Serotyping Coxiella burnetii isolates from acute and chronic Q fever patients by using monoclonal antibodies

Abstract Four mouse monoclonal antibodies reacting with Coxiella burnetii lipopolysaccharide antigens were produced and used in serotyping 17 C. burnetii isolates from acute Q fever and Q fever endocarditis patients in France. Two monoclonal antibodies (1B2 and 3B6) were considered specific for the Priscilla strain, a representative of Q fever endocarditis isolates, and did not react with the Nine Mile strain, which is representative of acute Q fever isolates. Monoclonal antibodies Nos. 1B2 and 3B6 reacted with 75% (3/4) acute Q fever isolates and 85% (11/13) of endocarditis isolates from France. It is reasonable to conclude that Priscilla-like strains cause both acute Q fever and Q fever endocarditis. The hypothesis that Priscilla-like strains only are associated with Q fever endocarditis should be reconsidered.     

A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever

Abstract Genetic studies of Coxiella burnetii strains suggested the possibility of differentiating new isolates according to their plasmid DNA content. Virulence and/or clinical manifestations ('chronic' and 'acute' Q fever) had been claimed to correlate with this plasmid typing. A new plasmid, named QpDV, was found to be common to C. burnetii isolates obtained from acute and chronic Q fever. According to the results obtained, plasmid usage for detection and differentiation of respective pathovars of C. burnetii and the correlation between gene specificity and pathovar has to be revised. Closer studies suggested a common origin of C. burnetii plasmids, but also showed some differences characteristic for each plasmid, probably reflecting divergent evolution.     

胶体金渗滤法检测贝氏柯克斯体的研究

本文建立一种快速、敏感、适于基层应用的贝氏柯克斯体（俗称Ｑ热立克次体）的检测方法。将Ｑ热立克次体多克隆抗体点于硝酸膜上,用以捕获待检标本中的Ｑ热立克次体抗原,通过胶体金标记的鼠抗Ｑ热立克次体单克隆抗体直接显色,阳性者出现红色斑点。结果表明,用该法检测Ｑ热立克次休实验感染豚鼠血液,小鼠肝或脾,蜱血淋巴等标本取得了较满意的结果,整个过程仅需５－６分钟。与其他病原体无交叉反应,敏感度不低于５０ｎｇ立克次     

Establishment of a genotyping scheme for Coxiella burnetii

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.     

Genome‐wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q‐fever patient sera and naïve controls in order to discover C. burnetii‐specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q‐fever cases than naïve controls. The remaining eight antigens were cross‐reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q‐fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof‐of‐concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.     

Isolation of Coxiella burnetii from Children with Influenza-Like Symptoms in Japan

The prevalence of Coxiella burnetii antibodies was investigated by indirect immunofluorescence (IF) test in 55 paired sera (acute and convalescent phases) of school children who had influenza-like symptoms. Of the convalescent serum samples examined, 18 (32.7%) sera reacted positively to phase II antigen of C. burnetii. Coxiella-like organism was isolated from the sera of 13 children after injection of the 18 acute phase sera into mice. The organism was identified as C. burnetii by Giemsa staining and the IF antigen test of mouse spleen smears, the polymerase chain reaction (PCR) method, electron microscopic observations of the mouse spleen cells, and the IF antibody test of mouse sera. This is the first report of isolation of C. burnetii from serum specimens of children having influenza-like symptoms. The evidence that C. burnetii was isolated from people indigenous to Japan at a considerably high incidence suggested that C. burnetii may be widespread as a cause of influenza-like symptoms in Japan.     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

灭活贝氏柯克斯体免疫小鼠抗登革病毒感染的实验研究

目的 研究灭活贝氏柯克斯体增强小鼠非特异性抗登革病毒感染的免疫效能。方法 用灭活贝氏柯克斯体全细胞疫苗(WCV)免疫BALB/c小鼠,每只小鼠皮下注射50μg WCV,初次免疫后第5周及第7周分别腹腔注射20μg WCV加强免疫。1周后,用10~5 TCID_(50)剂量的登革病毒2型经尾静脉注入感染小鼠,于感染后48、72h分别解剖小鼠.自血和脑组织提取RNA样本,用实时荧光定量聚合酶链反应(PCR)检测样本。结果 用登革病毒特异的定量PCR检测感染小鼠血RNA样本,结果显示感染72h血样本中病毒含量显著低于48h样本,但免疫小鼠与未免疫小鼠之间无显著差异。检测脑组织RNA样本,未免疫小鼠和免疫小鼠的感染48h样本检出病毒均为少量;但未免疫小鼠感染96h样本检出病毒量明显增加,显著高于免疫小鼠。结论 灭活贝氏柯克斯体可诱导机体产生非特异的免疫应答,具有一定增强小鼠抗登革病毒感染的能力,但具体机制有待进一步研究。     

Genetics of Coxiella burnetii

Abstract Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies. Little is known about their mechanisms for natural genetic exchange. Many genes from the bacterium Coxiella burnetii , an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli . Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C. burnetii .     

感染小鼠组织中Q热立克次体的分子病理学检测

腹腔注射Q热立克次体悬液感染BALB／c小鼠,发病后解剖,取主要组织脏器,应用免疫组化和原位杂交检测感染小鼠体内Q热立克次体的抗原分布和DNA在靶细胞中的表达,探讨Q热病变规律及致病机理。结果显示阳性信号多位于单核-巨噬细胞系统细胞胞浆内。结果提示免疫组化和原位杂交技术可以作为Q热特异性的诊断方法,并为致病机理的研究提供线索。     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.     

Seasonal Variations in the Presence of Antibodies against Coxiella burnetii in Dairy Cattle in Hokkaido,Japan

The prevalence and seasonal variations of infection by Coxiella burnetii in cattle were investigated seroepidemiologically on a farm in Hokkaido, Japan, by an immunofluorescent antibody test. A total of 364 serum samples from 28 cows were collected from August 1993 to October 1995 in two barns on the farm. It was found that the number of antibody-positive cows and their antibody titers were significantly elevated in winter and decreased in summer. In addition, antibodies were detectable in seroconverted cows for about five months.     

Expression of beta-lactamase in Coxiella burnetii transformants

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.     

Genotypic and phenotypic characterization of two Swedish isolates and two prototypic strains of Coxiella burnetii

Two Swedish isolates of Coxiella burnetii and the two prototype strains of the species, Nine Mile and Priscilla, were characterized with regard to their multiplication and cytopathic effect on BGM cells and by PCR-based amplification of repetitive element DNA and the C. burnetii -specific plasmids QpH1 and QpRS. Moreover, 1330 bp of each 16S rRNA gene were sequence-determined. All four strains multiplied at virtually the same rate and displayed the same type of vacuoles in the BGM cells. Genetic homogeneity was observed inasmuch as the 16S rDNA sequences were identical and the strains showed identical PCR amplification patterns using primers specific to enterobacterial repetitive intragenic consensus DNA sequences. The two Swedish strains and the Priscilla strain also showed identical patterns after PCR amplification of repetitive extragenic palindromic DNA sequences, whereas the Nine Mile strain demonstrated a similar, but not identical pattern. Thus, the investigated strains demonstrated very similar phenotypic and genotypic characteristics. This finding is discussed in view of the very rare occurrence of domestic Q fever in Sweden.     

Seroepidemiological Survey of Coxiella burnetii in Domestic Cats in Japan

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.     

Coxiella burnetii: international pathogen of mystery

Coxiella burnetii is an intracellular bacterium that causes acute and chronic Q fever. This unique pathogen has been historically challenging to study due to obstacles in genetically manipulating the organism and the inability of small animal models to fully mimic human Q fever. Here, we review the current state of C. burnetii research, highlighting new approaches that allow the mechanistic study of infection in disease relevant settings.