| Classical swine fever(CSF)is an acute or chronic,febrile and highly contagious infectious disease of pigs caused by classical swine fever virus(CSFV).It is characterized by acute onset,high fever retention and denaturation of small blood vessel walls,causing systemically punctate hemorrhage and spleen infarction.CSF is listed as a legally notifiable disease by World Organization for Animal Health(OIE)and class A of infectious diseases in China,which causes a huge threat to the pig industry.At present,vaccination is the most effective and economical measure to prevent and control CSF.The CSFV vaccine strains used in China all belong to Hog cholera lapinized virus and they can be divided into splenic organ origin,rabbit origin,cell origin and passage cell line origin according to different production processes.And CSF live vaccine(passage cell line origin)is the most widely applied.Antigen production is the key part of vaccine development.Antigen content and immunogenicity are the key factors for determining vaccine quality.At present,traditional roller bottle process is mainly used to produce vaccine antigen,which has the defects of easy contamination and great difference between batches in the production course.It is the mainstream trend of animal vaccine production to establish micro-carrier suspension culture technology based on bioreactor.In this study,we developed and optimized the key technological parameters for the production of CSF live vaccine by bovine testicular passage cells(BT cells)micro-carrier suspension culture process,and evaluated the safety and efficacy of three batches of products in the laboratory by the process,which set a technical foundation for the large-scale production of CSF live vaccine(strain C,passage cell origin)by BT cell micro-carrer suspension culture.1.Optimization of the proliferation of CSFV(C strain)by BT cell micro-carrier suspension cultureIn this study,the growth conditions of BT cells micro-carriersuspension culture and the proliferation conditions of CSFV(C strain)were optimized.The key parameters were determined as follows:cell inoculation density was 1.5×105?2.0×105 cells/mg,stirring speed was 40?45 rpm(for 2 L bioreactor),45?50 rpm(for 10 L bioreactor),MOI(Multiplicity of Infection)was 0.1?0.5.After inoculation,the virus was harvested and replaced maintenance medium for the first time on the fifth day.After that,the virus was harvested and replaced for new medium every four days,and it could be harvested for eight times.The average antigen of CSFV was above 107.2FAID50/ml.2.Inspection of laboratory products of CSF live vaccine(strain C,passage cell origin)developed by BT cells micro-carrier suspension cultureThree batches of CSF live vaccine(strain C,passage cell origin)developed by BT cells micro-carrier suspension culture in laboratory were tested.The results showed that the viral contents for three batches of products were 105.0,104.9 and 105.1 FAID50/dose respectively,and the each dosage was 7500 rabbit body infections(RID).The results of character test,aseptic test,mycoplasma test,exogenous virus test,identification test,residual moisture test and vacuum test all conform to the quality standard requirements of "Code for Manufacture and Inspection of Live Swine Fever Vaccine(?)".3.Safety evaluation of laboratory products of CSF live vaccine(strain C,passage cell origin)developed by BT cells micro-carrier suspension cultureTo evaluate the safety of CSF live vaccine produced by micro-carrier suspension culture,three batches of vaccine(batches:CSFXF001,CSFXF002,CSFXF003)were used in this study.Healthy piglets aged 4-6 weeks with CSF negative antibody were selected,and 15 for single dose inoculation test(1 dose per piglet),and 15 for single dose repeated inoculation test(14 days interval between inoculations).Fifteen healthy piglets aged 4-6 weeks with CSF negative antibody(Landrace,Dabai and Duroc)were inoculated with overdose vaccine(30 doses/piglet),while negative control was designed.15 healthy piglets aged 2 weeks with negative CSF antibody were inoculated with overdose(30 doses/piglet)at the same time,while negative control was designed.30 sows in the early pregnancy(20-30 days of gestation),the middle term pregnancy(50-70 days of gestation)and the late term pregnancy(80-95 days of gestation)were selected,10 sows for each stage.5 sows of each stage were inoculated with overdose(30 doses/sow),respectively,and 5 sows were set for the negative control group.The results showed that the 3 batches laboratory products of CSF live vaccine were all safe for single dose inoculation,repeated inoculation of single dose,over dose inoculation for different breeds of piglets,and over dose inoculation for 2-week-age piglets.Mental status and appetite were good,and no adverse side reactions were observed in all groups.And no abortion was found in vaccinated and control groups of over dose inoculation for sows in the early,middle and late pregnancy.The average survival rate of weaned piglets was over 97%.There was no difference in weight gain between the immunized and control groups from birth to weaning.4.Efficacy test of laboratory products of CSF live vaccine(strain C,passage cell origin)developed by BT cells micro-carrier suspension cultureIn order to evaluate the immune efficacy of laboratory products of CSF live vaccine(strain C,passage cell origin)developed by BT cells micro-carrier suspension culture,the batches of CSF live vaccine produced in laboratory was used in the study.To determine minimum immune dosage,15 healthy piglets of 4-6 weeks old with CSF antibody negative were immunized with 105.0 FAID50,101.5 FAID50 and 100.5 FAID50,five piglets in each group respectively.Negative control group was set.On the 14th day after immunization,all animals were challenged with 1 ml CSFV Shimen strain,and observed for 16 days.To compare the batch CSF-XF002 produced by suspension culture and similar product on market produced by roller bottle culture,10 healthy piglets aged 4-6 weeks were immunized by 101.5 FAID50/piglet,5 animals for each group.Negative control was set up.On the 14th day after immunization,all animals were challenged with 1 ml CSFV Shimen strain.And the immune protection effect between two processes was compared after 16 days observation.To analyze the immune interference of CSF maternal antibody to vaccine immunity,10 healthy piglets aged 4-6 weeks with positive CSF antibody were immunized with one dose CSF-XF003 batch vaccine.And CSF maternal antibody positive control group and CSF antibody negative control group were designed.On the 14th day after immunization,all animals were challenged with 1 ml CSFV Shimen strain.And the immune protection rates of all groups were calculated after 16 days observation.Getting data together,results showed that the minimum immune dose of CSFV was 100'5 FAID50/dose.5/5 was protected in the immunized group,and 5/5 died in the control group when challenged with virulent CSFV Shimen strain,indicating that there was no difference in the immune response between two production processes.10/10 of animals were protected in the immunized group with positive maternal antibody,and 5/10 of animals were protected in the non-immunized group with positive maternal antibody,while 5/5 of animals were infected and 5/5 died in the CSF antibody negative control group,indicating that CSF live vaccine(strain C,passage cell origin)produced by BT cells micro-carrier suspension culture could break through the maternal antibody and showed a good protective efficacy on experimental animals.In conclusion,the results above showed that CSF live vaccine(strain C,passage cell origin)produced by BT cells micro-carrier suspension culture was stable,safe and effective,which provided technical reference for the research of CSF live vaccine using suspension production process. |
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