Study On Inactivated Porcine Parvovirus Disease And Porcine Circovirus Disease Combined Vaccine

Porcine parvovirus (PPV) and Porcine circovirus type2(PCV2) are allthe serious epidemics that do harm to the pig industries.The epidemics are widespreadaround the world and endemic in most pig farms,seriously affecting the developmentof the swine industry.Nowadays,vaccination is a major measure for an effectiveprevention and control the two epidemics.However,the only use of Single unionvaccine is time-consuming and great stress and immune escape.Therefore,it isimperative to develop an efficient inactivated binary vaccine to prevent the twoepidemics with only one needle.Given this,the study screened one PCV2vaccinecandidate finally through the immunogenicity test and immune protection test.Toovercome the shortcomings of PCV2poor propagation in vitro and low proliferationtiter,the study primarly discussed the production of PCV2in PK-15cells which arecultivated in switch tube microcarriers,on this basis,PPV-PCV2bivalent inactivatedvaccine was successfully produced with the use of the PPV vaccine candidate-HNZKstrain which was screened earlier in our lab.The research contents are as follows:1. To screen strains which can meet the requirements of producing inactivatedPCV2vaccine, the immunogenicity of8strains of PCV2isolates in our lab wereanalyzed.The results showed that of the8isolates,the isolates of HNTP and HNYYcan induced higher antibody level after immunization,and the antibody level reachedhigher than1:800in1week after the second immunization,which has met the nationalstandard of the quality of inactivated PCV2vaccine.And the antibody level reachedthe peak of1:3200in3weeks after the second immunization,which was higher thanthat(1:1600) induced by the controlled commodity vaccine;Morever,the vaccineproduced with130times and463times diluted antigens can also reach the level of1:800in3weeks after the second immunization.The results showed that the HNTPand HNYY isolates of PCV2can be used as the ideal vaccine candidates for their highvirulence and immune efficacy. The study enriched the resource of seed virus ofPCV2inactivated vaccine and laid a foundation for further development of PCV2vaccine with high quality.2.The research studied the effect of protective immunity of the vaccine produced by the PCV2vaccine candidate,HNTP and HNYY isolates.The protective effect ofdifferent infection dosages was determined through observation of clinical symptomsand detection of viral load in the tissue from the immunized mice after4weeks sincethe second immunization.The results showed that no virus was detected in the tissueof mice from the immunized group,suggesting that the vaccine has providedprotection.Moreover, in the previous study,the existing time of antibodies induced byHNYY isolate could last longer.Consequently,the HNYY isolate was chosen as theinactivated vaccine candidate.The virus was detected in the negative groups whichwere challenged by the virus of6.072×106copies and6.072×107copies,suggestingthat the virus could replicate in the tissues of mice,with a relatively high level in theirhearts,livers,lungs and lymph nodes.3.To overcome the disadvantages of poor prolificacy in vitro and low titer ofproliferation of PCV2,taking the advantages of high specific surface area,high yield ofcells,convenience of monitoring,controlling and sampling,facility of scale-up,anddifficulty of contamination of microcarrier adherent cells,the researcher primarilyexplored the production of PCV2using the PK-15cells cultivated in the roller tubemicrocarrier.Results showed that cell population of PK-15utilizing roller tubecultivation was as much as10times of that cultivated in the ordinary flasks.Inaddition, the level of viral load reached the highest,5.53E+06copies/μL,in the groupinoculated of120.8MOI at the100hours of PI,which consequently enhanced thevirus titer as much as1.65times.This finding indicates that it is practicable to producePCV2in the PK-15cells cultivated on the microcarriers and that provided a technicalstrut for the improvement of the viral load and industrial mass production of potentialvaccine antigens.4. The virus suspension was harvested using the evaluated HNYY isolatesof PCV2and HNZK isolates of PPV isolated and identified in our lab respecti-vely,and viruses with high titer was achieved by concentration method throughdialysis bags.And then they were used as antigens of the bivalent inactivatedvaccine.The harvested virus suspension was inactivated by formaldehyde,and the bivalent inactivated vaccine of PPV and PCV2was produced by the two kindsof inactivated viruses which were mixed together with equal volume and emulsified with the oil adjuvant.The vaccine was evaluated by its physical properties,sterility,safety and protective immunity for animals.The results showed that thebivalent inactivated vaccine is safe to animals.Every mouse and guinea pig was immunized with0.3mL and0.5mL respectively,and the immune response was s-trengthened two weeks later.The ELISA antibody level of PCV2hit a peak of1:3200after the second immunization,which was higher than that of1:1600ind-uced by the single vaccine of PCV2.While the antibody level decreased at1:1600three weeks later after the second immunization,which was also equal tothe group of single vaccine.The HI antibody level of PPV had an upward tren-d both in the group of single PPV vaccine and the group of the bivalent vacci-ne after the first immunization of the guinea pigs,however the antibody level r-eached a peak of1:2048in the single vaccine group in four weeks after seco-nd immunization,by contrast,the bivalent vaccine group reached the same levelof1:2048in six weeks after second immunization.