Research, Development And Application Of New Technics Of Classical Swine Fever Living Vaccine

Classical swine fever (CSF) is a highly pathogenic disease of pigs, which is caused by Classical swine fever virus (CSFV). In this research, swine testis cells were cultured to apply to the production of Classical swine fever living vaccine, established the manufacture regulation and quality criteria. Classical swine fever living vaccine were batchly produced and gained satisfactory application from more than 30 swine farms of eight provinces in China. Further, this study developed substantial foundation for prevention of Classical swine fever.ST cell culture:ST cell line (Swine Testis Cell, ST cell) was employed to be cell matrix of Classical swine fever virus. firstly, ST cells were serially passaged 3 times, which proliferated sufficiently for the establishment of master cell bank; Secondly, master cells were serially passaged 4 times, which proliferated sufficiently for the development of working cell bank; last but not the least, there is no any contaminations of the cells from master cell bank and working cell bank, which was affirmed by sterility test, mycoplasma test and exogenous virus test. Additionally, studing the usage of ST cells in the supreme passage, we found that the cells rescuing from working cell bank still could proliferate well, maintaining the purity of cell and good ability for viral propagation, which had cultured 25 times. Cells of the 3 passages of master cell bank, the supreme limit passage, being superior 5 times to the supreme passage were assaied to be normal using soft agar colony forming test, tumorigenesis test in nude mouse, chromosome test. This kind of cell that have no mutagenicity, teratogenicity, carcinogenicity accord with every characteristic in the live attenuated vaccine manufacture, which could apply to the production of vaccine based on Classical swine fever virus.Classical swine fever virus Seed:Both the purity, safety and immunogenicity of Classical swine fever virus and the identification of virus from tissue culture seed lot using in the production are consistent with criterion of the Veterinary Pharmacopoeia of the People's Republic of China (2005 Edition).Research and Development of new vaccine:high density culture of ST cell and Classical swine fever virus were done by bioreactor. Compare tests were done to choose the optimal ST cell passages, virus-seed amount, seed harvest time, virus preservation temperature. The results show when the tissue virus seed amount is more than 105 RID/ml, the proportion of virus is 0.2-0.3% of the culture. When the cell-cultured virus seed amount is more than 5×105 RID/ml, the proportion of virus is 3-5% of the culture. First time to harvest the virus is the fifth day, and then change the culture and harvest the virus every 4 days. All the half-finished product's title kept unchanged after storaged 3 months below-15?.Safety and potency test:new Classical swine fever living vaccine (swine testide cell origin)'s virus content per dose is ten times higher than Classical swine fever living vaccine (bovine testide cell origin). The results of safety potency test show that 3 batches of vaccine made in lab are safe to swines which vaccinated 30 doses (7500RID/dose). Rabbits' potency tests show that all the rabbits which vaccinated 1/7500 dose passed the test. All the experimental swines which vaccinated 1/3000 dose have kept live 100% after challenged by high-virulence Classical swine fever virus. All the tests show that the new-type vaccine can induce better immunity protects to swine.Comparison with similar products:Refers to finished product testing methods of classical Classical swine fever vaccine (cell origin) from'The People's Republic of China Veterinary Pharmacopoeia'(2005 Edition), the comparison tests between classical swine fever vaccine (ST cell origin) developed in this study and the similar products (BT cell origin) shows that vaccinated swines of 1/5000 by three batches of classical swine fever live vaccine produced by ST cell could provide 100% protection. vaccinated rabbits of 1/7500 could produce fever-specific response, it's all qualified. The results of the experiment in virus amount?potency and safety indicate that the virus titer and immunization protection efficacy of new vaccine is ten-times of current classical swine fever vaccine standard produced by BT primary cell, three batches of classical swine fever live vaccine produced by ST cell are all qualified in safety test by using 30 doses.From the results made by nucleotide and amino acid sequence comparison of gp55 antigen protein encoded by E2 genes and difference analysis of antigenic structure and neutralization epitopes in ST-cell and BT-cell vaccine strains, we found that the protective antigen gene of HCLV vaccine strains cultured by ST and BT cell are no significant change in E2. Classical swine fever vaccine virus from ST cell show a high stability in heredity.Application and Extension:After obtaining the production permission of Classical Swine Fever Live Vaccine (Cell Line Origin) from The Ministry of Agriculture, we produced and tested strictly in accordance with the Classical Swine Fever Live Vaccine (Cell Line Origin) manufacturing and testing provisional statutes and the provisional regulations of quality standards which was promulgated by The Ministry of Agriculture. In 2008 the production of Classical Swine Fever Live Vaccine (Cell Line Origin) was total 5 batches of 5.17 million doses. In 2009 produced 22 batches which have passed the test 20 batches of 24.62 million doses. Until July 31 2009,7 batches of 7.33 million doses of Classical Swine Fever Live Vaccine (Cell Line Origin) have consumed in the 8 provinces. According to various immunization procedures on piglets, conservation pigs, fattening pigs and boars so far not aware of any adverse reactions and feedback of clinical infection and subclinical infection.In Guangdong, Hunan, Henan, Sichuan, Jiangsu, Shandong, Liaoning and Fujian provinces more than 30 standardized pig farms had been chosen to vaccination, at the same time the farms' Classical Swine Fever Live Vaccine (Cell Line Origin) protocol and immunization procedures was considered. Evaluating a 7 batches of 3.89 million doses of vaccine application by the follow up clinical application safety observation and immune effect of field experiment on sows, boars, reserve and commodity pigs with once and multiple immunization there are not any side-effects but well safety and immune effect.