布鲁氏菌PFGE分子分型的条件优化

[目的]将PFGE方法应用于布鲁氏菌分型研究,探索建立布鲁氏菌PFGE分型的最优化方法。[方法]在用PFGE方法进行布鲁氏菌基因分型的研究中,针对不同内切酶、酶切的浓度和时间、脉冲时间等影响PFGE分型结果的因素进行优化,利用优化后的条件对布鲁氏菌19株标准菌株进行分型研究。[结果]在布鲁氏菌的PFGE分型研究中,选择XbaI为内切酶、酶切浓度为96u/200μl、酶切时间为4h、脉冲时间为2～25s为PFGE电泳的优化条件,在分析的19株布鲁氏菌标准菌株中,6种布菌的PFGE图谱各不相同,但PFGE不能将种内不同生物型布菌完全区分开来。[结论]PFGE方法可用于布鲁氏菌的基因分型的研究,是对传统分型方法一种较好的补充。     

布鲁菌脉冲场凝胶电泳图谱分型方法建立

目的 建立我国95株布鲁菌的染色体DNA脉冲场凝胶电泳图谱,进行布鲁菌的分子遗传学分类鉴定。方法 选择我国不同地区、不同时限分离的布鲁菌100株(包括19株标准布鲁菌株),应用SeaKem Gold琼脂糖胶块纯化布鲁菌完整的染色体DNA,限制性内切酶XbaI消化后,进行脉冲场凝胶电泳分离(PFGE)。结果 XbaI酶切布鲁菌基因组DNA后,可产生20~500kbp范围的15~25条酶切片段,并可以在PFGE凝胶上被很好的区分开。PFGE可在种的水平上区分布菌各种标准株,76株中国分离株可被分为39种PFGE类型,并聚为4大类。PFGE与传统分型方法比较,2者的一致性达63%。结论 脉冲场凝胶电泳图谱可对布鲁菌进行遗传学分类鉴定,在布鲁菌的分子流行病学研究中具有重要意义。     

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.     

Rifampicin Resistance Phenotyping of Brucella melitensis by rpoB Gene Analysis in Clinical Isolates

Abstract

Rifampicin resistance of Brucella melitensis by rpoB gene analysis has not yet been performed in Turkey, where brucellosis is endemic. In this study, we investigated the efficacy of E-test and single nucleotide polymorphism (SNP) analysis of the B. melitensis rpoB gene, for the detection of mutations conferring rifampicin resistance, by sequencing 21 human B. melitensis strains from the Southeast and Marmara regions of Turkey. On CLSI slow-growing bacteria standards, all isolates were sensitive to rifampicin except for 6 which showed intermediate resistance to rifampicin. MIC₅₀ and MIC₉₀ values were 1 μg/ml and 1.5 μg/ml respectively (range 0.50 -1.5 μg/ml). The rifampicin-resistant phenotype was investigated at Cd 154 (GTT/TTT), Cd 526 (GAC/TAC, GAC/AAC, GAC/GGC), Cd 536 (CAC/CTC, CAC/TAC), Cd 539 (CGC/AGC), Cd 541 (TCG/TTG) and Cd 574 (CCG/CTG) of the rpoB gene in B. melitensis 16M and B115 strains, and in clinical isolates. No missense mutations were found in any of the B. melitensis isolates, which indicates that all isolates were rifampicin-susceptible. In conclusion, SNP analysis was useful as a molecular tool for rifampin resistance testing. Although resistance to rifampicin was not detected in our strains of B. melitensis; the presence of strains with intermediate resistance to rifampicin indicates that susceptibility testing should be performed periodically.     

Brucellosis acquired by eating imported cheese

Pyrexia of unknown origin is an important clinical presentation in both paediatric and adult medicine. We present a case of pyrexia of unknown origin in a 14 year-old boy which turned out to be due to infection with Brucella melitensis, despite the patient not having left Great Britain - an officially brucellosis-free country - in six years. Repeated history-taking provided a clue to the diagnosis.     

Spinal brucellosis: a review

Brucellosis is a zoonosis of worldwide distribution, relatively frequent in Mediterranean countries and in the Middle East. It is a systemic infection, caused by facultative intra-cellular bacteria of the genus Brucella, that can involve many organs and tissues. The spine is the most common site of musculoskeletal involvement, followed by the sacroiliac joints. The aim of this study was to assess the clinical, biological and imaging features of spinal brucellosis.