羊种布鲁氏菌疫苗株PCR-RFLP鉴定方法的建立

为建立一种羊种布鲁氏菌Rev.1疫苗株PCR-RFLP鉴定方法,利用羊种布鲁氏菌Rev.1具有链霉素抗性的特性,选取与链霉素抗性相关编码核糖体蛋白S12的rps L基因为模板设计引物,经PCR扩增后,将产物进行Nci I酶切,发现羊种布鲁氏菌疫苗株Rev.1出现约500 bp条带,而不具有链霉素抗性的羊种布鲁氏菌株16M则出现384 bp条带。结果表明,PCR-RFLP方法能够用于羊种布鲁氏菌Rev.1疫苗株的鉴定,这为今后区分羊种布鲁氏菌疫苗株Rev.1免疫与野株感染提供了基础。     

动物布鲁氏菌Rev.1疫苗研究进展

布鲁氏菌病（布病）是由布鲁氏菌引起的一种重要的人畜共患病,对公共卫生有着巨大的威胁。布鲁氏菌Rev.1疫苗研制于上世纪50年代中期,并在欧洲、中东和蒙古等地区被广泛应用于小反刍动物布病的防控,一度被认为是防控小反刍动物布病最为有效的疫苗。但是,由于Rev.1疫苗存在毒力偏强及毒力不稳定等现象,其安全性仍值得关注。本文主要从Rev.1疫苗背景及其菌株的生物学特性、疫苗使用情况以及存在的不足等几个方面进行阐述,以期为Rev.1疫苗的应用和布鲁氏菌相关疫苗的开发提供借鉴。     

评价牛型布鲁氏菌疫苗免疫保护力BALB/c鼠模型的建立

为建立评价流产布鲁氏菌疫苗株免疫保护力的小鼠模型,选取6周龄雌性BALB/c小鼠为试验动物,随机分为3组(n=40):A19免疫攻毒组、非免疫攻毒组和PBS对照组。A19免疫组腹腔接种BALB/c鼠A19 5.0×10⁴ CFU,非免疫攻毒组和PBS对照组均接种PBS液0.2 mL。免疫后45 d,A19免疫攻毒组和非免疫攻毒组BALB/c鼠以3.0×10⁴ CFU剂量的2308强毒株攻击,攻毒后15和45 d分别剖杀小鼠,取小鼠脾脏称重、细菌分离、病理组织学检测。结果表明,攻毒后15 d,A19免疫攻毒组与未免疫攻毒组和PBS对照组之间克脾指数差异显著(P<0.05);攻毒后45 d,A19免疫攻毒组与PBS对照组的克脾指数差异不显著(P>0.05),与未免疫攻毒组克脾指数差异显著(P<0.05);免疫攻毒组的小鼠组织病理变化明显轻于未免疫攻毒组。结果表明,用BALB/c鼠为试验动物,以A19为疫苗参考株建立动物实验模型可以应用于牛型布鲁氏菌疫苗免疫保护力评价。     

牛种布鲁氏菌clpP基因缺失株的毒力和免疫保护力评价

为研究布鲁氏菌clpP基因与细菌毒力的关系,采用同源重组的方法,构建牛种布鲁氏菌clpP基因缺失株,用巨噬细胞RAW264.7感染模型和小鼠感染模型评价clpP基因缺失株的毒力,同时测定该缺失株免疫小鼠后产生的免疫保护力。研究发现:牛种布鲁氏菌clpP基因缺失后,在细胞感染模型和小鼠感染模型中的毒力显著降低;clpP基因缺失株感染小鼠后,不引起脾脏肿胀,并且在脾脏内的持续期短于A19疫苗株,说明该基因缺失株具有更高的安全性;使用clpP基因缺失株免疫小鼠后,该基因缺失株无法抵御牛种布鲁氏菌2308株和羊种布鲁氏菌M28株的感染。本研究为揭示布鲁氏菌致病机制和新型布病疫苗研发提供了参考。     

布鲁菌疫苗株Rev.1全基因序列的测定与分析

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）冻干保护剂工艺的研究

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16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12×10^9CFUmL、12.44×10^9CFUmL、12.65×10^9CFUmL;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12.34×10^9CFUmL、12.62×10^9CFUmL、12.91×10^9CFUmL;使用蔗糖明胶的不同配比比例的冻干疫苗活菌数分别为12.22×10^9CFUmL、12.64×10^9CFUmL、12.8×10^9CFUmL。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。     

羊种布鲁氏菌Rev.1株VjbR基因的克隆、原核表达及生物信息学分析

本研究旨在构建表达载体pET-30a-VjbR,进而表达布鲁氏菌VjbR蛋白;利用生物软件分析该蛋白生物信息学信息,为进一步研究该蛋白奠定基础。以布鲁氏菌Rev.1株基因组为模板,扩增VjbR基因序列,将其克隆至原核表达载体pET-30a(+)中,获得重组质粒pET-30a-VjbR,并进行原核表达、Western-blot检测及该基因的生物信息学分析。结果显示:本试验成功克隆并表达了VjbR基因;经对表达产物进行SDS-PAGE分析纯化,发现本试验得到较纯蛋白;经Western-blot检测,发现该基因表达蛋白可与布鲁氏菌羊阳性血清发生特异性反应,具有良好的反应原性;通过生物信息学系列软件统计分析,发现VjbR蛋白无跨膜区,无信号肽,且该蛋白共有15个抗原决定簇,在二级结构中,α-螺旋的氨基酸有131个,占比为49.81%。布鲁氏菌VjbR基因的成功表达与纯化,为进一步制备该蛋白的单克隆抗体及iELISA诊断试剂盒的研制奠定了基础。     

4株布鲁氏菌弱毒株对小鼠毒力及免疫保护力的评价

为检测国内外已有的布鲁氏菌疫苗株S19、M5、S2和RB51的胞内存活力、毒力、免疫保护力以及抗体消长水平,将上述4种疫苗株,分别以100:1的MOI侵染小鼠巨噬细胞,结果发现RB51的胞内存活力最强;以1×10~6 CFU/只免疫昆明小白鼠,测定各疫苗株在小鼠脾脏中的定居力,结果发现M5的定居力最强;待疫苗株在小鼠体内被清除后,以1×10~5 CFU/只腹腔接种2308毒株,进行攻毒试验,检测各疫苗株的保护力,结果发现M5的保护效果最好;免疫后连续10周采集血清,用ELISA检测血清中的Ig G滴度、IFN-γ的表达水平及抗体消长水平,结果发现S2、M5组的Ig G水平高于其他组,而各组间的IFN-γ水平差异不显著(P0.05);分别用虎红平板凝集试验(RBPT)和标准试管凝集试验(SAT)检测血清凝集状况,结果发现S2、S19组的抗体持续时间较长;剖检各疫苗株对小鼠脾脏、肝脏、肾脏等组织引起的病理学变化,结果发现M5引起的病变程度最强。     

羊布鲁氏菌疫苗（S2株）口服免疫试验

为了解羊只布鲁氏菌疫苗(S2株)口服免疫的安全性及免疫效果,在甘肃省平凉市2个乡镇,各选1个行政村,选择散养户饲养的264只羊作为试验羊,进行小范围的布鲁氏菌S2株活疫苗口服免疫试验,其中3月龄以上羊只(包括怀孕母羊)每只口服活菌100×2亿,3月龄以下羊每只口服活菌100亿。分别在免疫后的30、60、180、210 d,采集试验羊血清样品,采用虎红平板凝集(RBT)+试管凝集联合试验(SAT)、酶联免疫吸附试验(ELISA)进行血清抗体检测。结果显示:试验组羊只免疫后均未出现异常反应,口服免疫30 d后,试验羊只2种检测方法检测的免疫抗体阳性率分别为47.3%和53.8%,不同时间2种方法的检测结果基本一致(P 0.05)。结果表明,布鲁氏菌S2株活疫苗按照试验剂量免疫安全有效,适用于所有月龄羊只以及怀孕母羊的整群免疫,可先行试点,逐步推广应用。     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.     

The characteristics of a variant strain of Brucella melitensis Rev 1

Circumstantial evidence is presented for the occurrence of a variant of a vaccine strain of B. melitensis Rev 1, designated "FSA" (foreign South African). FSA resembles Rev 1 in its reactions to penicillin and streptomycin but reacts closer to a field strain of B. melitensis as regards dye (thionine and basic fuchsin) sensitivity and colony size. Although colonies of Rev 1 were consistently smaller than other B. melitensis strains, their size was 0,75 mm as opposed to the 1-2 mm reported in the literature, while B. melitensis 16M colonies were 1,25-1,5 mm as opposed to the 3-4 mm previously reported. Rev 1 was found to be urease positive, unless a test of low sensitivity was applied.