Properties of phase I purified cellular antigen of Coxiella burnetii

An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests. Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient. The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C. burnetii in the following tests: complement fixation and microagglutination. Only fractions containing protein were serologically active. They proved to be of similar specificity and sensitivity as the antigens obtained by standard method. Moreover, it was found that after formalin treatment of C. burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.     

Sequencing and characterization of the cryptic plasmid QpRS from Coxiella burnetii

Plasmid QpRS from Coxiella burnetii, isolate Priscilla Q177, phase I, has been completely sequenced. DNA for sequencing was amplified with "extra-long" (XL) PCR. The size of the QpRS plasmid sequence was determined to be 39,280 bp, with a G + C content of 39.3%. Putative proteins associated with replication and recombination were identified. The sequence of QpRS plasmid was analyzed for shared and unique regions among C. burnetii plasmids.     

Dependence between penetration route and course of infection with Coxiella burnetii in mice]

This study was aimed at investigation of course of Coxiella burnetii infection in mice infected by these bacteria by different routes. The animals infected intranasally, perorally, intraperitoneally and intravaginally by suspension of C. burnetii cells. Mice were also infected via peritoneal and intravaginal route with spermatozoa derived from infected males. In all animals at the same time specific antibodies against phase I and phase II antigens of C. burnetii belonging to IgG and IgM classes of similar titers appeared and this was detected by dot-blot immunoenzymatic test. Independently of route of infection C. burnetii were present in the liver, spleen, testicles, prostate and spermatozoa of tested animals. The bacteria were detected in these organs for 18 days of infection, in the blood for 7 days only, whereas in urine they appeared as late as 14 days after infection. The course of infection with C. burnetii in mice in thus similar regardless of site of bacterial penetration. Infection with C. burnetii may be also transmitted by a sexual route from male to female animals. Infection of female mice occurs both after intravaginal application of live suspension of C. burnetii or spermatozoa derived from infected males.     

Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever]

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.     

Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.     

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.     

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.     

Sustained axenic metabolic activity by the obligate intracellular bacterium Coxiella burnetii

Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [(35)S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.     

Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase.

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.     

Detecting and measuring small numbers of viable Coxiella burnetii

Coxiella burnetii is an acidophilic, intracellular bacterium that causes the human disease Q fever. In some studies, it is important to distinguish between viable and nonviable C.?burnetii. We compared four methods for detecting and measuring viable C.?burnetii in biological samples as follows: growth in two different cell culture lines, infection of severe combined immunodeficient (SCID) mice (leading to death) and infection of SCID mice with detection of C.?burnetii in their spleen (after euthanasia at day 50 postinfection). Two isolates of C.?burnetii were used ('Henzerling' and 'Arandale'). Our in-house qPCR assay for C.?burnetii DNA was used as a control. SCID mouse inoculation was more sensitive than cell culture. The assay that detected C.?burnetii in SCID mouse spleens was slightly more sensitive than SCID mice deaths alone. Approximately one viable C.?burnetii cell could be detected by this method, making it suitable for determining the viability of C.?burnetii in a sample.     

Detection of Coxiella burnetii DNA on small-ruminant farms during a Q fever outbreak in the Netherlands

During large Q fever outbreaks in the Netherlands between 2007 and 2010, dairy goat farms were implicated as the primary source of human Q fever. The transmission of Coxiella burnetii to humans is thought to occur primarily via aerosols, although available data on C. burnetii in aerosols and other environmental matrices are limited. During the outbreak of 2009, 19 dairy goat farms and one dairy sheep farm were selected nationwide to investigate the presence of C. burnetii DNA in vaginal swabs, manure, surface area swabs, milk unit filters, and aerosols. Four of these farms had a positive status during the Coxiella burnetii bulk milk monitoring program in 2009 and additionally reported abortion waves in 2008 or 2009. Eleven farms were reported as having positive bulk milk only, and five selected (control) farms had a bulk milk-negative status in 2009 and no reported Q fever history. Screening by quantitative PCR (qPCR) revealed that on farms with a history of abortions related to C. burnetii and, to a lesser extent, on farms positive by bulk milk monitoring, generally higher proportions of positive samples and higher levels of C. burnetii DNA within positive samples were observed than on the control farms. The relatively high levels of C. burnetii DNA in surface area swabs and aerosols sampled in stables of bulk milk-positive farms, including farms with a Q fever-related abortion history, support the hypothesis that these farms can pose a risk for the transmission of C. burnetii to humans.     

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.     

Four-year evaluation of the effect of vaccination against Coxiella burnetii on reduction of animal infection and environmental contamination in a naturally infected dairy sheep flock

Vaccination is considered one of the best options for controlling Coxiella burnetii infection in livestock. The efficacy of a phase I vaccine was investigated over 4 years in a sheep flock with confirmed C. burnetii infection. Shedding was not detected in ewes and yearlings in the last 2 years, but C. burnetii still persisted in the environment.     

Comparative sensitivity of four different cell lines for the isolation of Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that causes the disease Q-fever. This is usually diagnosed by serology (immunofluorescence assay) and/or PCR detection of C.?burnetii DNA. However, neither of these methods can determine the viability of the bacterium. Four different cell lines were compared for their ability to amplify very low numbers of viable C.?burnetii. Two different isolates of C.?burnetii were used. For the Henzerling isolate, DH82 (dog macrophage) cells were the most sensitive with an ID (50) (dose required to infect 50% of cell cultures) of 14.6 bacterial copies. For the Arandale isolate, Vero (monkey epithelial) cells were the most sensitive with an ID (50) of less than one bacterium in a 100-μL inoculum. The Vero cell line appeared highly useful as vacuoles could be seen microscopically in unstained infected cells. The findings of this study favour the use of Vero and DH82 tissue culture cell lines for isolation and growth of C.?burnetii in vitro. The other cell lines, XTC-2 and L929, were less suitable.     

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.     

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.     

Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.     

Tick-borne infections as a cause of heart transplantation

Many bacterial species can be a cause of various heart diseases, such as: Borrelia burgdorferi sensu lato, Coxiella burnetii and Bartonella spp. The aim of the present studies was to establish if any tick-borne infections can contribute to serious heart disorders resulting in the need for heart transplantation. Myocardium, aortic and mitral valve samples from hearts removed from patients undergoing heart transplantation were tested. The presence of Bartonella spp., Borrelia afzeli and C. burnetii bacteria in malfunctioning human hearts has been shown. DNA of Bartonella spp., B. burgdorferi and C. burnetii were detected in various parts of tested hearts. DNA of B. afzelii and Bartonella spp. were found in the aortic valves. DNA of C. burnetii was detected in the myocardium. Mixed infections with Bartonella spp. and C. burnetii were also observed. Obtained results indicate that diagnosis of Bartonella spp., B. burgdorferi C. burnetii and Rickettsia spp. infections should be considered in cases of infectious endocarditis with negative blood cultures.