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Sensitization of mice to rickettsial toxin by Coxiella burnetii.

Intraperitoneal (ip.) inoculation with live or killed Coxiella burnetii (C.b.) rendered mice more sensitive to intravenous (iv.) administration of a toxic live suspension of Rickettsia typhi. Sensitization of mice by live and killed C.b. was time-and dose-dependent. Killed phase I and phase II C.b. cells possessed a similar degree of sensitization, which was increased slightly by their preincubation with corresponding immune sera. Lipopolysaccharide (LPS)-protein complex extracted from phase I C.b. cells exerted lower sensitization than whole phase I C.b. cells, and chloroform-methanol (CM) treatment of phase I C.b. cells reduced markedly their sensitizing effect. No toxic effect was observed either in C.b.-inoculated or in control mice upon i.v. administration of a heated R. typhi suspension. Specificity of rickettsial toxicity was demonstrated by its distinct reduction both in control and C.b.-inoculated mice after preincubation of R. typhi suspension with immune anti-R. typhi mouse serum.

Animals↗

Interaction between Dermacentor reticulatus cells and Coxiella burnetii in vivo.

By electron microscopy the distribution of Coxiella burnetii was followed in females of Dermacentor reticulatus injected intracoelomally in a dose of about 10(3) EID50 per tick. The heaviest infestation with coxiellae was noticed in the cells of haemolymph, fat body, Malpighian tubulus and tracheal complex. No rickettsiae were found in Gene's organ. Unexpected was the propagation of rickettsiae in muscle fibres. C. burnetii multiplied in all organs affected. Heavy infection resulted at the marked damage of cell components. Coxiellae were evident in the haemocytes; in organs they formed small and large cell variants; endospore formations were observed free in the haemolymph.

Animals↗

Changing of antigenic, imumnogenic and chemical properties of a Coxiella burnetii strain during chick embryo yolk sac passaging.

Coxiella burnetii (C.b.) strain 48 after an increasing number of chick embryo yolk sac (CEYS) passages had lost at egg passages (EP) 15--20 its antigenic, immunogenic and chemical properties typical of phase I. The change refers namely to phase I antibody-inducing ability in mouse, the inability to react in microagglutination (MA) test with the phase II-specific serum, the loss of phase I antibody-binding capacity as detected by immunofluorescence (IF), the capacity to induce an increased number of peritoneal exudate cells (PEC) in the mouse, the resistance to nonspecific phagocytosis by guinea pig macrophages, the polymer two-phase system. At the same time the phase I antibody-inducing ability in rabbit and phase I antibody-binding capacity as detected by the MA and CF tests were preserved up to EP 45, while the phase II antibody-binding capacity in IF and complement-fixation (CF) tests was determined as early as in EP 10 and EP 15, respectively. The changes of other properties, such as splenomegaly and hepatomegaly-inducing abilities as well as immunogenicity by resistance to challenge with phase I virulent cells were more gradual.

Animals↗

[Prevalence of antibodies against Coxiella burnetii in a healthy population in Lanzarote (Canary Islands)].

The Canary Islands area now appears to be a Q-fever endemic zone, especially the west side (La Palma island). The situation in the eastern islands in unknown. In order to evaluate the seric prevalence of Coxiella burnetii, 100 serum samples that were taken from the adult population of Lanzarote and, following strict criteria, were analysed using a complement fixation test; blood donors and patients who had suffered a recent infection were excluded. The study was carried out during November/1986. Three serum samples were positive, one had titers of 1/8 and the other two showed 1/64. This prevalence rate of residual Coxiella burnetii antibodies in Lanzarote (3%)--despite being low compared to other areas in Spain--together with te recent cases described, confirms the suspicion that the Canary Islands area is indeed a new endemic Q-fever zone.

Adolescent↗

The incidence of Coxiella burnetii antibodies in cattle in the Transvaal.

With the use of the complement fixation test, 8,900 cattle were tested for antibodies to Coxiella burnetii. These were randomly selected from 178 different farms in 37 districts in the Transvaal. The percentage of cattle in the sample with positive antibody titres was equal to 7.78%, with a standard error of 0.28%. Because of the large size of the sample, asymptotic normality can be relied upon and the population confidence interval calculated. This was found to be greater than or = 0.07 and less than or = 0.085 at a 99% confidence level. Hence we are 99% confident that between 7% and 8.5% of the cattle in the Transvaal had antibodies to Coxiella burnetii during the period March 1985 to July 1986. The proportion of cattle with C. burnetii antibodies was also estimated for each of the 37 districts tested. Every district tested had some evidence of C. burnetii. The percentage of positive titres ranged from less than 1%-30% per district. This suggests that C. burnetii is probably an endemic disease of the cattle population of the Transvaal. A higher proportion of cattle had antibody titres in the central and south-eastern parts of the Transvaal. This distribution may be linked to the distribution of Boophilus species ticks which occur in the same areas of the Transvaal.

Animals↗

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

The progression of Coxiella burnetii infection to acute or chronic Q fever has been attributed to biological characteristics of the bacterium and to the host immune response. We measured whether serum levels of total and specific subclasses IgA1 and IgA2 could be correlated with the course of disease in acute and chronic Q fever infections, and with the occurrence of endocarditis. In patients with chronic infection, total IgA2 levels were significantly increased. Q-fever-specific IgA1 antibodies were detectable in both acute and chronic infections, but only patients with endocarditis had IgA2 antibodies to C. burnetii phase II antigens. These findings indicate that the measurement of IgA subclasses may be a useful aid in the serological diagnosis of Q fever. Our results reinforce the idea that immunologically mediated host factors are important in the pathogenesis of Q fever and in the disease outcome of this infection.

Acute Disease↗

Immunogenicity and protective ability of corpuscular and soluble vaccines prepared from different Coxiella burnetii phase I strains.

BALB/c mice immunized intraperitoneally (ip) with killed purified Coxiella burnetii phase I corpuscular vaccines or trichloroacetic acid (TCA) extracts from phase I corpuscles (soluble vaccines) were protected against ip challenge with both homologous and heterologous C. burnetii phase I strains. Though the degree of protection, namely the inhibition of C. burnetii multiplication in the mouse spleen slightly varied, in general, corpuscular vaccines provided better protection than soluble ones. Cross-protection was accompanied by comparable levels of cell-mediated immune response as evaluated by lymphocyte transformation test (LTT). However, higher stimulation indices of LTT were obtained with homologous than with heterologous strains. The values of antibody response as determined by enzyme-linked immunosorbent assay (ELISA) were higher with homologous strains too. On average, both antibody-inducing and antibody-binding capabilities of the strains Priscilla and S were lower than those of the Nine Mile and Luga strains, except for values obtained with the antigens from homologous strains.

Animals↗

Evidence for the structural heterogeneity of the polysaccharide component of Coxiella burnetii strain Nine Mile lipopolysaccharide.

Highly purified lipopolysaccharide (LPS) preparation obtained from Coxiella burnetii strain Nine Mile in phase I was used to determine the structure and monosaccharide composition of the polysaccharide component. The procedure included sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by silver staining and gel chromatographic fractionation of acetic acid-hydrolyzed LPS. Five fractions (A-E) were analysed by GLC-mass spectrometry. D-Mannose and D-glycero-D-mannoheptose were present in an appreciable amount in all polysaccharide fractions (A-D), whereas the virenose and dihydrohydroxystreptose contents varied. The highest content of both rhamnose and ribose was found in the low-molecular weight polysaccharide fraction D. The former sugar is being reported for the first time to be a LPS constituent. D-Xylose and D-glucose content varied considerably in the individual fractions and was the highest in fraction A. Glucosamine and galactosaminuronic acid were present in all polysaccharide fractions and, surprisingly, L-glycero-D-mannoheptose was also found, but its presence was limited within the certain degree of polymerisation of the polysaccharide chains. Mild acid hydrolysis of LPS resulted in a partial release of dihydrohydroxystreptose and virenose residues, which were collected and identified in fraction E. The data presented indicate a strong microheterogenity within the individual polysaccharide chains with respect to their sugar composition, size, and shape. Thus, the chemical structure of Coxiella LPS appears to represent a significant departure from the structures described for enteric LPSs.

Carbohydrate Sequence↗

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.

Animals↗

Concomitant or consecutive infection with Coxiella burnetii and tickborne diseases.

BACKGROUND: Q fever is a worldwide zoonosis caused by Coxiella burnetii, which can be isolated from ticks. Reports of people with both Q fever and other tickborne diseases are rare. In this study, we describe 6 patients with Q fever who were infected with 1 of the following tickborne pathogens: Rickettsia conorii (2 patients), Rickettsia slovaca (2), Rickettsia africae (1), and Francisella tularensis (1). METHODS: Diagnoses were made on the basis of results of microimmunofluorescence assays for detection of C. burnetii, R. conorii, R. slovaca, R. africae, and F. tularensis antigens. Cross-adsorption studies and Western blots were used to confirm dual infections. RESULTS: Among the 6 cases presented, 3 were probably due to a concomitant infection after a tick bite, whereas the remaining 3 were more likely consecutive infections. CONCLUSIONS: Because acute Q fever is often asymptomatic, we recommend that patients infected with the tickborne pathogens mentioned above also undergo routine testing for concurrent infections with C. burnetii.

Adult↗

Molecular pathogenesis of Coxiella burnetii in a genomics era.

The agent of acute and chronic Q fever, Coxiella burnetii, occupies a unique niche among intracellular pathogens. The mechanisms the organism employs to cause disease are unclear but involve persistence in a parasitophorous vacuole and the subsequent host response. Studies designed to model molecular mechanisms of pathogenesis have relied upon indirect evidence for testing the role of virulence factors since methods for generation of defined mutations have not been developed. Evidence suggests replication involving a developmental lifecycle is critical for intra- and extracellular survival but this cycle is incompletely defined. It has been proposed that survival in the phagolysosomal-like parasitophorous vacuole requires specific iron uptake systems, secretion of enzymes to detoxify the compartment (catalase and SOD), and down-regulation of an oxidative burst (acid phosphatase). Studies to test these potential virulence mechanisms can be accelerated with the recent development of the complete genome sequence for the prototype acute disease isolate, Nine Mile. Proteins differentially expressed during the developmental cycle can more readily be identified with MALDI-TOF description of proteomic profiles. Genes encoding secreted Cu/Zn SOD, catalase, and acid phosphatase are predicted and can be tested for function and expression. An iron regulon is predicted based upon Fur-regulated open reading frames. The specific role the iron-regulated genes play in iron acquisition can be tested. Confirmation of the iron regulon and others can be tested using microarrays based upon the genomic ORF predictions. These are examples of how we are rapidly changing the experimental approaches used to investigate C. burnetii to improve our understanding of the biology of this unusual and highly adapted organism.

Coxiella burnetii↗

Isolation of Coxiella burnetii from heart valves of patients treated for Q fever endocarditis.

Coxiella burnetii was isolated from the valve material of two patients who underwent valvectomy because of progressive congestive heart failure due to endocarditis. In each case antibiotic therapy was administered for several months prior to valvectomy. Classical histopathological examination of the valves did not reveal an etiology. However, coxiella-like organisms were demonstrated in valvular material with Köster, Stamp, and Giemsa stains, and the organisms were grown in cell culture. Antibody titers were consistent with the diagnosis of chronic C. burnetii infection. This report illustrates the advantage of simple and fast staining techniques and cell culture for the demonstration and isolation of C. burnetii in the heart valve tissue of patients with Q fever endocarditis.

Antibodies, Bacterial↗

The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell.

The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24-48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation-induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.

Animals↗

The serologic prevalence of Q fever (Coxiella burnetii) complement-fixing antibodies in the Peninsular bighorn sheep of Southern California.

Q fever is a rare illness in the Southern California desert. During the past 34 years only 6 patients have been diagnosed with the disease at the Eisenhower Medical Center, a referral center for much of the desert and surrounding mountains. In all but 2 instances, Q fever was identified in patients who have been in contact with imported domestic sheep who are brought to the desert to graze and lamb in the fall and winter. The sheep are sent back to Idaho, Wyoming, and Montana in the spring. With frequent infection by Coxiella burnetii established in domestic sheep, we elected to study the prevalence of complement fixing antibodies to Coxiella burnetii in native bighorn sheep who reside in the lower levels of the mountains surrounding the desert. From 1992 to 1999, of 268 serum samples drawn from male and female lambs and adult sheep, 27 tested positive (10%), which is strikingly low when compared with Dall sheep in Alaska (12 of 15), kangaroos, wild rabbits, and brown rats. Because changes have been made in Peninsular bighorn sheep habitat since the animals were listed as endangered in 1998, further follow-up in Q fever serology testing will be of interest.

Animals↗

Detection of Coxiella burnetii from dust in a barn housing dairy cattle.

We attempted to detect Coxiella burnetii in dust samples collected from a barn housing dairy cattle by the polymerase chain reaction (PCR) method. Ten dust samples (five from ventilation fans and five from crossbeams) were collected from two areas in a barn on a farm near Sapporo, Hokkaido. C. burnetii was detected in 5 of the 10 dust samples. It was believed that aerial contamination by C. burnetii occurred in the barn.

Air Microbiology↗

Coxiella burnetii in polymorphic lymphocytes in tissue and blood of patients with polymorphic reticulosis.

Coxiella burnetii is a well-known causative agent of granulomatous inflammation and an inducer of morphological changes and transformation of human B lymphocytes in vitro. An association of the organism with polymorphic reticulosis (PMR), a malignant granulomatous inflammation characterized by polymorphic lymphocytes, was examined. The infection of C. burnetii was demonstrated in all cases tested, especially in polymorphic lymphocytes. Also the presence of morphologically transformed peripheral blood lymphocytes (PBLs) infected with C. burnetii was demonstrated. In cultures of blood lymphocytes, C. burnetii-infected polymorphic cells identified as B cells became immortalized in vitro. These findings implicate the role of C. burnetii in the process of PMR.

Adult↗