仔猪副伤寒活疫苗生产用菌株生长曲线的建立及合成培养基初步应用

Establishment of the Growth Curve of Paratyphoid Live Vaccine Strain for Production and Its Application in Synthetic Medium

关键词:  仔猪副伤寒活疫苗生产用菌株  培养参数  合成培养基

Key words:  strain for the production of paratyphoid live vaccine  cultivating parameters  synthetic medium

摘要:

建立了仔猪副伤寒活疫苗生产用菌株生长曲线，获取菌株培养参数，从而实现合成培养基初步应用。将猪霍乱沙门氏菌（CVCC79500株）运用试管和三角瓶分别静置和振荡培养30 h，期间取4、8、12、18、24、30 h等6个点进行活菌计数并建立生长曲线，确定37 ℃静置培养18～24 h，37 ℃、200 r/min振荡培养8～24 h为培养稳定期。不同接菌量比较结果表明，37 ℃静置培养24 h，37 ℃、200 r/min振荡培养12 h均可达到菌体稳定期。在稳定期参数条件下，比较合成培养基与常规普通肉汤培养基对该菌的增殖效果，结果表明，37 ℃静置培养24 h,试管及三角瓶培养菌数分别为27×108～31×108、33×108～41×108CFU/mL；37 ℃、200 r/min振荡培养12 h，试管及三角瓶培养菌数分别为58×108～66×108、78×108～88×108CFU/mL。而同条件3批普通肉汤培养菌数均低于合成培养基，且批次间稳定性较合成培养基差。将合成培养基振荡培养12 h的菌液接种小鼠均10/10存活；免疫后攻毒家兔达4/5～5/5保护，与普通肉汤基本一致。合成培养基保存期试验表明，25 ℃避光保存28 d与当天配制的液体合成培养基，振荡培养菌数基本一致。获取的培养参数适合用于合成培养基的初步评价，研制的合成培养基培养菌数优于普通肉汤，且不影响菌体安全性及免疫原性，室温保存期可达28 d。

Abstract:

In this study, we established the growth curve of the paratyphoid live vaccine strain and obtained the parameter for bacteria cultivation, resulting in its application on the production of paratyphoid live vaccine using the synthetic medium. Salmonella enterica serovar Choleraesuis (S. Choleraesuis) strain CVCC79500 was cultured for 30 h in the test tube or flask, via either static culture at 37 ℃ or shaking culture at 37 ℃/200 r/min. Culturing samples were taken out for live bacteria counting at 4, 8, 12, 18, 24 and 30 h, to establish the growth curve. It was shown that the growth of the strain CVCC79500 would reach its stationary phase by static culture at 37 ℃ for 18～24 h or shaking culture at 37 ℃/200 r/min for 8～24 h. While inoculated with different amount, the growth of the strain CVCC79500 could reach its stationary phase by static culture at 37 ℃ for 24 h or shaking culture at 37 ℃/200 r/min for 12 h. The effect for proliferation of strain CVCC79500 in synthetic medium and conventional common broth were compared using the obtained parameter. While static culture at 37 ℃ for 24 h in synthetic medium, the obtained bacteria count was 27～31×108 CFU/mL and 33～41×108 CFU/mL in test tube and flask, respectively. While shaking culture at 37 ℃/200 r/min for 12 h in synthetic medium, the obtained bacteria count was 58～66×108 CFU/mL and 78～88×108 CFU/mL in test tube and flask, respectively. However, the obtained bacteria count was lower when cultured in common broth at same condition. Using different batches of medium, the difference among obtained bacteria count was larger while cultured in common broth than in synthetic medium. The S. Choleraesuis strain CVCC79500 was cultured in synthetic medium at 37 ℃/200 r/min for 12 h. The mice was inoculated with the harvested bacteria and showed a survival ratio (number of alive species / number of vaccinated species) of 100% (10/10). The inoculated mice were challenged with virulent strain, and had a protective ratio (number of protected species / number of challenged species) of 80%～100% (4/5~5/5). The safety and the protective efficacy of the strain cultured in synthetic medium were coincident with the strain cultured in common broth. The obtained bacteria count via shaking culturing was similar while the synthetic medium was kept in dark at 25 ℃ for 28 days, compared to fresh prepared synthetic medium. Therefore, the synthetic medium was evaluated under condition of obtained optimum parameter for production of paratyphoid live vaccine. The synthetic medium was better than the common broth, as higher bacteria count was obtained in the former than in the latter. Moreover, the synthetic medium had no influence on safety and immunogenicity of harvested bacteria. The preservation period of the synthetic medium could reach 28 days at room temperature and away from light.