Overlapping deletion in two spontaneous phase variants of Coxiella burnetii

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.     

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 .     

Survival of Coxiella burnetii in soil

For the first time the survival of Coxiella burnetii of five types in soils has been studied. The survival of C. burnetii has been found to depend on the content of organic substances in black earth, as well as soil temperature. The method for the prevention of an epidemic outbreak of Q fever directly under the natural conditions has been proposed.     

Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.     

An immunoenzyme method for demonstrating Coxiella burnetii antigens

Materials on the development of an enzyme immunoassay (EIA) system for the detection of the antigens of C. burnetii, the causative agent of Q rickettsiosis, are presented. The system is highly specific and effective with respect to both corpuscular antigens of phases 1 and 2 and soluble antigen (lipopolysaccharide). The sensitivity of this method varies within the range 5-100 ng/ml. The effectiveness of EIA as a quantitative (semiquantitative) control test used in the process of the production of Coxiella preparations has been demonstrated.     

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.     

Quantitative Assay of Coxiella burnetii in Mice

Experimental data are presented which demonstrate that the complement-fixing antibody response in individual mice can be used for quantitative assay of Coxiella burnetii. The method allows the replacement of a single guinea pig with a single mouse, thus resulting in considerable savings in caging requirements and animal costs.     

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.     

Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis

Different tests performed on bulk tank milk samples (BTM) are available to determine the C. burnetii status of herds. However, these tests, which are based on the detection of either antibodies directed against C. burnetii (ELISA) or bacterial DNA (PCR), have limitations. A currently disease-free herd infected in the past may continue to test positive with ELISA due to the persistence of antibodies in animals that were infected and that subsequently cleared the infection. Infectious herds can also be misclassified using PCR because of the absence of bacteria in the BTM when the test is performed. Recently, PCR has been used for bacterial DNA detection in the farm environment, which constitutes the main reservoir of C. burnetii. The objectives of this study were to assess and compare the sensitivities and specificities of one commonly used PCR test in BTM (PCR BTM) and of a PCR applied to environmental samples (PCR DUST) in dairy cattle farms. BTM and dust samples were collected (using environmental swabs) in 95 herds. The evaluation of the performance of the 2 tests was conducted using latent class models accounting for within herd disease dynamics. Parameter estimation was carried out using MCMC, within a Bayesian framework. Two types of priors were used for the specificity of PCR DUST. A model with a uniform prior on 0–1 fitted the data better than a model with a uniform prior on 0.95–1. With the best model PCR DUST had a lower sensitivity than PCR BTM (0.75 versus 0.83) and a specificity of 0.72. The moderately low value for the specificity of PCR DUST suggests that the presence of bacteria on farm is not always associated with persistent infections and shedding of bacteria in milk.     

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     

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.     

The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii

Type-IV secretion systems are devices present in a wide range of bacteria (including bacterial pathogens) that deliver macromolecules (proteins and single-strand-DNA) across kingdom barriers (as well as between bacteria and into the surroundings). The type-IV secretion systems were divided into two subgroups and Legionella pneumophila and Coxiella burnetii are the only two bacteria known today to utilize a type-IVB secretion system for pathogenesis. In this review we summarized the available information concerning the icm/dot type-IVB secretion systems by comparing the two bacteria that possess this system, the proteins components of their systems as well as the homology of proteins from type-IVB secretion systems to proteins from type-IVA secretion systems. In addition, the phenotypes associated with mutants in the L. pneumophila icm/dot genes, their relations to properties of specific Icm/Dot proteins as well as the protein substrates delivered by this system are described.     

Glomerulonephritis associated with Coxiella burnetii endocarditis.

A patient with endocarditis associated with chronic Coxiella burnetii infection is described in whom glomerulonephritis developed with granular deposits containing immunoglobulins and complement in the glomeruli. The serum was notable for the variety of circulating antibodies detected, which included antibodies directed against native DNA.     

Biochemistry of Coxiella burnetii: Embden-Meyerhof pathway

Purified preparations of Coxiella burnetii were examined for enzymes of the glycolytic pathway. Glucose-phosphate isomerase, fructose-1,6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were shown to be present in C. burnetii extracts. Heat-killed C. burnetii purified with normal yolk sacs demonstrated no activity after disruption. Aldolase was shown to be of the class II type by complete inhibition of activity in the presence of 8 x 10(-3)m ethylenediaminetetraacetic acid. The host enzyme activity (normal and infected yolk sacs) was not affected by the same treatment. When cellulose acetate electrophoresis was performed on the extracts, aldolase from both normal and infected yolk sacs exhibited five isozyme bands, whereas aldolase from the C. burnetii extract appeared as a single band.