巴尔通体液体培养条件简化及生长曲线观察

【目的】应用一种昆虫细胞培养基作为基础成分培养巴尔通体(Bartonella species),建立一种操作方便、高效稳定的巴尔通体液体培养方法。【方法】昆虫细胞培养基中添加10%胎牛血清,以此为基础培养液分别添加蔗糖和谷氨酰胺,比较这两种成分对汉赛巴尔通体(B.henselae)和五日热巴尔通体(B.quintana)生长的影响并观察其他10种巴尔通体在简化后的培养液中的生长特性。【结果】添加蔗糖和谷氨酰胺不会明显促进巴尔通体的生长,10种巴尔通体在简化后的培养液中均生长良好。不同种巴尔通体生长曲线不同,汉赛巴尔通体和五日热巴尔通体的世代时间分别为5.2 h和4.3 h,生长速度快于固体培养。【结论】以昆虫细胞培养基作为基础成分的培养液适于巴尔通体液体培养,特别是对一些更难培养的巴尔通体提供了一种较好的培养方法。     

巴尔通体脂肪酸分析标准方法的建立

<正>巴尔通体(Bartonella species)是一群革兰氏染色阴性、难于培养的兼性胞内寄生菌,21个种及亚种,其中9种可致人类疾病。20世纪90年代以后,在欧美地区的一些流浪人群中出现了菌血症、心内膜炎,一些艾滋病人群中出现了杆菌性血管瘤等由巴尔通体引发的疾病,被世界卫生组织(WHO)确认为新发传染病,对巴尔通体及其疾病的研究也引起了人们的关注。由于巴尔通体生长缓慢、生化反应不活泼,表型鉴定的方法不能应用于巴尔通体的分类鉴定中,因此多基因序列系统发育分析是鉴定巴尔通体的唯一     

恒河猴五日热巴尔通体的分离和柠檬酸合成酶基因的序列分析

目的初步研究巴尔通体在恒河猴体内的存在情况,并分析其柠檬酸合成酶（gltA）的基因序列,判断巴尔通体的种属。方法 16只来自福建的恒河猴,用血琼脂培养基分离可能存在的巴尔通体。根据NCBI数据库上的巴尔通体gltA的基因序列,设计一对引物,以巴尔通体菌落为模板进行扩增,将获得的序列进行克隆测序。结果从3只恒河猴体内成功分离到了巴尔通体病原,并获得了巴尔通体gltA全长基因序列,测序结果表明该序列与五日热巴尔通体同源性99%。结论福建来源的恒河猴种群携带巴尔通体病原,巴尔通体流行地区可能存在鼠与灵长类动物之间病原体的流行和传播。     

巴尔通体细胞脂肪酸成分分析

【目的】分析影响巴尔通体脂肪酸成分的主要因素,建立适合巴尔通体脂肪酸成分分析的标准化方法,探讨脂肪酸图谱应用于巴尔通体分类鉴定的可能性。【方法】应用气相色谱技术分析不同培养条件下巴尔通体脂肪酸的组成与含量的变化;应用已构建的标准化方法获取10株巴尔通体标准菌株和9株来自不同地区的汉赛巴尔通体猫分离株脂肪酸图谱;应用SPSS16.0统计软件对获得的数据资料进行聚类分析。【结果】培养基、温度和传代次数主要影响巴尔通体脂肪酸的微量成分;10株巴尔通体标准菌株的成分相似,但也存在构成和含量上的差异;在所测巴尔通体中,检出可分辨脂肪酸成分有20种,共有成分为7种,其中C18:1ω7c、C18:0和C16:0累积含量达80%以上;猫分离株被准确鉴定为汉赛巴尔通体。【结论】巴尔通体脂肪酸成分受培养基、温度等培养条件影响,在脂肪酸提取方法标化后,可用于汉赛巴尔通体种水平分类鉴定。     

中国部分地区实验猕猴巴尔通体感染状况及其遗传特征

【目的】五日热巴尔通体(Bartonella quintana)由体虱在人群中传播,可引起多种人类疾病包括战壕热。为进一步搜集猕猴是五日热巴尔通体自然宿主的证据,本研究调查了国内4个地区实验用猕猴五日热巴尔通体的感染状况,对菌株遗传特征进行了分析。【方法】采集猕猴全血和血清样品各550份,用于菌株分离、核酸和血清IgG抗体检测。应用6个管家基因扩增及测序方法进行菌株鉴定、系统发育及核苷酸多态性分析;应用随机扩增多态性DNA标记(Random amplified polymorphic DNA,RAPD)技术分析不同宿主来源菌株RAPD指纹图谱差异;应用间接免疫荧光法(Indirect immunofluorescence assay,IFA)检测血清中抗五日热巴尔通体IgG抗体水平。【结果】从550只猕猴中分离到8株五日热巴尔通体菌株,带菌率为1.5%;直接PCR检测550份全血核酸的总感染率为8.2%。普通猕猴血清阳性率为19.0%,感染水平明显高于食蟹猕猴(5.6%)。五日热巴尔通体与汉赛巴尔通体RAPD指纹图谱的带型完全不同,猴源和人源五日热巴尔通体菌株Fuller带型基本一致。不同宿主来源菌株核苷酸多态性分析显示,猴源菌株之间差异小,其与人源菌株差异较大。【结论】中国猕猴五日热巴尔通体感染水平较高,普通猕猴自然感染率及抗体水平明显高于食蟹猕猴,猴源与人源菌株的基因型有明显差异。     

巴尔通体在滇西南蝙蝠中高度流行并具有丰富的遗传变异特征

蝙蝠是很多病原微生物的自然宿主, 全球多项研究表明蝙蝠是巴尔通体(Bartonella species)的主要宿主。为了解滇西南地区蝙蝠中巴尔通体的流行特征, 我们于2015-2017年间在云南省4个地区应用网捕法捕获蝙蝠3种305只。经种类鉴定后采集肝脾组织, 提取核酸, 通过TaqMan实时荧光定量PCR方法检测巴尔通体的tmRNA基因ssrA, 并进行测序鉴定和系统发育分析。结果发现172只蝙蝠检出该基因, 总感染率为56.4%; 其中临沧、西双版纳、保山和瑞丽4个采样点的蝙蝠感染率分别为50.0% (22/44)、61.7% (29/47)、62.1% (18/29)和55.7% (103/185)。中菊头蝠(Rhinolophus affinis)、小菊头蝠(R. blythi)和棕果蝠(Rousettus leschenaultii)的感染率分别为50.0% (22/44)、62.1% (18/29)和56.9% (132/232), 差异没有统计学意义(χ² = 1.135, P = 0.567), 表明巴尔通体在云南当地的蝙蝠种群中高度流行。定量PCR扩增产物2次扩增后测序获得37个巴尔通体ssrA序列, 属于10个系统发育分支, 其中1个为伊丽莎白巴尔通体(B. elizabethae)、特利波契巴尔通体(B. tribocorum)和克拉斯诺夫巴尔通体(B. krasnovii)的近缘种。其余序列与已知巴尔通体距离较远, 与亚洲、欧洲和美洲等其他地域来源于蝙蝠的巴尔通体近缘。遗传多样性分析显示, ssrA基因的核苷酸多样性指数(π)为0.11381 ± 0.00928, 基因型多样性指数(Hd)为0.985 ± 0.010, 形成29个基因型(单倍型), 说明云南蝙蝠巴尔通体具有丰富的遗传多样性。通过对本研究标本与全球相关序列的系统发育网络重建, 分析全球蝙蝠巴尔通体的地理和宿主分布特征, 可以看出巴尔通体与蝙蝠之间存在显著的宿主特异性关联。因此可初步确定蝙蝠-巴尔通体具有协同进化特征, 同时受到地理隔离的影响。     

巴尔通体的液体培养

巴尔通体(Bartonella species)是一群革兰氏染色阴性、营养条件要求苛刻、兼性细胞内寄生的需氧杆菌,主要寄生在动物宿主和人的血管内皮细胞和红细胞内,20世纪90年代以后,在欧美地区一些流浪人群中出现了由巴尔通体引起的菌血症、心内膜炎,一些艾滋病人群中出现了杆菌性血管瘤等,由于这些疾病以往不曾为人们所认识,被WHO确认为新发传染病,因而引起广泛关注。     

巴尔通体感染性心内膜炎的研究进展

感染性心内膜炎一直是威胁人类健康的重要疾病之一。近年来人类正面临着此病发病率持续上升的局面,其诊断、治疗和预防依然是目前需要解决的重要临床和公共卫生问题。本文介绍了感染性心内膜炎疾病的最新研究进展,分析了国内外报道的538份巴尔通体感染性心内膜炎的病例,重点阐述了巴尔通体和相关心内膜炎的流行病学、实验室诊断、治疗以及发病的危险因素和预防控制措施。预测这些研究将对人类理解和控制巴尔通体感染性心内膜炎具有重要的指导意义。     

巴尔通体分离培养特性观察

从鼠类血液中分离巴尔通体(Bartonella),观察其分离培养特性。被检鼠血为2004年收集自云南省的4个县,采用含5%去纤维兔血脑心浸液琼脂培基置于35℃含5%CO2培养箱中分离培养巴尔通体,进行观察,涂片革兰染色镜检,疑似菌落用巴尔通体属特异性引物进行聚合酶链反应(PCR)扩增特异基因片段[枸橼酸合酶基因(gltA)的379 bp片段],电泳图中出现目标带即判断为阳性菌株。从397份鼠血分离到巴尔通体47份,分离率为11.8%。阳性菌落长出时间最早为3 d,多数为1~2周,占70.2%(33/47)。阳性菌落形态随培养时间延长而改变,特点多样。PCR阳性的菌经革兰染色,镜下均可见革兰阴性小杆菌。结果可见,可先用涂片革兰染色镜检,对疑似菌落进行初筛,巴尔通体种类多、形态多样,培养时间延长,菌落形态可能发生变异,有待进一步探索。     

实时荧光定量TaqMan-MGB探针法检测杆菌样巴尔通体

【目的】应用Taq Man探针实时荧光定量PCR技术建立特异性强、敏感性高和稳定性好的快速杆菌样巴尔通体检测方法。【方法】应用生物信息学方法查找杆菌样巴尔通体特有基因,从中筛选出一段特有的基因序列为模板设计探针和引物。通过比较Ct值和荧光强度确定扩增反应的最佳退火温度、探针和引物浓度;将扩增产物连接到p EASY-T载体上制备标准品,绘制标准曲线,分析扩增效率和线性关系;评估方法的特异性、敏感性及重复性。【结果】优化后退火温度为60°C,探针和引物浓度均为200 nmol/L,反应体系20μL。特异性实验显示只有杆菌样巴尔通体扩增出荧光信号,其他种属细菌均未见荧光信号;标准曲线线性关系良好(R2=1),扩增效率E=98.18%;最低检出限为每个PCR反应3个拷贝;组内和组间的变异系数CV值分别为0.21%–0.42%和0.29%–0.59%,在允许范围内。【结论】研究建立的实时荧光定量Taq Man-MGB探针法特异性强、灵敏度高、稳定性好,可快速检测鉴定杆菌样巴尔通体,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

Association of Bartonella with the fleas (Siphonaptera) of rodents and bats using molecular techniques.

Bartonella spp. are putatively vector-borne bacterial agents of humans and animals. Fleas have been incriminated as vectors of Bartonella spp. and are suspected of transmitting Bartonella of rodents and bats, but some of these Bartonella spp. have not yet been directly detected in wild caught fleas. We report the molecular detection of Bartonella tribocorum, Bartonella vinsonii subsp. vinsonii, and two novel genotypes of Bartonella from the fleas Xenopsylla cheopis, Ctenophthalmus pseudagyrtes, Sternopsylla texanus, or Orchopeas howardi.     

Optimization of pulse-field gel electrophoresis for Bartonella subtyping

Bartonella is a significant human pathogen and is the world's most common bacterial zoonosis acquired from companion animals. However, there is no uniform method for Pulse-Field Gel Electrophoresis (PFGE) for Bartonella population genetics studies. Further, some genes of Bartonella can mutate frequently and may affect the use of PFGE for Bartonella. Here we designed methods to solve these problems. We standardized the bacterial concentration, selected the appropriate digestion enzyme, optimized the electrophoretic parameters and characterized reproducibly two Bartonella species strains. Thus we optimized the PFGE procedure and determined how often Bartonella mutated. Our data shows a practical protocol for inter- and intra-species identification of Bartonella and was reproducible using two species strains that showed no mutation occurred after two passages for B. elizabethae; but mutation did occur in B. henselae.     

Pathogenic mechanisms of Bartonella quintana

Bartonella quintana is an epi- and intracellular gram-negative rod responsible for both acute and chronic clinical manifestations. We review the literature about pathogenic mechanisms of B. quintana and discuss our data. Our efforts to clarify Bartonella quintana pathogenesis run on two parallel tracks. The first one concerns interactions between Bartonella quintana and endothelial cells by evaluation and modulation of apoptosis, signal transduction pathways and inflammation. The second one concerns some biological activities of Bartonella quintana endotoxin on human whole blood and endothelium. The elucidation of the mechanisms regulating the inflammatory/proliferative pattern of chronic clinical manifestations of Bartonella quintana infections may offer a contribution for addressing the pathogenesis of intracellular bacterial persistence.     

Mixed infections, cryptic diversity, and vector-borne pathogens: evidence from Polygenis fleas and Bartonella species

Coinfections within hosts present opportunities for horizontal gene transfer between strains and competitive interactions between genotypes and thus can be a critical element of the lifestyles of pathogens. Bartonella spp. are Alphaproteobacteria that parasitize mammalian erythrocytes and endothelial cells. Their vectors are thought to be various biting arthropods, such as fleas, ticks, mites, and lice, and they are commonly cited as agents of various emerging diseases. Coinfections by different Bartonella strains and species can be common in mammals, but little is known about specificity and coinfections in arthropod vectors. We surveyed the rate of mixed infections of Bartonella in flea vectors (Polygenis gwyni) parasitizing cotton rats (Sigmodon hispidus) in which previous surveys indicated high rates of infection. We found that nearly all fleas (20 of 21) harbored one or more strains of Bartonella, with rates of coinfection approaching 90%. A strain previously identified as common in cotton rats was also common in their fleas. However, another common strain in cotton rats was absent from P. gwyni, while a rare cotton rat strain was quite common in P. gwyni. Surprisingly, some samples were also coinfected with a strain phylogenetically related to Bartonella clarridgeiae, which is typically associated with felids and ruminants. Finally, a locus (pap31) that is characteristically borne on phage in Bartonella was successfully sequenced from most samples. However, sequence diversity in pap31 was novel in the P. gwyni samples, relative to other Bartonella previously typed with pap31, emphasizing the likelihood of large reservoirs of cryptic diversity in natural populations of the pathogen.     

Phylogenetic analysis of Bartonella detected in rodent fleas in Yunnan, China

Previous studies have demonstrated a diversity of Bartonella spp. in rodent populations in Yunnan Province, China. Although Bartonella spp. have been isolated from cat fleas and cattle ticks collected from their animal hosts, little is known about Bartonella carried by rodent fleas. In this study, Bartonella DNA was detected by polymerase chain reaction (PCR) in two of five species of rodent fleas. These included Xenopsylla cheopis and Ctenophthalmus lushuiensis, which were collected from Rattus tanezumi flavipectus and from the nests of voles, respectively, during 1997 from two sites in western Yunnan Province, China. Sequence analysis of the Bartonella citrate synthase gene (gltA) amplicons obtained from six of 65 grouped flea samples showed that Bartonella genetic variants were clustered in four groups. One from Xenopsylla cheopis was identical to Bartonella tribocorum, whereas the other three genotypes from Ctenophthalmus lushuiensis were related to the vole-associated Bartonella isolates and cat-associated Bartonella clarridgeiae. This is the first detection of this Bartonella variant from fleas in China. Therefore, further investigations are needed to clarify the distribution of Bartonella in rodents and their ectoparasites in China to define the role of these arthropods in the transmission routes of Bartonella.     

Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.     

Vector transmission of Bartonella species with emphasis on the potential for tick transmission

Bartonella species are gram-negative bacteria that infect erythrocytes, endothelial cells and macrophages, often leading to persistent blood-borne infections. Because of the ability of various Bartonella species to reside within erythrocytes of a diverse number of animal hosts, there is substantial opportunity for the potential uptake of these blood-borne bacteria by a variety of arthropod vectors that feed on animals and people. Five Bartonella species are transmitted by lice, fleas or sandflies. However, Bartonella DNA has been detected or Bartonella spp. have been cultured from numerous other arthropods. This review discusses Bartonella transmission by sandflies, lice and fleas, the potential for transmission by other vectors, and data supporting transmission by ticks. Polymerase chain reaction (PCR) or culture methods have been used to detect Bartonella in ticks, either questing or host-attached, throughout the world. Case studies and serological or molecular surveys involving humans, cats and canines provide indirect evidence supporting transmission of Bartonella species by ticks. Of potential clinical relevance, many studies have proposed co-transmission of Bartonella with other known tick-borne pathogens. Currently, critically important experimental transmission studies have not been performed for Bartonella transmission by many potential arthropod vectors, including ticks.     

Investigation of Bartonella acquisition and transmission in Xenopsylla ramesis fleas (Siphonaptera: Pulicidae)

Bartonella are emerging and re-emerging pathogens affecting humans and a wide variety of animals including rodents. Horizontal transmission of Bartonella species by different hematophagous vectors is well acknowledged but vertical transmission (from mother to offspring) is questionable and was never explored in fleas. The aim of this study was to investigate whether the rodent flea, Xenopsylla ramesis, can acquire native Bartonella from wild rodents and transmit it transovarially. For this aim, Bartonella-free laboratory-reared X. ramesis fleas were placed on six naturally Bartonella-infected rodents and six species-matched Bartonella-negative rodents (three Meriones crassus jirds, two Gerbillus nanus gerbils and one Gerbillus dasyurus gerbil) for 7 days, 12-14h per day. The fleas that were placed on the Bartonella-positive rodents acquired four different Bartonella genotypes. Eggs and larvae laid and developed, respectively, by fleas from both rodent groups were collected daily for 7 days and molecularly screened for Bartonella. All eggs and larvae from both groups were found to be negative for Bartonella DNA. Interestingly, two of five gut voids regurgitated by Bartonella-positive fleas contained Bartonella DNA. The naturally infected rodents remained persistently infected with Bartonella for at least 89 days suggesting their capability to serve as competent reservoirs for Bartonella species. The findings in this study indicate that X. ramesis fleas can acquire several Bartonella strains from wild rodents but cannot transmit Bartonella transovarially.     

Variability of Bartonella genotypes among small mammals in Spain

In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas-247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)-by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.