为制备禽结核菌素国家标准品,研究禽结核菌素生产用菌株禽分枝杆菌的菌种特性,对1992年冻干保存的3株菌CVCC68201、CVCC68202和CVCC68203进行复苏,采用SPF鸡复壮,分离培养后冻干了新批次菌种,对菌种形态、生化特性、培养特性、毒力和抗原性进行了鉴定.结果表明,制备的菌种纯粹,形态、生化和培养特性符合禽分枝杆菌的生物学特征,免疫原性符合的兽用生物制品质量标准的规定.本研究为禽分枝杆菌的鉴定提供参考,也为制造禽结核菌素的工艺改进奠定基础.

目的 通过研究1株粗糙型布鲁氏菌诱导株RB71的毒力及免疫效果,进一步验证其生物学特性,探讨其作为疫苗株的潜力.方法 通过药敏片扩散法检测诱导株RB71对β-内酰胺类及非β-内酰胺类抗生素的耐受程度;通过对小鼠脾脏载菌量测定,评价诱导株RB71对感染小鼠的免疫保护率并确定其最小免疫剂量;利用小鼠感染模型进行毒力试验,比较诱导株RB71与亲本株的毒力差异.结果 诱导后的布鲁氏菌RB71株与亲本株相比,对β-内酰胺类抗生素和非β-内酰胺类抗生素的敏感性未发生改变;诱导株RB71对感染小鼠具有一定的保护效果,最小免疫剂量为109CFU/只;诱导株RB71与亲本株相比毒力更小,安全性更高.结论 本研究验证了布鲁氏菌诱导株RB71对抗生素的敏感性,其具有良好的免疫原性且具有较低的毒力,具备成为疫苗株的潜力.

为了研究布鲁氏菌病温敏凝胶疫苗对羊不同黏膜途径免疫的效果,本研究采用冷溶法制备了用于黏膜免疫的布鲁氏菌病温敏凝胶活疫苗(S2株),将该疫苗以不同黏膜免疫途径(阴道灌注、点眼)、注射免疫途径免疫绵羊,同时以常规S2疫苗作为对照,无菌采集绵羊的免疫部位拭子后培养,通过菌落计数方法计算黏膜免疫部位排菌数,结果显示:凝胶疫苗免疫部位从免疫后第30 min开始停止排菌,常规疫苗的排菌持续到免疫后第2d.免疫后采集各时间点绵羊血血清,采用微量试管凝集试验(MSAT)和攻菌保护试验分别测定不同免疫途径(阴道灌注、点眼、注射)免疫后产生的抗体水平和保护效果的差异,抗体检测结果显示,黏膜免疫后45 d左右抗体即降至1∶50或以下,黏膜免疫后产生的抗体水平明显低于注射免疫产生的抗体,且两种黏膜免疫下降速度均比注射免疫的快;攻菌后的免疫保护结果显示,在对照组出菌指数为4.76情况下,免疫组的单位保护(对照出菌指数-免疫出菌指数)分别达到2.19、2.42、2.78,表明3种免疫方式均具有良好的免疫保护效果,其中阴道灌注的单位保护高于点眼,与注射免疫组更接近.以上结果表明:与常规疫苗相比,温敏凝胶疫苗可有效提升疫苗的安全性.相比点眼免疫,阴道灌注的免疫保护效果更好、安全性更好,虽然点眼抗体反应更弱、持续时间更短,但与阴道灌注相比,对诊断的干扰差异不大.综合以上结果表明,两种黏膜免疫方式中,阴道灌注是较理想的黏膜免疫方式,但黏膜免疫抗体持续时间短于注射免疫.因此,采用布鲁氏菌病温敏凝胶疫苗进行黏膜免疫更有利于鉴别诊断,同时也为新型布鲁氏菌病疫苗的研究提供新的思路.

本试验旨在研究布鲁氏菌病疫苗的新型研制方法,并通过op诱导剂成功诱导出一株粗糙型牛种布鲁氏菌弱毒株,命名为RB71.试验检测了RB71相关基因的缺失情况,并对其脂多糖完整性、生长特性、遗传稳定性以及在小鼠巨噬细胞(RAW264.7)中生存能力等与光滑型菌株进行了比较研究.结果 显示,试验成功获得了诱导突变株,其缺失片段大小为15 070 bp;热凝集试验阳性,能被结晶紫染色,吖啶黄凝集试验阳性;对提取脂多糖进行银染,结果显示O链缺失,诱导株RB71脂多糖不完整;连续传代30次,PCR检测未发现基因回复突变;在体外相同培养条件下,诱导株RB71生长速度显著低于亲本株A19;入侵RAW264.7细胞72 h时,其胞内存活率与亲本株相比极显著下降(P＜0.01).综上所述,本试验开发出一种能高效诱导光滑型布鲁氏菌变异为粗糙型布鲁氏菌的试剂及诱导方法,成功获得一株具有良好遗传稳定性的粗糙型减毒布鲁氏菌RB71诱导株,该诱导株在RAW264.7细胞内的存活能力显著变弱,这为新型弱毒布鲁氏菌粗糙型疫苗的研制奠定技术基础.

试验旨在分析糖基转移酶编码基因WadC影响布鲁氏菌胞内存活的作用.以羊种布鲁氏菌Rev.1基因组为模板,通过同源重组方法获得WadC基因上、下游同源臂融合片段,并与载体pUC19-SacB连接,构建pUC19-SacB-ΔwadC重组载体,电转至羊种布鲁氏菌Rev.1,构建△wadC缺失株(Rev.1ΔwadC),检测菌株Rev.11ΔwadC的遗传稳定性,比较分析亲本株Rev.1和缺失株Rev.1ΔwadC的生长特性及其在BMDC和RAW264.7细胞中的生存能力.结果显示,试验成功构建基因缺失株,连续传代30次未发现基因回复突变;在体外相同培养条件下,缺失株Rev.1ΔwadC与亲本株Rev.1生长趋势相似,均在20 h到达对数生长期,44 h进入平台期;侵染BMDC细胞48和72 h时,其胞内存活率显著低于亲本株(P＜0.05);而侵染小鼠RAW264.7巨噬细胞试验显示,亲本菌株和基因缺失株无显著性差异(P＞0.05).综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌WadC基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在BMDC细胞内的存活能力显著变弱,为深入研究布鲁氏菌WadC基因功能奠定基础.

布鲁菌是一种革兰阴性胞内寄生菌,能引起动物和人类急性和慢性感染.疫苗免疫是布鲁菌病控制的关键.由于粗糙型布鲁菌毒力低,且接种后不会刺激机体产生针对O抗原的抗体,不干扰临床诊断,因此粗糙型布鲁菌疫苗有望成为布鲁菌病防控中的优良疫苗.论文就与光滑型与粗糙型布鲁菌之间的联系、粗糙型布鲁菌突变体获得方法及其疫苗的研究进展做一综述,以期为布鲁菌粗糙型疫苗的开发及合理应用提供参考和借鉴.

Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.

为了比较布鲁氏菌病温敏凝胶疫苗经不同黏膜途径免疫的效果,本研究采用BABL/c小鼠为实验动物模型,制备了用于黏膜免疫的布鲁氏菌病温敏凝胶活疫苗(S2株),比较了该疫苗与常规疫苗黏膜免疫(阴道灌注、点眼、口服、直肠灌注)后的体表排菌,研究了黏膜免疫与注射免疫的抗体反应规律差异及保护效果.结果 显示:凝胶疫苗能够在34℃～36℃完成由液态到凝胶态的相转变,适合所选小鼠黏膜途径的免疫;凝胶疫苗从免疫后第2d开始停止排菌,常规疫苗的排菌持续到免疫后第4d;黏膜免疫小鼠抗体效价比注射免疫低25％～80％,并且在免疫后45 d左右微量试管凝集试验(MSAT)结果显示前者抗体效价即下降至1:50以下或接近1:50,下降速度显著快于注射免疫;对阴道灌注、点眼、口服、直肠灌注及注射免疫的小鼠攻毒结果显示对照组小鼠的出菌指数为6.47,免疫组的单位保护(对照出菌指数-免疫出菌指数)分别为3.89、3.81、3.32、3.25、4.08,其中阴道灌注、点眼2种黏膜免疫小鼠的单位保护更接近注射免疫的单位保护(4.08).研究结果提示,凝胶疫苗比常规疫苗在免疫后更可以显著减少体表排菌,抗体持续时间更短、使得对疫病的诊断干扰更低,此外,凝胶疫苗免疫也具有接近注射免疫的单位保护.因此,采用布鲁氏菌病凝胶疫苗进行黏膜免疫更有利于布鲁氏菌病的鉴别诊断.

Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M.

To investigate the effects of different mucosal immunization approaches on humoral immunity response,cellular immunity response and brucella excretion situation of dairy cattle vaccinated with live brucellosis vaccine, 80 cows (10~12 months old) were divided into four groups (20 for each group).The cows were inoculated with Brucella A19(B.abortus)by eye infusion (group A), oral injection (group B), vaginal perfusion (group C) and subcutaneous injection(group D) respectively with same full standard dose.The brucella excretion situation within 7 days after immunization for group A, B and C, the antibody changes within 180 days and cytokines secretion (IFN-γ, IL-2, IL-4) within 60 days for group A , B , C and D were checked.The results showed that brucella excretions of three mucosal immunization approaches were lasted for 5 days and reached highest within the first day;the brucella excretion of group C was the lowest compared to group A and B;the antibody titer and the duration of antibody positive rates of group A, B, C were significantly lower than group D, for all the cows the antibody titer reached the peak on 20 dpi, furthermore group C had the highest antibody level compared to other two mucosal immunization groups;the antibody positive rates of group A, B, C were all 0% on 120 dpi, 120 dpi, 150 dpi respectively while the antibody positive rate of group D was 35% on 180 dpi;tested groups' IFN-γ excretions reached the peak on 7 dpi (group B and group C) or 20 dpi (group A and group D) while the IL-2 and IL-4 secretion had no significantly change, IFN-γ excretion of group C was the most among all the mucosal immunization groups and a little lower than group D.Our results indicate that vaginal perfusion is a much better approach for vaccination of dairy cattle with live attenuated brucellosis vaccine.

In this study, an isolated strain of Brucella was identified by biological feature and genotype, and determined the minimum infected dose ( MID) and bacteria content of spleen in experimental animals ( beagle) . This isolated strain is Brucella cains which was identified by dyeing properties, culture characteristics, serological characteristics, biochemical characteristics, and further identified by Multi-PCR and 16S rRNA. The MID is 105 CFU/mL in beagle, and the content is 4×105~3×106 CFU/g in spleen.

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Immunization with attenuated vaccines has proved to be an effective method of prevention; however, it may also interfere with diagnosis. Brucella abortus strain A19, which is homologous to B. abortus strain S19, is widely used for the prevention of bovine brucellosis in China. For effective monitoring of the control of brucellosis, it is essential to distinguish A19 from field strains. Single-nucleotide polymorphism-based assays offer a new approach to such discrimination studies. In the current study, we developed a cycleave PCR assay that successfully distinguished attenuated vaccine strains A19 and S19 from 22 strains of B. abortus and 57 strains of 5 other Brucella species. The assay gave a negative reaction with 4 non-Brucella species. The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 7.6 fg for the A19 strain and 220 fg for the single non-A19/non-S19 Brucella strain tested (B. abortus 104M). The assay was also reproducible (intra- and interassay coefficients of variation: 0.003-0.01 and 0.004-0.025, respectively). The cycleave assay gave an A19/S19-specific reaction in 3 out of 125 field serum samples, with the same 3 samples being positive in an alternative A19/S19-specific molecular assay. The cycleave assay gave a total of 102 Brucella-specific reactions (3 being the A19/S19-specific reactions), whereas an alternative Brucella-specific assay gave 92 positive reactions (all also positive in the cycleave assay). Therefore, this assay represents a simple, rapid, sensitive, and specific tool for use in brucellosis control.

为了解全国布氏菌病活疫苗生产企业活菌计数能力,2015年对该项目进行了能力验证分析,每名参比企业发放布氏菌活疫苗(S2株)样品3份,要求在规定时间内对其进行活菌计数并提交结果报告.结果显示,参比的17家企业中,15家结果“满意”,2家企业结果“不满意”,表明全国大部分布氏菌病活疫苗生产企业具备可信任的布氏菌病活疫苗活菌计数检测能力.

In order to evaluate the effect of control measures on brucellosis in Inner Mongolia and to further polish con-trol strategy, an evaluation was conducted on different control measures that being implemented in Siziwangqi county. The targeted control measures included tracing and culling strategy, vaccination, surveillance, quarantine and so on. The result showed that the tracing and culling strategy has distinctive effect in general, but the effect might vary along with different infection statuses. Single tracing and culling was unable to remove all the infected individuals when the prevalence was high, while“vaccination without tracing and culling strategy” had a certain effect on brucellosis control in that situation.

To investigate the pattern of increasing or decreasing of serum antibody in sheep and goats immunized with brucellosis vaccine,RPST and tube agglutination test were used in the study to determine the level of serum antibody in sheep and goats before and after immunization with Brucella strain S2 vaccine. The results indicated that the serum antibody positive rate in sheep reached the highest level 20 DPI,then started to decrease and remained relatively low level 90-360 DPI,mostly below 6%,and relatively higher in few individuals,up to 14%. The positive rate in goats reached the highest 20 DPI,then decreased and remained low or relatively low level 90-360 DPI,but with decreasing or increasing sometimes. Comparatively,antibody positive rate in goats was generally higher than in sheep,and the descending and ascending range was bigger. It was also shown that there was no significant difference in antibody posi-tive rate between 10 billion CFU dose group and 20 billion CFU dose group in same species.

Brucellosis is a worldwide zoonosis. Vaccination is the most efficient means to prevent and control brucellosis. The current licensed attenuated vaccines for animal use were developed by sequential passage in nonnatural hosts that decreased virulence in its original hosts. The attenuation mechanism of these strains remains largely unknown. In the present study, we sequenced the genome of Brucella melitensis vaccine strain M5-10. Sequence analysis showed that a large number of genetic changes occurred in the vaccine strains. A total of 2854 genetic polymorphic sites, including 2548 SNP, 241 INDEL and 65 MNV were identified. Of the 2074 SNPs in coding regions, 1310 (63.2%) were non-synonymous SNPs. Gene number, percent and N/S ratios were disproportionally distributed among the cog categories. Genetic polymorphic sites were identified in genes of the virB operon, flagella synthesis, and virulence regulating systems. These data indicate that changes in some cog categories and virulence genes might result in the attenuation. These attenuation mechanisms also have implications for screening and development of new vaccine strains. The genetic changes in the genome represent candidate sites for differential diagnosis between these vaccine strains and other virulence ones. Transcription analysis of virulence genes showed that expression of dnaK, vjbR were reduced in M510 strain when compared with that in 16M. A duplex PCR targeting virB6 and dnaK was successfully used to differentiate between M5-10 and the virulent 16M strain. The genome re-sequencing technique represents a strong strategy not only for evaluation of vaccines, but also for development of new vaccines.

To distinguish Brucella abortus vaccine strain A19 from field strains, the single nucleotide polymorphism (SNP) signatures for B. abortus A19 were initially analyzed using bioinformatics method and gene sequencing. Then, the specificity of several SNPs was verified, by comparing the nucleotide sequences of these SNPs with common species and biovars of Brucella and three Brucella vaccines. The results showed 29 SNPs were successfully screened from genome of B. abortus A19. Furthermore, ClpX G825-C825,LysR A605-C605and Omp2b G503-A503were confirmed to be specific to B. abortus A19 (or B. abortus S19). Our study systematically revealed the SNP distribution of B. abortus A19, which provided a molecular basis for differentiating B. abortus A19 from field strains.

In order to identify different smooth Brucella strains, we prepared the monospecific serum against B.abortus O-chain A epitope and B.melitensis O-chain M epitope. Hyperimmune serum was collected from five rabbits, which were inoculated with heat-inactivated B.abortus (2308 strain) and heat-inactivated B.melitensis (16M strain) respectively. The hyperimmune serum was treated by serum antigen cross-absorption to get mono-specific antiserum. Monospecific serum against B. abortus O-chain A epitopes were obtained which at a 1/320 dilution agglutinated the homologous antigen and at a 1/2. 5 dilution did not agglutinated the the heterologous antigen. Monospecific serum against B. melitensis O-chain M epitopes were obtained which at a 1/160 dilution agglutinated the homologous antigen and at a 1/2.5 dilution did not agglutinated the the heterologous antigen. Then the monospecific serum was lyophilized and stored at-20℃. The slide agglutination test showed that monospecific serum against B. abortus O-chain A epitope and B. melitensis O-chain M epitope had no agglutination with homologous antigen. The results showed that the obtained monospecific serum were of high specificity and high titer, which could assist the identification of smooth Brucella species.

Objective The objective of this study is to explore the biological characteristics and assess the virulence of a B. abortus isolate (strain 343).[Method]The culture of B. abortus 343 was diluted and cultivated to single clone on Tryptic Soy Agar (TSA) for morphological observation, and it was then stained with Gram’s and Koch’s methods. Biochemical characteristics tests (including the growth ability test on thionine and alkaline azaleine, the dependence on CO 2 , and H 2 S release test) , single phase specific serum (B. abortus specific anti-serum A, B. melitensis specific anti-serum M and Brucella rough-type specific anti-serum R) agglutination tests and AMOS-PCR were used to identify its comprehensive characteristics. Mice and guinea pigs were infected with a certain dose of B. abortus 343 to determine the virulence of the isolate. The survival time of B. abortus in mice, the isolated number per gram of the spleen from the infected guinea pigs, and the minimum infecting dose (MID) for guinea pigs were tested.[Result]B. abortus 343 is a smooth-type, Gram-negative, Koch's staining red strain, which can reflect glaucous radiance from its surface culture. It releases H 2 S during its metabolism, and grows on TSA containing thionine or alkaline azaleine, and the growth of which was not dependent on CO2. The antigen prepared from B. abortus 343 was positive with A-type serum in plate agglutination test, and it could induce the immunized mice to produce the specific antibody. The bacteria were isolated from the spleen of the tested mice 29 weeks post infection with a dose of 1×105 CFU. While infected with a dose of 1×109 CFU to the female guinea pigs, 2.4×105 CFU-1.2×106 CFU could be cultured from spleen per gram 14 days post infection. It could induce specific smooth-type antibody from all guinea pigs infected with a dose of 1×105 CFU, and 1 month post infection the titers of serum were high from 320 to 1 280 in agglutination test. To guinea pigs, the MID is about 40 CFU. [Conclusion]A middle virulence B. abortus strain, which can be used as a reference strain in the further study was identified. As a new isolate, the detailed identified B. abortus 343 strain is a good supplement to the brucella resources.

To analysis the sensibility, specificity and coincidence rate of the complement fixation enzyme linked immunosorbent assay kit ( CF-ELISA kit ) for detecting brucellosis antibodies, 400 bovine and sheep serum specimens were collected from brucellosis-affected areas selecting CF-ELISA kit to detect brucellosis antibodies, where cases had already been identified. Another total of 200 bovine and sheep serum specimens for detecting antibodies were collected from no brucellosis-affected areas, where no cases had been identified. Results showed that there was no significant difference (P>0.05) among the CF-ELISA kit, iELISA kit and CGS for detecting antibodies of brucellosis-affected areas serum. Kappa statistics result showed that the method of CF-ELISA and iELISA had highly coincidence rate. The specificity and coincidence rate were 100% between the CF-ELISA kit and iELISA kit for detecting antibodies of no brucellosis-affected areas serum.