Isolation and characterization of a plasmid from phase I Coxiella burnetii.

The DNA from the Nine Mile phase I strain of Coxiella burnetti, the etiological agent of Q fever, has been isolated and purified by cesium chloride-ethidium bromide density gradient centrifugation. A fraction of this DNA has a density characteristic of plasmid DNA. The plasmid DNA was cut with 20 different restriction endonucleases and shown to be a discrete entity. The plasmid, designated QpH1, is approximately 36 kilobases in size and has a molecular mass of 2.4 x 10(7) daltons. A partial restriction map of QpH1 has been constructed by using the restriction endonucleases SalI, KpnI, PstI, and XbaI. QpH1 DNA radioactively labeled by nick translation was used to show that sequences similar to the plasmid are also present in the phase II antigenic variant of C. burnetii.     

Histological changes in mouse liver and spleen caused by different Coxiella burnetii antigenic preparations

The effect was examined on mouse liver and spleen (inbred line A) of intraperitoneal (i.p.) inoculation of phase I Coxiella burnetii (C.b.) cells either untreated or treated with chloroform-methanol (CM) mixture in comparison to the trichloracetic acid extract (TCAE) from phase I C.b.) cells. The phase I C.b. cells were highly toxic as manifested by marked hepatosplenomegaly accompanied with hyperplastic, degenerative and necrotic changes in the liver. By contrast, phase I CM--treated C.b. cells and TCAE were nontoxic as evidenced by the absence of any distinct pathological changes in mouse viscera.     

Inhibition by Coxiella burnetii of ascites tumour formation in mice.

Mice injected intraperitoneally (i.p.) with killed purified Coxiella burnetii organisms were protected from ascites development and death caused by i.p. inoculation of sarcoma-180 cells. The extent of protection was a function of the relative dose of C. burnetii and tumour cells, and of the time of injection of C. burnetii. Phase II C. burnetii organisms exerted an antitumour protection at least as high as phase I C. burnetii organisms.     

Role of antibody in Coxiella burnetii infection.

BALB/c mice infected with Coxiella burnetii phase I developed a state of acquired resistance which could be detected during week 2 postinfection. Immune serum, administered to normal mice 24 h before challenge with C. burnetii, appeared to accelerate the development of resistance. An increased clearance rate could be measured in these serum recipients 1 week postinfection. Simultaneous administration of immune serum and C. burnetii did not affect the normal clearance rate of rickettsiae from the spleens of infected mice during week 1, but enhanced clearance of the organism by 14 days postchallenge. Passive transfer of immune serum 24 h after challenge of normal mice with viable C. burnetii had no effect on rickettsial growth within the spleens of animals treated in this fashion. Treatment of athymic mice with immune serum 24 h before challenge with C. burnetii had no effect on rickettsial multiplication within the spleens of these T-cell-deficient animals.     

Physical mapping of the Coxiella burnetii genome.

Coxiella burnetii isolates from different genomic groups contain restriction fragment polymorphisms that were easily distinguishable using pulsed field gradient electrophoresis (PFGE). Conversely, isolates that belong to the same genomic group yield identical patterns indicating that PFGE can be used to identify the genomic grouping of new C. burnetii isolates. Intact C. burnetii cells were embedded in agarose and lysed in situ. The genomic DNA was digested with low-frequency cutting restriction endonucleases, and subjected to PFGE analysis. NotI and SfiI cut C. burnetii DNA least often and produced the largest fragments. ApaI, MluI, SalI, XbaI or XhoI produced only small DNA fragments (+/- 50 kbp). When PFGE was used to analyse C. burnetii genomes for the presence of plasmid-related sequences, all the plasmid sequences in Nine Mile and Priscilla were associated with their 36 kbp or 39 kbp plasmid bands, respectively. If these isolates contained plasmid sequences which had integrated into their chromosomes those sequences would have been visible as additional bands. These same studies also showed that plasmid sequences in the plasmidless-Ko isolate were completely contained within two NotI fragments, indicating that the integrated plasmid is localized to a concise region of the C. burnetii genome. Since it is difficult to conduct genetic analyses of obligate intracellular parasites using standard techniques, a physical map is being developed using PFGE. In addition to providing a means for determining gene loci, the physical maps provide a means for comparing genetic organization among the different strains of C. burnetii.     

DNA probes for detecting Coxiella burnetii strains.

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological cross-reactions between Coxiella burnetii and Legionella micdadei.

Coxiella burnetii and Legionella micdadei are both gram-negative bacteria potentially responsible for identical clinical syndromes resembling upper respiratory infections. These infections, quite common in immunocompromised patients, are usually diagnosed by serology with a microimmunofluorescence assay. We found that 34.5% of Q fever patients had a significant titer of antibodies against L. micdadei. Cross-reactions involved immunoglobulin G antibodies and were demonstrated by a cross-adsorption study and protein immunoblotting. Western blot analysis performed after treatment with proteinase K indicated that cross-reactions were probably due to both protein and lipopolysaccharide antigens. It is critical that the existence of this cross-reaction be recognized, as misdiagnosis of either condition may lead to incorrect and ineffective treatment.     

Prevalence of antibodies to Coxiella burnetii in Japan.

We evaluated the prevalence of Coxiella burnetii antibodies in 626 human serum samples (275 from veterinarians, 107 from meat-processing workers, 184 from respiratory-disorder patients, and 60 from healthy humans) by the indirect immunofluorescence test. Of the serum samples examined, 54 (8.6%) and 103 (16.5%) reacted positively to phase I and II antigens, respectively, of C. burnetii. The rates differed for healthy humans and respiratory-disorder patients. Antibody prevalence was high for healthy humans living in close contact with animals (e.g., veterinarians and meat-processing workers).     

Phagocytosis of Coxiella burnetii by Hyalomma dromedarii tick haemocytes

Haemocytes of laboratory bred half-engorged Hyalomma dromedarii ticks phagocytized intracoelomally inoculated Coxiella burnetii organisms. Significantly higher phagocytosis of phase II than phase I C. burnetii was observed, irrespective of whether live, killed untreated or killed organisms treated with chloroform-methanol (CM) mixture were used. However, HCl- or KIO4-treated phase I cells were phagocytized to a similar extent as phase II cells. More consistent results were obtained with haemocytes of male than of female ticks. Phagocytosis of phase I killed and live organisms was significantly increased by their preincubation with phase I but not with phase II immune rabbit sera.     

Reduced transendothelial migration of monocytes infected by Coxiella burnetii

The migratory properties of THP1 monocytes infected by Coxiella burnetii were determined in a transmigration assay across a human microvascular endothelial cell monolayer. Transendothelial migration of monocytes infected by virulent, but not avirulent, C. burnetii was inhibited. This inhibition was observed in spite of conserved adherence properties of infected monocytes.     

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.     

Characterization of an endotoxic lipopolysaccharide from Coxiella burnetii.

Phase I Coxiella burnetii antigen isolated by phenol extraction from purified suspensions of C. burnetii in phase I is a complex lipopolysaccharide (LPS) molecule containing substances typical of the bacterial LPS. Some endotoxic properties of this C. burnetii LPS, namely pyrogenicity and skin epinephrine reaction in rabbits, hypothermia in white rats, lethal effect on chicken embryos or on actinomycin-D-treated mice are similar to those of LPS isolated from other Gram-negative bacteria.     

Antigenicity of chloroform-methanol-treated Coxiella burnetii preparations

Phase I Coxiella burnetii (C.b.) cells untreated (Cb I) or treated with chloroform-methanol (CM) mixture (Cb I-CM) were compared as to their capacity to induce antibodies in laboratory animals and cattle, their ability to elicit delayed type hypersensitivity (DTH) reaction in mice and rabbits and protective effect in mice. In all animal species (mice, guinea pigs, rabbits, cattle) tested, the same doses of Cb I-CM cells induced lower levels of both phase I and phase II microagglutinating (MA) antibodies than Cb I cells at different intervals post-immunization (p.i.). Though for elicitation of DTH reaction in rabbits immunized with different C.b. preparations lower doses of Cb I than of Cb I-C M cells were necessary, C.b. cells caused inflammatory reaction at lower doses also in control rabbits. In mice immunized with Cb I and Cb I-CM cells, but not with trichloracetic acid extract (TCAE) from intact Cb I cells, DTH reaction was elicited by the same doses of Cb I and Cb I-CM cells. Higher immunizing doses of Cb I-CM than of Cb I cells were required, however, to induce DTH reaction (as tested by TCAE) as well as protection to phase I virulent challenge. TCAE from intact Cb I cells was protective in mice also at lower doses than TCAE from Cb I-CM cells (TCAE-CM). In humans who suffered from Q fever one year ago, higher proportion of positive skin test (ST) reactions and antibody recalls with higher mean geometric titres (MGT) of phase II MA antibodies was noticed following intradermal administration of TCAE than of TCAE-CM. When humans with no evidence of Q fever in past were vaccinated with TCAE or TCAE-CM, the former preparation not only caused higher proportion of both local and general post-vaccination reactions, but also of phase II MA antibody response and positive ST reactions as tested by TCAE 3 months post-vaccination in addition to higher proportion of phase II MA antibody recalls.     

Interaction between Coxiella burnetii and guinea pig peritoneal macrophages.

The phagocytosis and subsequent degradation of phase I and II Coxiella burnetii by macrophages obtained from immune and nonimmune guinea pigs were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. There was no significant difference in the percentage of phagocytosis of either phase of rickettsiae by macrophages from immune or nonimmune animals. After ingestion, phase I and II organisms pretreated with normal serum multiplied and destroyed normal macrophages as well as macrophages obtained from guinea pigs immunized with phase II rickettsiae. In contrast, only phase I organisms were degraded by macrophages from phase I-immunized animals in the presence of normal serum. Immune serum rendered rickettsiae more susceptible to phagocytosis and also potentiated the destruction of organisms by all types of macrophages. The specificity of macrophages from phase I animals to degrade only phase I rickettsiae was demonstrated by the ability of Rickettsia rickettsii to replicate in these macrophages.     

Induction of splenomegaly in mice by killed Coxiella burnetii cells

Splenomegaly induced in mice inoculated intraperitoneally (i.p.) with purified formalin-killed phase I and phase II Coxiella burnetii (C.b.) cells was dose-dependent. The phase I cells induced higher splenomegaly than phase II cells. The splenomegaly-inducing ability of phase I cells was reduced upon incubation with phase I but not with phase II antiserum, whereas the phase II cells preincubated with phase I or phase II immune sera induced higher splenomegaly than the phase II cells alone. Phase I cells caused lower splenomegaly in mice previously immunized with C.b. The splenomegaly-inducing ability of phase I cells was abolished by mild acid hydrolysis, by treatment either with phenol-chloroform-petroleum ether (PCP) or with a chloroform-methanol (CM) mixture. However, either the CM or the PCP-treated phase I cells retained their capacity to protect mice challenged with virulent phase I C.b.     

Immunological properties of the lipopolysaccharide-protein complex of Coxiella burnetii.

Purified lipopolysaccharide-protein complex (LPS-PC) extracted by trichloroacetic acid from phase I Coxiella burnetii organisms induced in mice and rabbits fair levels of antibodies directed to antigen 1 and antigen 2, as detected by complement-fixation (CF), microagglutination (MA), opsonization-phagocytosis (OP) and serum protection (SP) tests. In guinea pigs only very low levels of MA antibodies against antigen 2 were demonstrated. In rabbit serum, MA antibodies directed to antigen 2 were found exclusively in the IgM fraction after the primary immunizing dose; the second dose was followed by gradual shift of MA antibodies to the IgG class. Two immunizing doses of the LPS-PC were more effective when testing antibody response in mice or protection of mice and guinea pigs against phase I virulent challenge.     

Differences in buoyant-density properties of Coxiella burnetii and Rickettsia rickettsii.

The pronounced change in the buoyant density of Coxiella burnetii in CsCl gradients that was caused by treatment with formalin or ultraviolet radiation was not observed with Rickettsia rickettsii.     

Evaluation of Coxiella burnetii antibiotic susceptibilities by real-time PCR assay

Coxiella burnetii is an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantify C. burnetii cells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing of C. burnetii Nine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions of C. burnetii and was significantly (P < 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 microg of chloramphenicol per ml, 4 microg of tetracycline per ml, 4 microg of rifampin per ml, 4 microg of ampicillin per ml, or 1 microg of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P < 0.01) inhibited the replication of C. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics against C. burnetii Nine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities of C. burnetii.     

Induction of hyperreactivity to endotoxin in mice by Coxiella burnetii

Intraperitoneal inoculation of mice with live or killed Coxiella burnetii phase I or phase II cells induced a marked hyperreactivity to the lethal effect of bacterial endotoxin and was accompanied by a marked hepatosplenomegaly. The degree and duration of hyperreactivity depended on the dose of C. burnetii administered and were higher with phase I than with phase II cells. Sensitization to the lethal effects of endotoxin and induction of splenomegaly by phase I C. burnetii cells also proceeded in the endotoxin-resistant C3H/HeJ strain of mice. Preincubation of C. burnetii cells with the corresponding immune serum significantly diminished the ability of phase I but not phase II cells to induce hyperreactivity to endotoxin.