布鲁氏杆菌LPS抗原制备及其免疫原性测定

采用热酚水法提取布鲁氏杆菌脂多糖,分离、纯化并SDS-PAGE银染鉴定。纯化的细菌内毒素免疫小鼠,通过斑点ELISA测定血清抗体效价,检测细菌内毒素免疫原性。采用热酚法能获得较高纯度的细菌内毒素,其免疫原性较高,斑点ELISA测血清效价达到1∶1 000 000,与全菌免疫原性接近。本研究为制备布鲁氏杆菌单克隆抗体和建立布鲁氏杆菌病快速诊断方法奠定研究基础。     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

Pathological changes,distribution and detection of Brucella melitensis in foetuses of experimentally-infected does

BackgroundBrucellosis of goats is caused by Brucella melitensis. It is a re-emerging zoonotic disease in many countries due to transmission from domestic animals and wildlife such as ibex, deer and wild buffaloes.ObjectiveTo describe the pathological changes, identification and distribution of B. melitensis in foetuses of experimentally infected does.MethodsTwelve female goats of approximately 90 days pregnant were divided into 4 groups. Group 1 was exposed intra-conjunctival to 100 µL of sterile PBS while goats of Groups 2, 3 and 4 were similarly exposed to 100 µL of an inoculum containing 10⁹ CFU/mL of live B. melitensis. Goats of these groups were killed at 15, 30 and 60 days post-inoculation, respectively. Foetal fluid and tissues were collected for bacterial identification (using direct bacterial culture, PCR and immuno-peroxidase staining) and histopathological examination.ResultsBilateral intra-conjunctival exposure of pregnant does resulted in in-utero infection of the foetuses. All full-term foetuses of group 4 were either aborted or stillborn, showing petechiations of the skin or absence of hair coat with subcutaneous oedema. The internal organs showed most severe lesions. Immune-peroxidase staining revealed antigen distribution in all organs that became most extensive in group 4. Brucella melitensis was successfully isolated from the stomach content, foetal fluid and various other organs.ConclusionVertical transmission of caprine brucellosis was evident causing mild to moderate lesions in different organs. The samples of choice for isolation and identification of B. melitensis are stomach content as well as liver and spleen tissue.     

一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定

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。     

犬种布鲁氏菌研究进展

犬种布鲁氏菌属于6个经典种布鲁氏菌之一，为天然粗糙型，毒力较弱。长期以来，由于犬种布鲁氏菌对人类致病性较低，其危害一直被忽视。自1966年在美国发现该菌以来，已传播至世界多地。近年来，随着养犬业的不断发展及宠物犬数量的增多，犬布鲁氏菌病发病率不断上升，在某些地区已成为流行性疫病，且对人类公共卫生安全的威胁也日益加重。目前，犬种布鲁氏菌感染导致的犬布鲁氏菌病尚无可靠的诊断方法及有效的疫苗。鉴于此，在查阅相关文献的基础上，笔者对犬种布鲁氏菌的病原学、流行病学、致病机理、检测方法、疫苗研究等方面的相关研究进展进行综述，以期为犬布鲁氏菌病的深入研究和防控提供参考。     

Isolation and sero-genomo-epidemiological studies on Brucella infection in dairy cattle in Meghalaya,India

In this study, we report the serological, bacteriological and whole genome sequencing data of a 6 years study of Brucella abortus in Meghalaya, India. Investigation of 3060 sera samples indicated overall prevalence of 6.4% by Rose Bengal Plate Test and 10.7% by ELISA. Considerably higher prevalence was observed among milk samples (17.5%, n = 362) and in blood samples (37.7%, n = 262) by direct PCR. Clinical samples (n = 94) from late abortion cases yielded 11 B. abortus isolates. Multi-locus sequence typing indicated circulation of single sequence type, ST1. Whole genome sequencing (n = 8) and phylogenomic analysis revealed close clustering of majority of isolates in two clusters alongwith genomes from other countries, indicating global relatedness among B. abortus. Taken together, the results of our study revealed the putative hotspot of infection in the dairy-dominant districts of the state and also calls for concerted One Health based action for prevention and control of this zoonotic disease.     

制品生产用及地方疑难布氏菌DNA基因同源性的研究

本文以布氏菌19株国际标准菌种为对照,对国内外实验室参考菌种14株及地方疑难菌种11株结合常规方法进行了DNA G+Cmol％测定,同时以羊种菌M16 DNA为标准,与布氏菌所有种和型的24个代表株及8株地方疑难菌种进行了DNA-DNA杂交测定,布氏菌及地方疑难菌种的G+Cmol％值范围为54.1～59.3,M16株与布氏菌参考菌种的杂交率81.7～109％,与地方疑难菌种的杂交率除一株为72.1外,其余为84.2～109％,属高度同源,鉴定为布氏菌。本试验首次使用进口新仪器“PEKIN LAMBDA 17UV/Vis SPECTROPHOTOMETER”采用热变性法测DNA G+Cmol％,复性速率法进行DNA-DNA杂交,使杂交的DNA样品变性和复性在同一比色杯中进行,省去半导体点温计操作带来的麻烦,减小了实验误差,缩短测时,结果精确、操作简便、重复性好。     

绵羊血液中布氏杆菌DNA提取方法的比较研究

本研究旨在比较5种DNA提取方法对绵羊血液中布氏杆菌DNA的提取效果及对PCR检测的影响。将不同浓度的疫苗株布氏杆菌加入绵羊全血中,采用3种DNA提取试剂盒和酚/氯仿法以及碘化钠法等5种方法提取模拟的绵羊血液样品中的DNA,评价所获得DNA的浓度、纯度和完整性,并采用布氏杆菌特异性PCR进行检测。同时,对各提取方法所需时间及经济成本进行了比较。结果表明,各方法均能提取获得绵羊全血中布氏杆菌DNA,3种试剂盒和碘化钠法获取布氏杆菌DNA的效果相同,而酚/氯仿法获取布氏杆菌DNA的效率最低或存在PCR抑制剂而不适合用于绵羊血液中布氏杆菌的PCR检测。碘化钠法具有耗时较短、成本低、方便的优点,是从绵羊血液中提取布氏杆菌DNA的良好方法。本研究结果为临床绵羊血液中布氏杆菌DNA提取方法的选择提供了参考。