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Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii.

Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29 NotI restriction fragments. The average resolution is 72.5 kb, or about 3. 5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative oriC has been located on a 27.5-kb NotI fragment. Gene organization upstream the oriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.

Bacillus subtilis↗

Isolation of a protein antigen from Coxiella burnetii.

Antigens prepared from Coxiella burnetti, strain Frankfurt, phase II, propagated in persistently infected Buffalo Green Monkey (BGM) cell cultures were purified by guanidinium hydrochloride treatment and chloroform/methanol extraction. By ELISA analysis, chloroform/methanol residues (CMR) proved to be free of host cell antigens. The CMR were sensitive to trypsin, pronase E and proteinase K, as determined by absorption-kinetics of CMR suspensions at 600 nm and release of protein. Coomassie blue stained SDS polyacrylamide gels of proteinase hydrolysates from CMR revealed only a single component of apparently 27,000 D. Silver stained gels, however, showed a second component of apparently 12,000 D. In contrast, from untreated native C. burnetii a large variety of proteins, most of them protease-sensitive, were released by detergents at low temperatures, but the 27,000 D component was only solubilized at 60-100 degrees C. The 27,000 D component was obviously the major protein of CMR as well as of whole cells. Antigenicity of this 27,000 D protein could be demonstrated by agargel precipitation test, ELISA and immunoperoxidase techniques applying antisera raised against whole cells and against the extracted component. The component was also recognized in a dot immunobinding assay by sera from guinea pigs infected with cloned C. burnetii stain Nine Mile, phase I, thus indicating an important role of this antigen in C. burnetii specific immune response.

Animals↗

Detection of Coxiella burnetii in ticks collected in Slovakia and Hungary.

A total of 235 adult ticks collected from vegetation in Slovakia and Hungary in 1998-2000 were tested for Coxiella burnetii by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). C. burnetii was identified in six ticks of Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis concinna species.

Animals↗

Detection of Coxiella burnetii in cow's milk by PCR-enzyme-linked immunosorbent assay combined with a novel sample preparation method.

The use of an adequate concentration of Triton X-100 enhanced immunomagnetic separation of Coxiella burnetii from milk. PCR-enzyme-linked immunosorbent assay (PCR-ELISA) could detect coxiellas more sensitively than could conventional PCR. PCR-ELISA is therefore thought to be suitable for the simultaneous assay of a large number of samples. However, the number of cows from which raw milk tested positive for coxiellas by PCR-ELISA was inconsistent with that found with the antibody to coxiella by indirect immunofluorescence assay. The inconsistency is thought to be associated with the differences in the infectious route, infectious dose, or the timing of yielding the antibody and the period of duration of the antibody.

Animals↗

Pathomorphological studies in European bison (Bison bonasus Linnaeus, 1758) with seropositive reaction to Coxiella burnetii.

Comprehensive serological and histopathological examinations of 47 free living European bison (Bison bonasus Linnaeus, 1758) were performed. Of these animals, 36 were serologically positive due to Coxiella burnetii, which confirmed the presence of Q fever epizootic foci in this population of wild animals in Poland. The presence of multiple foci of mononuclear cells typical for Q fever was a consistent finding in all tissues of the majority of C. burnetii seropositive animals under study. Pathomorphological changes observed in myocardium as the focal coagulation necrosis, and in kidneys, resembled the glomerular lesion observed in humans with Q fever, as well as in the experimental Q-fever infections in laboratory animals. These changes were absent in bison showing a C. burnetii seronegative reaction.

Animals↗

Seroprevalence of Coxiella burnetii infection in dairy cattle and non-symptomatic people for routine health screening in Korea.

We report results on the seroprevalence of antibodies to Coxiella burnetii in cattle and healthy people in Korea. Upon agreement with dairy owners, serum samples from 414 dairy cattle were collected between March and June 2001 and samples from 205 people for health screening were collected between April and December 2002. The sera were analyzed for the presence of anti-C. burnetii phase II antibodies using an indirect microimmunofluorescence test; strong fluorescence at a 1:32 dilution was regarded as positive. The overall seroprevalence of C. burnetii in cattle in Korea was 25.6%, with regional variation from 8.9 to 59.3%. Of the positive serum samples, 75.5% had antibody titers >or=1:256. By contrast, only 1.5% of people in a rural area were seropositive, and most of the positive samples had low antibody titers. In conclusion, this study showed that relatively high seropositivity of C. burnetii in dairy cattle, accordingly, the studies on the high-risk groups are needed to evaluate the seroprevalence for this organism in Korea.

Adult↗

Quantification of Coxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA).

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination of Coxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horse-radish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification of C. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.

Bacterial Proteins↗

[Clinical and serologic study of Czecho-Slovak citizens working in developing countries from the aspect of risk of infection with Coxiella burnetii].

In the years 1984-1988 we observed, besides the routine examinations of history, clinical state, and laboratory tests, also the presence of antibodies to Coxiella burnetii antigens by means of microagglutination reaction (MAR). The examinations were performed in our citizens returning to Czecho-Slovakia from a long termed business stay in the developing countries. Antibodies to C. burnetii antigen 2 have been detected entirely in 91 serum samples, out of which 48 cases following their stay in Libya, 42 cases following their stay in Iraq, and 1 in Syria. Diagnostically significant antibody titres (64) were detected in 44 serum samples, in which the serological positivity was proved by complement fixation test (CFT) examination. Clinical symptoms in the history, responding to the acute form of "Q" fever, occurred in 44 serologically positive cases, 37 of which developed high antibody titres, which is considered to be a significantly high incidence. The results imply the necessity to become more concerned by the incidence of "Q" fever, its diagnostics and therapy, eventually prevention in our citizens working in a more exacting climate of some developing countries. (Tab. 1, Ref. 15.).

Antibodies, Viral↗

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.

Adenosine Triphosphate↗

Identification and characterization of Coxiella burnetii strains and isolates using monoclonal antibodies.

We evaluated 6 monoclonal antibodies (MAbs) for their usefulness in identifying and characterizing recognized laboratory strains as well as field isolates of Coxiella burnetii. Five had been generated in response to strain Nine Mile (3 IgM class, 1 IgG class, 1 light chain producers only) and were polypeptide-specific, and 1 was anti-Priscilla (IgG class) and was lipopolysaccharide (LPS)-specific. Initially, the MAbs were used in conjunction with a dot blot assay with which we could differentiate C. burnetii from rickettsiae or chlamydiae. Confirmation of the specificity of these MAbs was provided by demonstrating that only C. burnetii antigens were recognized by certain combinations of antibodies used for immunoblotting proteins of various C. burnetii strains. Subsequently, we characterized antigens of 11 C. burnetii field isolates and 3 reference strains by Western blotting with individual MAbs. MAb 921 and 922 (IgG class), MAb 241, 242, 384, 386, 614 (IgM class), and 7A5, 7A1 (light chain) consistently recognized a protein. Staining intensity differed, depending on the strain tested, and there was variability in the size of the antigen immunoreactive with MAb 14H (IgG class, LPS-specific). The most reactive region was at about 249 kD. Variability of reactivities with field isolates was seen in both the distribution of individual bands and their intensities. We conclude that an extensive immunoblotting technique may be useful for C. burnetii strain differentiation and routine identification of C. burnetii can be accomplished using this MAb-based dot blot assay.

Animals↗

Analysis of phospholipids from Coxiella burnetii by fast atom bombardment mass spectrometry. A rapid method for differentiation of virulent phase I and low virulent phase II cells.

Phospholipids extracted from the Coxiella burnetii strain Nine Mile virulent phase I and low-virulent phase II cells were directly analyzed by fast atom bombardment mass spectrometry (FAB-MS). Constant neutral loss (CNL) scanning mass spectra (MS) were acquired to identify various phospholipids within phospholipid classes. Phospholipids from the phase I C. burnetii cells were much more complex than those from the phase II cells. Moreover, in the latter, the absence of phospholipids of the phosphatidylinositol class could be noticed. The results indicate that CNL scanning of phospholipid samples provides a rapid and simple method for identification of the phase state of the bacterium.

Coxiella burnetii↗

Unexpected antibiotic susceptibility of a chronic isolate of Coxiella burnetii.

Evidence is mounting in support of the idea that different isolates of Coxiella burnetii, the etiologic agent of Q fever, are responsible for the distinct disease syndromes observed clinically. Recent studies have shown distinct antibiotic susceptibilities of different isolates of C. burnetii implicated in distinct clinical Q fever syndromes. With this in mind, we performed antibiotic susceptibility testing of the "S" isolate, a chronic-type isolate retrieved from a human patient with chronic disease. Antibiotics with differing efficacies upon the Nine Mile and Priscilla isolates were tested for their abilities to control acute and persistent "S" isolate infection of L-929 cells in vitro. The efficacies of doxycycline, rifampin, and three fluoroquinolone drugs--ciprofloxacin, ofloxacin, and norfloxacin--were tested. Compared to the chronic Q fever-implicated Priscilla isolate, which has been shown to exhibit a significant resistance to these antibiotics, the "S" isolate was much more susceptible. In persistently infected cells (greater than 300 d), the "S" isolate proved to be significantly more resistant to doxycycline, slightly more resistant to ciprofloxacin, slightly more susceptible to rifampin, and equally sensitive to ofloxacin and norfloxacin compared to the acute Q fever-implicated Nine Mile isolate. As with both the Nine Mile and Priscilla isolates, the "S" isolate was more susceptible to doxycycline, rifampin, and ofloxacin in recently infected cells (22 d) compared to cells having been persistently infected. With respect to the resistant nature of the chronic Q fever-implicated Priscilla isolate, as well as the lack of success in treating the "S" isolate in vivo, these results were unexpected. Such data supports an evolving hypothesis that the distinct C. burnetii isolates which may be responsible for the clearly different Q fever syndromes exhibit a spectrum of antibiotic susceptibility ranging from very susceptible (acute-implicate), Nine Mile isolate), to moderately susceptible (chronic-implicated "S" isolate), to moderately resistant (chronic-implicated Priscilla isolate).

Anti-Bacterial Agents↗

Electron-microscopic study of the effect of various extractants on the morphology of Coxiella burnetii.

This paper presents the results of the effect of trichloroacetic acid (TCA) at two different temperatures and the effect of a mixture of detergents (D) on Coxiella burnetii. Both TCA and D caused a large destruction of C. burnetii cells. In both cases complexes of high-molar-mass components from the outer membrane were extracted. In the D case not only proteins but a mixture with other high-molar-mass structures were released from the destroyed cells. By extraction of TCA, the antigenic complex composed of lipopolysaccharides (LPS), proteins and phospholipids was released. The effect of the D mixture on C. burnetii causes their complete destruction. The TCA causes a surface destruction of cells but it does not cause complete disintegration. The small-cell variants (SCV) in both cases were shown to be more stable compared to the large-cell variants (LCV).

Antigens, Bacterial↗

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence.

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.

Apoptosis↗

Histologic, immunofluorescence, and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii.

A histologic, immunofluorescence, and electron microscopic study of the intracellular parasitism of Coxiella burnetii (the Q fever agent) in mouse lungs after intranasal challenge was undertaken. It was shown that this microorganism invades type I and, rarely, type II pneumocytes as well as pulmonary fibroblasts and histiocytes. The infectious process can be described as a focal intra-alveolar inflammation with the macrophages prevailing in the exudate. It is self-limited, with a complete resolution. The inflammation is associated with atelectases and with increased secretory activity by type II pneumocytes. Alveolar macrophages and granulocytes degrade C. burnetii. This degradation is followed by damage to and eventual disintegration of some macrophages and by damage to some bacterium-free pneumocytes and vascular endothelial cells in the vicinity of macrophages degrading organisms. The cell damage might be caused by lipopolysaccharide released from degraded organisms. The infectious process is also associated with the influx of T cells in the pneumonic foci, T-cell attachment to the macrophages degrading organisms, and fusion of some macrophages. These are considered a morphologic expression of cell-mediated immunity involved in the infectious process.

Administration, Intranasal↗

Differentiation of Coxiella burnetii by sequence analysis of the gene (com1) encoding a 27-kDa outer membrane protein.

The gene (com1) encoding a 27-kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene-specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.

Amino Acid Sequence↗

[Studies of Coxiella burnetii infections in dairy herds with special regard to infections in men].

Investigations of 1167 dairy cows out of 105 herds with fertility disorders on secretion of Coxiella burnetii (C. burnetii) by the genitals as well as serological studies of these animals using ELISA demonstrated that C. burnetii infections are significantly associated with abortions but not with repeated inseminations without success or vaginal excretions. The demonstration of an increased risk of infections for persons caring for those dairy herds could be shown by a total of 253 serological studies. A comparison of these studies of farmers caring for dairy herds suffering from abortions a seroprevalence of > or = 20% showed that these persons revealed significantly more frequent antibodies against C. burnetii than farmers of the group compared with. Further studies showed that in herds suffering from abortions a seroprevalence of > or = 20% means an additional risk of infections of farmers. Vice versa abortions of the cows in herds with a seroprevalence of > or = 20% imply an additional infectious risk.

Abortion, Veterinary↗

Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue.

The gene encoding a protein that reacted with antibodies specific for Legionella pneumophila macrophage infectivity potentiator (LpMip) was cloned from Coxiella burnetii, the obligate intracellular rickettsia that causes Q fever in humans. Nucleotide sequencing analysis revealed an ORF encoding a gene product of 230 amino acids with a molecular mass of 25.5 kDa and a predicted pI of 10.7. The predicted amino acid sequence from the ORF shows similarity with Mip/Mip-like proteins of Legionella (46%) and Chlamydia (30%). Moreover, like LpMip, the amino acid sequence of the C terminus of this protein has over 35% identity to prokaryotic and eukaryotic FK506-binding proteins (FKBPs) that belong to a superfamily of immunophilins and are peptidyl-prolyl cis-trans isomerases (PPIases). When overproduced in Escherichia coli, the C. burnetii protein also exhibited PPIase activity. Taken together, these results demonstrate that C. burnetii encodes a Mip analogue (CbMip). A putative leader peptide at the N terminus of CbMip was detected by computer analysis. Furthermore, TnphoA mutagenesis demonstrated that in E. coli CbMip was secreted. In view of the role of Mip/Mip-like proteins in the pathogenesis of Legionella and Chlamydia, CbMip may be a C. burnetii virulence factor.

Amino Acid Isomerases↗