer of GPV agar diffusion antibody from the breeding geese via intramuscular injection at 7?8-month-old with lml per bird,is not less than 1:8 on week 4 p.i.When the 7-8-month-old breeding geese were immunized with no less than 0.5 ml inactivated vaccine,90%of their offspring can effectively resist the attack from GPV challenge based on the minimum immune dose examination.In terms of the virus titer,the minimum immune dose test and the complexity in clinic,the standard of the semi-finished antigen before inactivation is 105.5 ELD50/0.2mL and the routine immunization dose of vaccine is 1.0 ml per goose.According to the antibody titer sustain period both from the breeding geese and goslings received 1 or 2 immunization of these 3-batch vaccines,and the protective efficacy of the hatching goslings from the immunized breeding geese,the immunization program of the vaccine was defined as follows:Prime vaccination of breeding goose at 5 weeks before egg-production with 1.0 ml per bird through intramuscular injection,boosting with the same dosage and route 3 weeks later.With this immunization schedule,the immune efficacy could sustain 5 months and protect the goslings,hatched from the breeding geese during the 6-month egg production period,against the GPV infction.Due to the instable source and quality of the healthy breeding geese with negative serum against GPV,the stable and repetitive potency tests for the inactivated gosling plague vaccine can not be guaranteed when geese were used as the target animal for vaccine immunization.Therefore,the SPF chickens were immunized with this inactivated vaccine in this study,and the titer and specificity of the antibody from the vaccinated chickens was analyzed to assess the possibility of the chicken as the alternative animal for vaccine potency test.Twenty-one-day-old SPF chickens were immunized with 3 batches of inactivated vaccines against the goose plague at different doses,and the AGP antibody titer of serum from the chickens,injected with the same dose,was almost the same at different time points,indicating that the SPF chicken could generate a stable immune response to the vaccine.The neutralization test of the positive serum from the SPF chicken immunized with the inactivated gosling plague vaccines showed that the specific antibody against the GPV could be generated.The alteration of the AGP antibody titer from the chicken serum was in accordance with that from the 7?8-month-old healthy geese immunized with different immune dose at different time,which confirmed that detection of AGP antibody titer from the vaccinated chicken serum is an alternative method to evaluate the vaccine efficacy of the goose plague inactivated vaccine.According to the correlation of neutralization antibody and AGP antibody titers between the SPF chickens and breeding geese post-immunization,the neutralization antibody titer was 2.87 log10 when the AGP antibody titer from goose serum was 1:8,and the corresponding AGP antibody titer of the SPF chicken serum was 1:16.Based on the positive correlation between the immunization dose and the antibody titer,and the oil-emulsion vaccine absorption,the test criteria of the vaccine efficacy of the goose plague inactivated vaccine(TZ10 strain)in 21-day--old SPF chicken was determined as follows:the geometric mean titer of AGP antibody from the chickens immunized with this vaccine via subcutaneous injection with 0.25ml per bird should not be less than 1:18 on 21 day p.i.