Brucellosis is one of the zoonotic diseases of animal origin that seriously threatens human health.The prevention and control of brucellosis in animals is mainly based on prevention.At present,there are S2,A19 and M5 vaccines for brucellosis in China,and S19 and Rev.1 vaccines in the world,among which M5 and Rev.1 strains of sheep are the least stable.TSA is Tryptone soy agar medium(also known as Tryptone soy agar medium),a commercial medium that is suitable for the growth of Brucella Rev.1 strain.After TSA medium plate with different agar concentration was placed under different conditions(temperature and time),Brucella Rev.1 strain was inoculated.After 96 h culture,colony growth was observed and colony crystal violet staining was performed.Through comparative analysis,it is found that the humidity of TSA medium plate is an important factor affecting the accuracy of variation test results.At the same time,the preparation method of TSA medium for Brucella Rev.1 strain was clarified.

为促进布鲁菌病Rev.1疫苗及相关疫苗的研发,该研究提取Rev.1疫苗株核酸,应用PacBio平台进行全基因序列测定与分析.结果 表明,Rev.1疫苗株基因组大小约3 299 187 bp,G+C含量为57.2％,组装为染色体1、染色体2两条环状基因组,大小分别为2 121 370、1 177 817 bp,G+C含量分别为57.2％、57.3％.将其Ery、BLS和VirB10基因序列与9株GenBank上发表的布鲁菌参考菌株的Ery、BLS和VirB10基因序列进行比较分析,存在不同程度的差异,同源性为97.4％～100％.

布氏菌病活疫苗(Rev.1株)的冻干工艺借鉴、采纳布氏菌病活疫苗(S2株)成熟的冻干工艺及菌液的配比,对冻干保护剂的选择进行了优化.我们选择蔗糖明胶保护剂和蔗糖脱脂乳保护剂,保护剂与布鲁氏菌Rev.1株菌液以1:6的比例混合,冻干后测定其活菌含量.结果显示,3批使用蔗糖脱脂乳保护剂的不同配比比例的冻干疫苗活菌数分别为12×109CFU/mL、12.44×109CFU/mL、12.65×109CFU/mL;使用蔗糖明胶再加入终浓度约1％硫脲的不同配比比例的冻干疫苗活菌数分别为12.34×109CFU/mL、12.62×109CFU/mL、12.91×109CFU/mL;使用蔗糖明胶的不同配比比例的冻干疫苗活菌数分别为12.22×109CFU/mL、12.64×109CFU/mL、12.8×109CFU/mL.3种冻干保护剂对冻干疫苗的活菌含量影响无明显差异,但使用蔗糖明胶保护剂制备冻干疫苗时加入适量硫脲可适当增加冻干疫苗的耐热性,因此我们选择10％明胶、20％蔗糖冻干保护剂与菌液以1:6配比比例混合,再加入终浓度约为1％的硫脲作为布氏菌病活疫苗(Rev.1株)的冻干工艺.

在相同饲养条件下,用布氏菌病活疫苗(Rev.1株)和布氏菌病活疫苗(S2株)进行羊只的免疫,按照常规免疫剂量分别接种健康绵羊30只,并设空白对照10只,共计70只.免疫前和免疫后14、21、28、49、60、90、120、150 d采血,采用虎红平板凝集试验(RBT)和试管凝集试验(SAT)2种检测方法进行血清抗体检测.结果证实2种疫苗的免疫效果,为指导布鲁氏菌病的防治提供科学依据.

To prepare antiserum for the identity test of classical swine fever(CSF) live vaccine and its seed virus,healthy pigs were immunized with CSF live vaccine and boosted with CSFV virulent Shimen strain.Four lots of antiserum was prepared by filtration and lyophilization.All serum candidates passed the quality control tests.The specificity of the sera was tested and shown negative against foot and mouse disease virus,porcine pseudorabies virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus and bovine viral diarrhoea-mucosal disease virus.The CSFV residuals in the sera were also negative tested by IFA and nested RT-PCR.The neutralization titers of the sera were 1∶800,1∶800,1∶800 and 1∶1,600,respectively.The application of the sera was verified by testing seed virus of CSF vaccines and 5 vaccines.Therefore all 4 lots of sera could be used for the identity test of CSF live vaccine and its seed virus.