用Western印迹法鉴别布氏菌和O:9型耶氏菌抗体

本文用Western印迹法证实O:9型耶氏菌外膜蛋白中分子量为36、22、12kda等多肽特异于布氏菌,几乎和所有静脉接种、皮下感染O:9型耶氏菌家兔血清抗体反应。用YOMPs为抗原的Western印迹法,结合布氏菌试管凝集试验(STA),可以应用于布氏菌和O:9型耶氏菌血清学鉴别诊断。该技术具有敏感、可靠的特点。     

广西家狗自然感染犬种布鲁氏菌的病理学研究

本文观察结果表明,犬种布病的特征性病变是在肝、脾、淋巴结、肺、子宫及骨髓出现慢性肉芽肿和血管机能亢进性类,伴实质不同程度损害。超微结构的特征性基本病变是在上述器官内大量巨噬细胞增生,并转化成上皮样细胞形成肉芽肿。有明显吞噬现象,胞内大量的溶酶体,并有残缺不全的球杆菌。     

神经型布鲁菌病临床特点和实验室诊断分析

目的总结神经型布鲁菌病（NB）的临床及实验室诊断过程,为提高临床和实验室对该病的认识和早期诊疗提供经验。 方法回顾性分析2015年1月至2017年1月于首都医科大学宣武医院确诊的12例神经型布鲁菌病患者的临床表现、流行病学资料及脑脊液（CSF）培养等病原学检查、影像学表现和其他实验室检查结果,并与非神经型布鲁菌病患者比较平均发病年龄,CSF白细胞数、蛋白含量、氯化物、葡萄糖水平等。 结果12例神经型布鲁菌病患者中男性9例,女性3例,平均年龄为（43 ± 14）岁;10例患者来自流行地区,9例有明确的流行病学接触史,其中3例既往诊断布鲁菌病;12例患者均出现不同程度发热,6例有头痛伴脑膜刺激征表现;3例患者表现为复视、视力下降;伴有听力减退伴步态不稳,肢体麻木无力,精神状态改变者各4例。血清布鲁杆菌凝集试验阳性（100%）较CSF凝集阳性率（58.3%）和CSF培养阳性率（46.1%）对临床诊断神经型布鲁菌病价值较大,差异具有统计学意义（χ² = 52.68、P = 0.005,χ² = 73.79、P = 0.005）;12例NB患者CSF白细胞数（单核为主）和蛋白均不同程度升高,葡萄糖、氯化物正常或减低,与非NB感染者相比,CSF葡萄糖检测具有重要价值（P均< 0.05）。给予多西环素或米诺环素+利福平+头孢曲松治疗后12例患者均预后良好。 结论神经型布鲁菌病临床表现多样,易误诊或漏诊,非疫区医务人员应重视CSF血瓶培养及布鲁杆菌凝集,加强多学科协作交流以及早确诊及早治疗。     

巨噬细胞-布鲁氏菌感染的靶点

布鲁氏菌是兼性胞内寄生菌,它们主要定居在巨噬细胞和胎盘滋养层细胞这两类宿主细胞,.在巨噬细胞内,布鲁氏菌在长期的胞内微生态环境的定居中经历重重考验,使自己成为适合生存和复制的胞内部分,即包含布鲁氏菌的早期吞噬体通过与内质网适当融合而获得内质网的部分膜成分而发展为有利于布鲁氏菌胞内生存和复制的布氏小体.     

布鲁氏菌PCR鉴定方法的研究与应用

目的采用PCR、PCR SSCP方法对布鲁氏菌进行快速鉴定,并对其种、型鉴别。方法分析已发表布鲁氏菌属、种、型基因序列,寻找出不 同布鲁氏菌的种、型特异性碱基分布规律,设计特异性引物,用于布鲁氏菌属、种、型的鉴定;根据聚合酶链反应 单链构象多态性（PCR SSCP）分析原理,建立PCR SSCP方法,用于区分牛种A19疫苗株与其他型布鲁氏菌。结果所建立的PCR方法能高效、准确鉴定出布鲁氏菌,并能 对其种、型进行区分;PCR SSCP方法可将A19疫苗株从其他型布鲁氏菌中区分出来。结论利用PCR、PCR SSCP方法能快速、准确地进行布鲁氏 菌属、种、型以及疫苗株A19的鉴别,且简便、可靠,便于临床应用。     

Analysis of Brucella cellular fatty acids

To acquire data of Brucella cellular fatty acids (CFAs) and probe into the possibility of utilizing CFAs information in typing Brucella, 19 reference strains were subjected to CFAs study. After all strains were inoculated on Brucella Agar plates, the cells were harvested, saponificated, methylated and extracted to provide fatty acid methylesters for gas chromatography (GC) analysis. Based on the CFAs data matrix, a dendrogram of 19 reference strains was generated by SPSS11.5 software package. The results showed that 19 reference strains were divided into five clusters: cluster 1 included B. suis (bv. 1, 2, 3, 5) and B. ovis; cluster 2 included B. abortus (bv. 3, 4, 5, 6) and B. melitensis (bv. 1, 2, 3); cluster 3 included B. abortus (bv. 1, 2, 7, 9) and B. neotomae; cluster 4 was B. suis (bv. 4); and cluster 5 was B. canis. Typing Brucella by GC analysis of CFAs is a good method to reflect drug resistance of Brucella, and the classification is beneficial for clinical therapy. It also provides a new result of typing and demonstrates that the traditional classification is not completely reasonable. CFAs analysis may identify B. suis (bv. 4) and B. canis.     

Improved Immunogenicity of Tetanus Toxoid by Brucella abortus S19 LPS Adjuvant

Background: Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. Objectives: To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. Methods: LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for the conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund’s adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Results: Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. Conclusion: These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.     

布鲁氏菌外膜蛋白 VirB4在感染胚胎滋养层细胞中的作用分析

目的 筛选布鲁氏菌侵染牛胚胎滋养层细胞过程中与Ⅳ型分泌系统VirB4蛋白结合的潜在靶蛋白。方法 设计引物并PCR扩增布鲁氏菌的virB4基因,构建表达载体pGBKT7-VirB4,酶切鉴定,测序分析正确后,转化酿酒酵母菌感受态细胞Y187,进行自激活和毒性检测;建立布鲁氏菌侵染牛胚胎滋养层细胞模型,构建布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;采用酵母双杂交技术筛选与VirB4相互作用的滋养层细胞蛋白,实时定量PCR检测靶蛋白表达量的变化。结果 成功构建了pGBKT7-virB4诱饵质粒,转入Y187后无毒性,不能自激活;获得了布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;筛选到了13个阳性质粒,其中蛋白辅酶Q₁₀和SLC3A2在布鲁氏菌侵染后mRNA表达量均增加。结论 本试验对VirB4蛋白与宿主细胞的互作研究为进一步阐明布鲁氏菌感染宿主细胞的发病机制奠定了基础。     

布鲁氏菌疫苗株S2全基因组测序及牛羊布鲁氏菌比较基因组学研究

目的 为了了解布鲁氏菌S2疫苗株全基因组结构及分子生物学功能。方法 对布鲁氏菌疫苗株S2进行全基因组测序,并与GenBank公布的6株牛羊布鲁氏菌进行比较基因组学研究。结果与结论 通过全基因组测序发现S2基因组大小为3 331 982 bp,预测基因有3 243个,GC含量为57.23 %。S2预测基因中大多数基因功能主要与糖代谢﹑氨基酸代谢﹑氨基酸转运和膜转运有关。通过比较基因组学研究发现S2有20个特有基因。另外S2与GenBank公布的S2序列进行比对发现有19个差异基因。