Biosafety concerns and Coxiella burnetii.
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Coxiella burnetii↗
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Mechanisms of resistance to fluoroquinolones in Coxiella burnetii.
Mechanisms of resistance to quinolones were investigated in two in vitro DNA gyrase mutant Coxiella burnetii strains. Accumulation of pefloxacin was found to be lower in resistant strains than in susceptible strains. Pre-incubation with carbonyl cyanide m-chlorophenylhydrazone had no apparent effect on intracellular quinolone fluorescence, indicating that an active efflux mechanism is not responsible for the decrease of pefloxacin accumulation. No differences were detected between the strains after outer membrane protein analysis. Decreased accumulation was found in all strains at acidic pH. It is possible that, apart from the mutation-induced mechanism of resistance, a pH-related mechanism is involved in the drug accumulation process.
Animals↗
Search for correlates of resistance to virulent challenge in mice immunized with Coxiella burnetii.
Mice immunized with live phase I or phase II Coxiella burnetii, with killed phase I or phase II organisms or with trichloroacetic acid (TCAE) or phenol (PE) extracts were resistant to intraperitoneal infection with phase I C. burnetii irrespective of whether or not they displayed phase I antibody response at the time of virulent challenge. Increased phagocytosis of purified phase I organisms by blood leukocytes or peritoneal exudate cells (PEC) was noticed only in mice with phase I agglutinating antibodies in their sera or peritoneal washings. Passive transfer of resistance was made possible only by sera containing phase I agglutinating antibodies. Adoptive transfer of immunity by spleen cells, but not by PEC, was achieved providing that these cells were taken from mice immunized with live phase I C. burnetii.
Animals↗
Antibodies are generated during infection to Coxiella burnetii macrophage infectivity potentiator protein (Cb-Mip).
Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C. burnetii macrophage infectivity potentiator protein (Cb-Mip) cloned in Escherichia coli. Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb-Mip by immunoblot analysis. In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb-Mip. This study indicates that Cb-Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip-like proteins of intracellular bacteria in human sera. Cb-Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.
Animals↗
Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm.
Most infections due to intracellular bacteria respond poorly to antibiotic treatment. The chemical conditions within the subcellular site of bacteria may change antibiotic activity. Coxiella burnetii multiplies within phagolysosomes. The antimicrobial activity of antibiotics combined with the lysosomotropic agents amantadine (1 microgram/mL), chloroquine (1 microgram/mL), and ammonium chloride (1 mg/mL), which alkalinized Coxiella burnetii-containing phagolysosomes from pH 4.8 to 5.3, 5.7, and 6.8, respectively, was evaluated. Percentages of residual viable bacteria (RVB) in cell cultures were significantly reduced after exposure to combinations of doxycycline (4 micrograms/mL) with amantadine (RVB = 18.2% +/- 8.7%, P < .05), chloroquine (RVB = 0.64% +/- 0.38%, P < .01), or ammonium chloride (RVB = 0.29% +/- 0.17%, P < .01); the same was seen with pefloxacin (1 microgram/mL) with chloroquine (RVB = 27.6% +/- 10.8%, P < .05) or ammonium chloride (RVB = 3.72% +/- 1.1%, P < .05). Such bactericidal activity correlated with increased phagolysosomal pH, as determined by Pearson's correlation coefficient, suggesting that phagolysosomal alkalinization is critical for the bactericidal effect of antibiotics.
Amantadine↗
Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain.
After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.
Antigenic Variation↗
NEW METHOD FOR PURIFYING COXIELLA BURNETII BY DEXTRAN SULFATE PRECIPITATION FOLLOWED BY CENTRIFUGATION.
This paper describes a simple method for purifying Coxiella burnetii cultivated in the chick embryo. The procedure is based upon the preliminary clarification of infected whole-egg suspensions by precipitating the low-density lipoproteins and other extraneous solids with dextran sulfate, calcium chloride, and sodium chloride solutions, and then concentrating the rickettsiae by centrifugation.
Animals↗
Lipid A component of lipopolysaccharides from Coxiella burnetii.
Free lipid A from phase I and phase II Coxiella burnetii lipopolysaccharides was isolated and studied serologically. Antisera against lipid A from phase I and phase II C. burnetii cross-reacted with the lipid A preparations from both phases as well as with Salmonella lipid A.
Animals↗
Host-parasite interaction and development of infraforms in chicken embryos infected with Coxiella burnetii via the yolk sac.
Two phase I strains of Coxiella burnetii of different virulence were injected into the yolk sacs of chicken embryos, and the yolk sacs and livers were examined at intervals by light, fluorescent, and electron microscopy. The high absorptive and digestive capacities of the yolk endoderm contributed to he entrance of the organisms into endodermal epithelial cells where C. burnetii multiplied. Organisms multiplied not only inside specific vacuoles originating from phagolysosomes but also in the cytoplasm itself. Lysis of the limiting membrane of some phagolysosomes, a normal function of endodermal cells, as well as rupture of vacuoles, provided the release of C. burnetii into the cytoplasm. The C. burnetii strain of greater virulence infected 100% of the endodermal cells, whereas the strain of lesser virulence infected only 60%. Budding of very small particles from the C. burnetii bodies was demonstrated. The particles were regarded as filterable forms of the organism. Despite the enormous multiplication of C. burnetii in the endodermal cells, organisms were only rarely detected in the vitelline blood vessels and liver sinusoids of the embryos. Peculiarities of the infectious process of C. burnetii in chicken embryos and possible mechanisms of limitation of spread of the infection are discussed.
Animals↗
Experimental Coxiella burnetii infection in pregnant goats: a histopathological and immunohistochemical study.
Pregnant goats were inoculated subcutaneosly with Coxiella burnetii and the course of infection was studied. Abortion in the last third of pregnancy occurred in all infected animals. Tissues from the placenta and other organs were studied before and after abortion by immunohistochemistry and PCR analysis. After infection, mild lesions were observed in several maternal organs, mainly the mammary gland but also the lung and the liver. The trophoblast cells of the choriallantoic membrane were the first target cells of the placenta; there was, however, a substantial delay between initial infection and placental colonization. In the last weeks of pregnancy, just before abortion, massive bacterial multiplication was detected in the placenta. In this stage of infection a necrotic and suppurative placentitis separated the fetal trophoblast cells from maternal syncytial epithelium. Vasculitis was observed in the fetal mesenchyme. A strong maternal T-cell response was detected in the inter-placentomal areas but not in the placentomes, where only neutrophils and smaller numbers of macrophages were associated with the lesions. Neither lesions nor C. burnetii DNA were found in maternal organs in animals maintained until day 120 post-abortion.
Aborted Fetus↗
Immunoblot analysis of antibody response in mice infected with Coxiella burnetii phase I.
Mouse sera collected from day 4 to day 133 postinfection (p.i.) with phase I Coxiella burnetii strain Nine Mile were analysed by immunoblotting with phase I C. burnetii cell lysate. Antibodies of IgG class protein antigens were revealed already on day 10 p.i. (60, 49 and 27 kD proteins), followed by those to 77 kD protein from day 18 p.i. and to further 7 (from 42 to 70 kD) proteins from day 28 p.i. IgG antibody reaction was observed also with 5 antigens in 14-20 kD region corresponding to lipopolysaccharides from day 22 p.i. Surprisingly, antibodies of IgM type appeared later (from day 22 p.i.) and were directed only to protein antigens, most markedly to 60 and 77 kD proteins. Differences in immunoblot patterns observed with the serum collected on day 72 p.i. before and after absorption to phase I and phase II C. burnetii cells, and to phase I cells treated by trichloroacetic acid (TCA) or KIO4, indicate the surface localization of protein phase I antigenic epitopes, which can be destroyed partly by TCA and almost completely by KIO4 treatment.
Animals↗
[Experimental research on organotropism and elimination of Coxiella burnetii].
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Coxiella burnetii↗
Coxiella burnetii plasmid types QpDG and QpH1 are closely related and likely identical.
Strains and isolates of Coxiella burnetii, an obligate intracellular bacterium, carry a single plasmid or a plasmid-homologous sequence integrated into the chromosome. The plasmids QpH1, QpRS, QpDV and the chromosome-integrated plasmid-homologous region have been completely sequenced, whereas no sequence data are available for the QpDG plasmid. In this study, we used total genomic DNA from reference strain C. burnetii Dugway 5J108-111 to demonstrate and characterize the QpDG plasmid by pulsed-field gel electrophoresis (PFGE) and Southern hybridization. Primers derived from regions shared among C. burnetii plasmids were used to construct a physical map of the QpDG plasmid by extra long (XL) PCR. Both approaches, Southern hybridization and XL PCR indicated that QpDG and QpH1 represent a closely related and likely identical plasmid.
Animals↗
[Seroprevalence of Coxiella burnetii and Rickettsia conorii infection in the province of Soria].
BACKGROUND: Determine the antibodies prevalence against Coxiella burnetii and Rickettsia conorii in Soria (Spain). METHODS: By an indirect immunofluorescence test we examined sera samples for antibodies (IgG, IgM and IgA) against C. burnetii (phase II, formolized antigen) and R. conorii (moroccan strain). The test has been applied to 298 sera (150 men and 148 women) from Soria (Spain). The age of the study population ranged between 2 and 91 years. The results were considered positive when the titres were greater than or equal to 1/80 for C. burnetii and 1/40 for R. conorii. RESULTS: Antibodies against C. burnetii were present in 20.8% of the examined sera, 8.8% for women (13/148) and 32.7% for men (49/150). We demonstrated titers of antibodies upper to 1/40 in all age groups, and a greater number of positive results in the persons older than 40 years old (p = 0.0001). In all areas we detected positive results and in many of these areas, the positivity was high (38%). We demonstrated positive results against R. conorii in 5% of the people (4.7% for women and 5.3% for men). More than 90% of the positive cases was present in the eastern part of Soria, and in an small area, the positivity was 20%. CONCLUSIONS: These results confirm a high prevalence of antibodies against C. burnetii in the Soria people, and the existence of R. conorii infection in this area of Spain with a particular distribution. We believe that it is very important to establish an epidemiological surveillance for the detection of clinic cases.
Adult↗
Q fever pneumonia: virulence of Coxiella burnetii pathovars in a murine model of aerosol infection.
Q fever is a worldwide zoonosis caused by Coxiella burnetii, a strictly intracellular bacterium that is a potential bioweapon. Humans usually acquires Q fever after inhalation of dust infected by subclinical animals. We used an aerosol exposure apparatus to challenge immunocompetent (BALB/c) and severe combined immunodeficient (SCID) mice with two different strains (strain Nine Mile and strain Q 212) of C. burnetii at two different inocula. Pathological lesions and dissemination of the bacteria were related to the size of the inoculum. SCID mice showed major pulmonary lesions, whereas similarly infected BALB/c mice were more able to eliminate the bacteria. Pathological differences were found between the strains, with Nine Mile showing more severe histological lesions and quantified spread of bacteria. Our animal model could provide a new tool for the study of acute Q fever pneumonia, the development of Q fever in immunodeficient hosts, and the differentiation of pathogenicity among C. burnetii isolates.
Aerosols↗
Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.
Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.
Acetylation↗
Enhancement of the immunogenicity of phase I antigen of Coxiella burnetii.
The immunogenicity of the soluble phase I antigen of Coxiella burnetii for guinea pigs was enhanced by a nuclease-resistant complex of polyriboinosinic-polyribocytidylic acid, poly-L-lysine, and carboxymethyl cellulose.
Animals↗
Phase variation of Coxiella burnetii strain Priscilla: influence of this phenomenon on biochemical features of its lipopolysaccharide.
During the phase variation of Coxiella burnetii, modifications in its lipopolysaccharide (LPS) component were investigated. The cloned phase I C. burnetii cells were passed serially in chicken embryo yolk sacs up to the egg passage (EP) 90. The LPSs from the cells in EPs 3, 12, 21, 40, 60, and 90 were all separated by steric exclusion chromatography into three major populations: the high, intermediate, and low molecular weight fractions, differing one from another in size and chemical composition. No noticeable shortening of the O-polysaccharide chains was observed in the LPSs isolated during the C. burnetii cultivation. However, a redistribution of the existing LPS populations has been observed due to an increasing prevalence of those cells in the whole cell population that express LPS molecules with truncated O-chains and those being of R-type. In the high and intermediate molecular weight LPS populations, virenose and dihydrohydroxystreptose are lost gradually with the progress in phase variation. This occurs more readily with the former sugar. At present, the molecular mechanisms influencing the LPS modifications during the C. burnetii phase variation remain unclear.
Animals↗