The intramacrophagic environment of Brucella suis and bacterial response

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival.Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen’s stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0–4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome–lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly.How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis.Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.     

Type IV secretion and Brucella virulence

The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate.     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other -proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

猪布鲁菌S2omp10基因缺失株的构建及特性

现有的布鲁菌减毒活疫苗存在一定毒力,且野强毒株和减毒活疫苗株间缺少可供鉴别的抗原,导致在血清学检测上自然感染与疫苗接种很难区分,限制了现有的减毒活疫苗的广泛应用.本文拟对布鲁菌的减毒活疫苗株S2进行遗传改造,克服上述缺陷.本研究利用同源重组的方法,得到了布鲁菌S2株omp10基因缺失株.分别用基因缺失株和疫苗株感染小鼠,比较基因缺失株小鼠体内的存活能力.结果成功构建了布鲁菌S2株omp10基因缺失株,动物试验结果表明,基因缺失株仍能在小鼠体内存活,具备作为减毒活疫苗的特性.与原始S2株比较,基因缺失株的感染力进一步减弱.表明omp10基因在布鲁菌的毒力及体内生存方面发挥了作用,为基因标记疫苗的研制奠定了基础.     

Fun stories about Brucella: the "furtive nasty bug"

Although Brucella is responsible for one of the major worldwide zoonosis, our understanding of its pathogenesis remains in its infancy. In this paper, we summarize some of the research in progress in our laboratory that we think could contribute to a better understanding of the Brucella molecular virulence mechanisms and their regulation.     

Brucella suis in three dogs: presentation,diagnosis and clinical management

Brucella suis is an emerging, zoonotic disease predominantly affecting dogs and humans that engage in feral pig hunting in Australia and other countries. Although B. suis infection in dogs shares some clinical similarities to the host-adapted species (B. canis), B. suis remains an incompletely understood pathogen in dogs with limited published data on its pathogenesis and clinical features. This case series describes the presentations, diagnosis, and clinical management of B. suis infection in three dogs: (1) a bitch with dystocia, abortion and mastitis; (2) an entire male dog with septic arthritis and presumptive osteomyelitis; and (3) a castrated male dog with lymphadenitis. Unique features of these cases are reported including the first documented detection of B. suis from milk and isolation from lymph nodes of canine patients, as well as the follow-up of pups born to a B. suis-infected bitch. Consistent with previous reports, all three dogs showed a favourable clinical response to combination antibiotic therapy with rifampicin and doxycycline. Individually tailored drug regimens were required based on the clinical presentation and other factors, including owner expectations and compliance with therapy as well as a zoonotic risk assessment (generally considered low, except around time of whelping). The authors include their recommendations for the clinical management of dogs that are at-risk or seropositive for B. suis with or without clinical signs or laboratory-confirmed infection.     

小鼠阴道黏膜接种布鲁菌S_2疫苗株树突状细胞动力学变化

将25只小鼠随机分为5组,1~4组经阴道接种布鲁菌,第5组阴道滴注PBS(对照组),分别在处理后12、24、487、2 h摘取髂内淋巴结,用S-100抗体进行免疫组织化学染色,观察小鼠树突状细胞(Dendritic cells,DCs)在淋巴结的分布变化;同时收集外周血用ELISA法进行IFN-γ检测。用布鲁菌负载DCs,分别在2、4、6、8、10、12 h收集细胞悬液制备涂片,姬姆萨染色。结果显示:阴道接种布鲁氏菌疫苗12~48 h髂内淋巴结中DCs的数量明显增多,成群分布,呈现由浅层皮质向副皮质区迁移的趋势;72 h后大量DCs呈单在分布,分布范围增大;接种疫苗后血清中IFN-γ含量逐渐升高,至48 h,与对照组相比差异显著(P0.05),72 h后,血清中IFN-γ含量达到最大值,与对照组相比差异极显著(P0.01);布鲁菌负载DCs后,在4 h仅有少量DCs吞噬布鲁菌,至12 h吞噬布鲁菌的DCs数量明显增加。结果表明,DCs具有较强捕获布鲁菌的能力,并在抗原刺激下向引流淋巴结迁移,刺激淋巴结免疫细胞产生IFN-γ应答。     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

布鲁氏菌致病及免疫机制研究进展

布鲁氏菌病是由布鲁氏菌属的细菌引起的人畜共患传染病 ,目前布鲁氏菌属的细菌主要有七个种。随着分子生物学技术的发展 ,对布鲁氏菌的致病机制在分子水平上有了更进一步的理解 :布鲁氏菌自身一些与致病有关的基因使得布鲁氏菌逃避了巨噬细胞的杀伤作用 ,在巨噬细胞内存活和定居。在布鲁氏菌病免疫过程中 ,先天性免疫应答主要通过补体、巨噬细胞、天然杀伤细胞来参与 ,获得性免疫应答中 ,CD4 、CD8 、rδT细胞起很重要的作用。文章围绕这些与致病、免疫有关的基因及参与免疫反应细胞的研究进展进行了探讨 ,可为今后进行布鲁氏菌病预防及开发新型基因工程苗提供理论依据。     

布鲁氏菌诊断学研究进展

布鲁氏菌病是一种人兽共患传染性疾病,分布广泛,严重威胁公共卫生事业和畜牧业的发展,布鲁氏菌的诊断是防控该疫病的关键。本文对细菌学、常规免疫学、酶联免疫吸附（ELISA）、免疫胶体金层析法（GICA）、荧光偏振法（FPA）、聚合酶链式反应（PCR）和基因探针等布鲁氏菌检测技术及其优缺点和应用现状进行综述,并对诊断技术的发展前景进行了展望。     

New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis

Rapid and specific identification of Brucella suis at the biovar level is necessary because some of the biovars that infect animals are pathogenic for humans. None of the molecular typing methods described so far are able to discriminate B. suis biovars in a single test and differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows the correct discrimination of all known Brucella species is also described.     

布鲁菌进化和分类学研究进展

布鲁菌属革兰氏阴性兼性胞内寄生菌,能感染多种宿主动物和人。该属可分为6个典型种,包括羊种、牛种、猪种、沙林鼠种、绵羊附睾种以及犬种布鲁菌等。此分类是基于其致病性以及宿主偏好性的差异划分。尽管6个种通过传统表型试验能区分,但布鲁菌种内采用DNA-DNA杂交证明DNA同源性高度一致（相似性大于90%）。因此有人提议布鲁菌由单一种组成,即布鲁菌属中只有羊种布鲁菌,其他种都是羊种菌的生物亚型之一。然而基于其他分子技术的基因分型表明其DNA多态性表现明显,说明目前对这个种的分型还是比较准确。而最近分离的海洋种布鲁氏菌分离株（鳍型和鲸型）采用传统分型标准和一些特异的分子标记也证明这种分型比较正确。本文对目前布鲁菌种属进化和分类学进行综述,希望对研究其进化和分类有所帮助。     

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 μg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to β-lactam antibiotics. PBP2 complements the activity of PBP1. New β-lactam antibiotics and improved vaccines might be developed based on this property.     

粗糙型布鲁菌M111菌壳的安全性和免疫原性

以粗糙型布鲁菌M111株和重组裂解质粒制备出布鲁菌菌壳,利用小鼠模型对布鲁菌菌壳、布鲁菌M111活菌和福尔马林灭活菌的安全性和免疫原性进行比较研究。结果显示,与布鲁菌弱毒菌株M111比较而言,布鲁菌菌壳具有更好的安全性,免疫小鼠后能产生与弱毒菌株相似的血清抗体水平、脾CD3+和CD4+T淋巴细胞反应,甚至产生更高水平的IFN-γ。这些结果表明,布鲁菌菌壳具有与弱毒菌株相似的体液免疫和细胞免疫能力,将来可能作为预防布鲁菌感染的新型候选疫苗,但布鲁菌菌壳疫苗的有效性和特异性免疫机制还有待深入研究。     

羊种布鲁氏菌生物3型的分离和鉴定

本实验对新疆某羊场小尾寒羊5只流产胎儿进行布鲁氏菌病原分离、鉴定,结果从5只流产胎儿胃内容物中均分离到羊种布鲁氏菌生物3型。表明新疆羊群中存在羊种布鲁氏菌生物3型病原。     

Brucella evolution and taxonomy

The genus Brucella contains alpha-Proteobacteria adapted to intracellular life within cells of a variety of mammals. Controversy has arisen concerning Brucella internal taxonomy, and it has been proposed that the DNA–DNA hybridization-based genomospecies concept be applied to the genus. According to this view, only one species, Brucella melitensis, should be recognized, and the classical species should be considered as biovars (B. melitensis biovar melitensis; B. melitensis biovar abortus; etc.). However, a critical reappraisal of the species concept, a review of the population structure of bacteria and the analysis of Brucella genetic diversity by methods other than DNA–DNA hybridization show that there are no scientific grounds to apply the genomospecies concept to this genus. On the other hand, an enlarged biological species concept allows the definition of Brucella species that are consistent with molecular analyses and support the taxonomical standing of most classical species. Both the host range as a long-recognized biological criterion and the presence of species-specific markers in outer membrane protein genes and in other genes show that B. melitensis, B. abortus, B. ovis, B. canis and B. neotomae are not mere pathovars (or nomenspecies) but biologically meaningful species. The status of B. suis is, however, less clear. These approaches should be useful to define species for the marine mammal Brucella isolates, as illustrated by the grouping of the isolates from pinnipeds or from cetaceans by omp2 gene analysis. It is shown that a correct Brucella species definition is important to understand the evolution of the genus.     

实时荧光定量PCR检测布鲁菌方法的建立

旨在建立实时荧光定量PCR检测布鲁菌的方法。经选取高度保守的具布鲁菌属特异性的BSP31基因设计了一对引物及探针,并采用矩阵法优化反应体系,筛选出引物、探针、dNTP和Mg2+最优浓度配比,同时进行了特异性和灵敏性试验,建立了实时荧光定量PCR检测布鲁菌的方法。该方法可用于对布鲁菌病普查监测及进出境动物及其产品的检验检疫。     

布鲁菌免疫分子研究进展

布鲁菌病是布鲁菌引起的人兽共患传染病,包括7种21型,其中感染人的主要有牛、羊、猪布鲁菌3种,其免疫涉及体液免疫和细胞免疫.在布鲁菌的免疫过程中,先天性免疫应答主要通过补体、自然杀伤细胞、巨噬细胞、细胞因子等的参与.获得性免疫应答中,CD4 、CD8 、γβT细胞起重要作用.布鲁菌重组亚单位疫苗是近年研究的热点,而且发现的免疫分子也很多.文章围绕布鲁菌免疫机理、免疫相关分子进行探讨,为筛选疫苗候选分子奠定基础.     

布鲁氏菌检测技术的研究进展

布鲁氏菌病是由布鲁氏菌引起的,以流产和发热为特征的一种全世界广泛分布的人畜共患慢性细菌传染病.严重威胁着人和多种动物的生命健康.布鲁氏菌可感染人类、多种家畜和野生动物.导致巨大的经济损失和严重的公共卫生问题.因此,该病的检测工作也是预防其发生的重要组成部分.笔者综述了免疫学ELISA、胶体金、PCR等布鲁氏菌的检测技术,这些技术均能准确、快速、特异性地检测出布鲁氏菌.