Further characterization of a lipopolysaccharide from Coxiella burneti

The lipopolysaccharide previously isolated from the rickettsial agent of Q fever, Coxiella burneti, phase I, has been further characterized. The sugar residues ribose, mannose, gluclose, D-glycero-D-mannoheptose, and L-glycerto-D-mannoheptose are present. Two sugars remain unidentified, one of which is a minor and the other a major constituent. Isomyristic, palmitic, and beta-hydroxymyristic acids are the major fatty acid residues of the 15 identified. The nature and content of other lipopolysaccharide constituents are presented.     

Serological cross-reactions between Coxiella burnetii and Legionella micdadei

PURPOSE: The bronchodilator effect of salbutamol formulated in hydrofluoroalkane-134a (HFA-134a), a chlorofluorocarbon (CFC)-free propellant for metered dose inhalation (MDI) devices, was compared with that of salbutamol formulated in CFC in anesthetized dogs. METHODS: Bronchospasms were induced by the intravenous injection of histamine, and bronchial resistance was measured by the method of Konzett and Rossler. RESULTS: While the placebo vehicles (HFA-134a and CFC propellants) had no significant effect on histamine-induced bronchospasms, the salbutamol/HFA-134a and salbutamol/CFC MDI formulations had equivalent dose-related inhibitory effects. CONCLUSIONS: These data indicated that salbutamol formulated in HFA-134a and that in CFC propellant are bioequivalent.     

Codon usage and nucleotide composition in Coxiella burnetii

The serum of many teleosts, including turbot, contains chitinolytic and proteolytic enzymes. In the present study, the possible role of these enzymes in nonspecific immune responses to microsporidian infection was investigated. The rate of phagocytosis of Glugea caulleryi spores by turbot splenic macrophages was significantly reduced after pretreatment of spores with proteolytic or chitinolytic enzymes, suggesting that alteration of surface glycoproteins affects spore recognition. However, intracellular superoxide production by macrophages was significantly higher after stimulation with protease-treated spores, or with untreated spores plus normal turbot serum (NTS), than after stimulation with untreated spores in the absence of NTS. These results support the view that the chitinolytic and proteolytic activities in teleost serum may play a role in defence against microsporidian infection.     

Seroepidemiology of Coxiella burnetii among wildlife in Nova Scotia

We used an indirect immunofluorescence assay to determine antibody titers to phase I and phase II Coxiella burnetii antigens in serum samples from a variety of wild animals in Nova Scotia. Forty-nine percent of the hares, 16.5% of the moose, 7.1% of the raccoons, and 1.5% of the white-tailed deer tested had antibodies to phase I antigen. We conclude that there is extensive infection of the hare population by C. burnetii, with lesser degrees of infection of the moose, raccoon, and deer population.     

A cationic antimicrobial peptide enhances the infectivity of Coxiella burnetii

The pulmonary arteriole remodeling in Wistar rats with respiratory infection induced by mycoplasma pneumoniae was observed using light microscopy and morphometry. The pulmonary artery pressure (PAP) and index of right ventricular hypertrophy (RVHI) were measured. The intimal and medial hypertrophy can be seen in the pulmonary arterioles, leading to vessel wall thickening and narrowing of the lumina. The total number of the pulmonary arterioles decreased (P < 0.01), and both pulmonary hypertension (Ppa 4.11 +/- 0.19 kPa) and right ventricular hypertrophy (RVHI = 34.96 +/- 3.91%) occurred. In addition, an interstitial pulmonary fibrosis (IPF) was found, in which the content of collagen in the lung tissue changed, i. e., type I collagen increased whereas type III one decreased, and the ratio of type I collagen to type III one increased. It suggested that respiratory infection induced by repeated MP may result in remodeling of pulmonary arterioles and are closely related to pulmonary hypertension.     

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.     

A developmental stage-specific histone H1 homolog of Coxiella burnetii

Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.     

Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils

Supernatants prepared from disrupted Coxiella burnetii possess acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M(r) of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 degrees C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 degrees C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-linked glycan residues since its treatment with N-glycosidase F (PNGase F) decreased its M(r) to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phosphocompounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP > 5'-AMP. The ACP also dephosphorylated (32)P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C. burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells.     

Serological evidence of Coxiella burnetii infection in wild animals in Japan

One hundred and thirty-four (26%) of 511 sera from 11 wild animal species in eight prefectures in Japan had antibody titers to Coxiella burnetii by the enzyme-linked immunosorbent assay. High prevalences were observed in Japanese black bears (Ursus thibetanus) (78%), Hokkaido deer (Cervus nippon yesoensis) (69%), Japanese hares (Lepus brachyurus) (63%), Japanese deer (Cervus nippon centralis) (56%), and to some extent in Japanese monkeys (Macaca fuscata) (28%). A low prevalence (13%) was observed in nutrias (Myocastor coypus). Japanese serows (Capricornis crispus), wild rats (Muroides sp.), raccoon dogs (Nyctereutes procyonoides viverrinus), wild pigs (Sus scrofa leucomystax), and masked palm civets (Paguma larvata) had no detectable antibodies to C. burnetii. Thus, six of 11 wild animal species in Japan were exposed to C. burnetii. Based on the high prevalences in some species, they may be a potential source of infection to both domestic animal and human populations.     

Prevalence of Coxiella burnetii infection in dairy cattle with reproductive disorders

The prevalence of Coxiella burnetii infection in 207 cattle with reproductive disorders was studied by using an indirect immunofluorescence (IF) test, nested polymerase chain reaction (PCR) and isolation. IF antibodies to phase I and phase II antigens of C. burnetii were found in 122 (58.9%) and 125 (60.4%) of the sera, respectively, and PCR-positives were found in 8 (3.9%) of the sera and in 51 (24.6%) of the milk samples. In addition, C. burnetii was isolated from 51 (24.6%) of the milk samples by inoculating laboratory mice. The results indicate that the IF test plus PCR are useful in the diagnosis of bovine coxiellosis. It is difficult to deny that dairy cattle with reproductive disorders would be one of the important reservoirs of C. burnetii responsible for infection in both animal and human populations in Japan.     

Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes

Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetii organisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetii may therefore play a critical role in the internalization strategy of this bacterium.     

Isolation of Coxiella burnetii from dairy cattle and ticks, and some characteristics of the isolates in Japan

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

The characteristics of the spread of Coxiella burnetii in the Carpathian region

The paper presents data of the long-term (1975-1994) study of Q fever in Carpathian region [correction of Forecarpathia] (Ivano-Frankivs'k and Chernivtsi provinces) and dynamics of epidemiologic manifestations of this disease during the recent years. It is shown that after Q fever outbreaks in 1975-1977, people sick for it have been recorded sporadically, and a considerable decrease of epidemiological manifestations of Coxiella burnetii circulation is observed in the late years. This is accompanied by the decrease of the immune stratum among the population from 10.1% to 1.6-2.8%. In the authors opinion there is the interepizootic period now when the agent population remains in the phase of reservation that requires devoting much attention to the future appearance of its epidemiologically dangerous variants. The expediency of the purposeful examination of the profile therapeutic patients for Q fever and a necessity of special study of the chronic form of this infection are emphasized.     

IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF

The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages. Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-gamma. While C. burnetii multiplied in untreated monocytes, IFN-gamma reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h. IFN-gamma also affected the viability of infected cells. Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-gamma, it reached a maximum after 48 h and was followed by necrosis. Reactive oxygen intermediates were not required for C. burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-gamma. The role of cytokines was also investigated. IFN-gamma elicited a moderate release of IL-1beta in infected monocytes. Moreover, the IL-1 receptor antagonist did not affect C. burnetii survival, suggesting that IL-1beta was not involved in the bacterial killing induced by IFN-gamma. TNF was involved in IFN-gamma-induced killing of C. burnetii and cell death. IFN-gamma induced mRNA expression and sustained secretion of TNF. Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-gamma-dependent killing of C. burnetii and cell death. These results suggest that IFN-gamma promotes the killing of C. burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.     

Prevalence of antibodies to Rickettsia conorii Ricketsia africae, Rickettsia typhi and Coxiella burnetii in Mauritania

A 24-year-old male developed cytogenetic relapse of chronic myeloid leukemia (CML) four years after allogeneic BMT. After a year of treatment with IFN-alpha, he achieved a partial cytogenetic response. Treatment with donor leukocyte infusions (DLI) was given (total dose 1 x 10(8) T lymphocytes/kg). Two months later, he developed acute GVHD (skin and liver), that improved with CsA and methylprednisolone and resulted in cytogenetic remission with complete donor chimerism. One month later he developed rhinocerebral mucormycosis and was successfully treated with surgical debridement and liposomal amphotericin B (total dose 12 g). This is the first case of mucormycosis described after DLI.     

Distribution of IgA subclass response to Coxiella burnetii in patients with acute and chronic Q fever

BACKGROUND: In some general practice intervention trials, patients must be randomized in practices rather than individually, and this must be taken into account in the analysis. OBJECTIVES: In this article we aim to show how failure to do this may lead to spurious statistical significance and CIs which are narrower than they should be, and to describe the use of summary measures for each practice as a simple method of analysis. METHOD: The statistical issues are demonstrated by an example of a trial in general practice. DISCUSSION: The choice of unit of analysis will be most important where there are large numbers of patients recruited from each practice or a high degree of variability between practices.     

Detection of Coxiella burnetii in cow's milk using the polymerase chain reaction (PCR)

A PCR approach (transposon PCR) with primers based on repetitive transposon-like sequences, which--depending on the isolate--were found at a minimum frequency of 19 on the C. burnetii genome, was established for the highly sensitive and specific detection of C. burnetii. This study describes the analytical detection of C. burnetii in milk, which requires a special preparation method prior to PCR. Because of the low level of C. burnetii particles in milk samples, template DNA was concentrated by a factor of 200, using cetyltrimethylammonium bromide as the precipitation reagent. Using this particular preparation method, even a single C. burnetii particle could be detected in 1 ml milk.     

Evaluation of a recombinant 27-kDa outer membrane protein of Coxiella burnetii as an immunodiagnostic reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.