Study Of Porcine Pseudorabies Virus Gene Deletion Live Vaccine With Heat Protective Agent

Pseudorabies is an acute infectious disease caused by pseudorabies virus,which is classified as 2th level of animal diseases in china.The main clinical manifestations are elevated body temperature,reproductive disorder and neurological symptoms.The mortality rate is very high.Adult pigs often manifeste as latent infection.By the widespread use of the disease gene deleted vaccine,the epidemic has been well controlled befor 2011.However,since the end of 2011,the outbreak of the disease happened in many provinces again,which resulting in huge economic losses to China's pig industry.Our lab isolated a high virulent PRV ZJ01 strains,and the successful constructed a gE,gI and TK gene deletion strains:ZJ01 ?gE/gI/TK strain.In this study,we developed a freeze-dried vaccine with ZJ01 ?gE/gI/TK strain and determined its immune protection against PRV.The manin contents includes as following:1.Choice of heat resistant freeze drying protective agent for ZJ01 ?gE/gI/TK vaccine:We designed five kinds of heat resistant freeze drying protective agent(S151,S152,S153,S154,S155),mixed with PRV ZJ01 gE/gI/TK virus,and freeze-drying.The vaccines were placed at 37? and-20 ? for 1 weeks and the virues TCID50 were determined individually.The results showed that the protective rate of S151,S152,153,154 and S155 was 93%,95%,98%,88%and 95%respectively.After placed for a week at 37?,the average titer of vaccines with S151,S152,S153 and S155 were decreased by 1.88,0.33,1.13,and 2.06,respectively,compared with the original vaccines.After placed at-20? a week,the average titer of vaccine viruses with S151,S152,S153 and S155 was decreased by 0.52?0.21?0.47 and 1.16,respectively.The average titer of virus was decreased by 0.39,which was prepared with S152 for 10 months at 4?.It indicated that S152 agent has the best protective effect to PRV-ZJ01 gE/gl/TK strains.The main composition of the protective agent included 4%gelatin,2%bovine serum albumin,6%polyvinylpyrrolidone(K30),2%sorbitol,0.05%Vc,15.2%sucrose and 4%ABC preparation,which can be used for vaccine production.2.Determination of the protective effect of the freeze-dried vaccine:Thirsty 30-days old pigs free of PRV were divided into 6 groups each with 5.First group was inoculated with Bartha vaccine,group 2-5 were inoculated with dose of 102 0,103 0,104 0 and 105 0TCID50 ZJ01 ?gE/gl/TK vaccine,sixth group was PBS control group.At 14 days post immunization,Anti-PRV gB ELISA antibody and neutralizing antibody levels weredetermined.After 21 days of immunization,10 " TCID50 PRV-ZJ01 strains were used for chanllenge.After challenge,the body temperature was measured and the clinical symptoms were observed eneryday for 14 days.The pathological changes were observed at 14 days post challenge.The results showed that gB antibody was positive in all vaccinated pigs,gE antibody was negative.And the neutralizing antibody titer in 105 0TCID50 group is the highest at two weeks after immunization.After challenge,the incidence in PBS group is 100%.The incidence in Bartha group is 40%.But there were no obvious clinical symptoms in ZJ01 ?gE/gI/TK vaccine group.Pathological observation showed that the lung of all pigs in group PBS was atrophic,with severe cerebral hemorrhage.Bartha vaccine group,ZJ01 vaccine group gE/gl/TK did not show significant pathological changes.In conclusion,the freeze-dried vaccine with 102.0TCID50/ml could provide protection against ZJ01 challenge in clinical.And the immune effect with 105.0TCID50/ml vaccine is the best.3.Determination of immune routes of the freeze-dried vaccine:Fifteen 30-days old pigs free of PRV were divided into 3 groups,the first group was PBS immune group,2th,3th groups were treated with intramuscular injection and intranasal inoculation of 105.0TCID50 injection dose of ZJ01 gE/gl/TK vaccine.Anti-PRV gB ELISA antibody and neutralizing antibody level were determined at 14 days after immunization.At 21 days post immunization,107.0TCID50 PRV-ZJ01 strain was used for nasal challenge.After challenge,the body temperature was measured and the clincal symptoms and the pathological changes were observed after 14 days.The results showed that at two weeks after immunization,gB antibody was positive in all vaccinated pigs,gE antibody was negative,and 105.0TCID50 immune dose groups could induce high levels of neutralizing antibodies with no significant difference.After challenge,the incidence of pigs in PBS group is 100%.ZJ01 vaccine group gE/gI/TK did not show significant pathological changes.Pathological observation showed that the lung of all pigs in group PBS was atrophic,with severe cerebral hemorrhage.Meanwhile,ZJ01 vaccine group gE/gl/TK did not show significant pathological changes.In conclusion,intranasal and intramuscular injection can provide adequate protection against PRV infection in swine.