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.     

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.     

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.     

Differential effects of cyclophosphamide on Coxiella burnetii infection in mice

Treatment of mice with a single dose (200 mg/kg body weight) of cyclophosphamide (CPA) 3 days before intraperitoneal (i. p.) administration of Coxiella burnetii did not substantially affect either the yield of C. burnetii from mouse spleen or the splenomegaly induced by the infection. Administration of the same CPA dose 2 days after i. p. infection resulted in an increased yield of C. burnetii from the spleen and in a reduction of the spleen size as compared to CPA-untreated and CPA-pretreated infected mice. The yields of C. burnetii from the blood and peritoneum were similarly affected. The reduction of splenomegaly was accompanied by a marked decrease in the number of spleen lymphocytes especially in white pulp. Agglutinating antibody response to C. burnetii antigens was inhibited regardless whether CPA was given before of after infection. Whereas delayed hypersensitivity reaction to phase I soluble C. burnetii antigen was not influenced significantly by CPA pretreatment, it was markedly inhibited in mice treated with CPA after C. burnetii administration. The results stress the importance of cell-mediated immunity in C. burnetii infection.     

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).     

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.     

Ultrastructure of viable and inactivated Coxiella burnetii after inoculation of mice.

C3HA mice were inoculated intraperitoneally (i.p.) with viable Coxiella burnetii (C.b.) (strain Apodemus flavicollis-Luga, phase I) or C.b. antigen (formalin-inactivated C.b.); pieces of spleen, liver, omentum, kidney, and suspension of peritoneal macrophages were examined by electron microscopy at 1, 7, 14, 21, 28, and 42 days post-infection (p.i.). Two main types of viable C.b. cells--small dense (DC) and bacteria-like (BC)--were found in omentum, spleen, and peritoneal exudate cells beginning from the 1st day p.i. but maximal amount they reached in spleen at day 14. Some parasitophorous vacuoles contained damaged BC or lysing C.b. cells. C.b. corpuscular antigen was found in phagosomes in ultrathin sections of spleen macrophages until 28 days p.i. By 7 days p.i. in omentum 3 types of parasitophorous phagosomes were described which were supposed to be the sites of the antigen utilization. It is assumed that most BC are destroyed in omentum macrophages during the 1st week after i.p. injection of the antigen, and DC can persist in spleen at least for one month.     

Influence of Coxiella burnetii infection of male mice on their offspring.

The dramatic spread of Q fever in Poland among cattle kept in isolation from natural environment (ticks, wild animals) has suggested the possibility that the infection may also be transmitted sexually. To test this hypothesis series of experiments have been performed in controlled laboratory conditions. Male mice infected with C. burnetii were allowed to mate with healthy female mice. On day 18 of pregnancy serum IgM antibodies to C. burnetii antigens and bacteria in spleen, liver and placenta were detected. The influence of C. burnetii transmission between parents of their offspring was investigated. It has been found that C. burnetii infection in males diminish the number of fertilized females. Their litters are fewer in number and the number of dead embryos is increased.     

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.     

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.     

Q热立克次体单克隆抗体的产生及其特性的研究

Q热立克次体存在相变异的现象。随着相变异的出现,Q热立克次体的生物学特性、理化性质和抗原性等方面均有变化。这种变化的发生与Q热立克次体的表面结构和抗原组分的改变有密切关系。为了在分子水平上研究Q热立克次体,阐明相变异的机制或研究Q热立克次体的致病性、血清免疫学     

Adaptive immunity to the obligate intracellular pathogen Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes the zoonosis Q fever. While an effective whole-cell vaccine (WCV) against Q fever exists, the vaccine has limitations in being highly reactogenic in sensitized individuals. Thus, a safe and effective vaccine based on recombinant protein antigen (Ag) is desirable. To achieve this goal, a better understanding of the host response to primary infection and the precise mechanisms involved in protective immunity to C. burnetii are needed. This review summarizes our current understanding of adaptive immunity to C. burnetii with a focus on recent developments in the field.     

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.     

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.     

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.     

Kinetics of phospholipid extraction from Coxiella burnetii

Kinetics of phospholipid extraction from purified suspensions of Coxiella burnetii in phase I by various chloroform--methanol mixtures at various temperatures were evaluated based on the amount of phosphorus extracted. Extraction by a boiling (53 degrees C) 2:1 chloroform:methanol mixture proved to be the most efficient.     

Direct Identification of Coxiella burnetii Plasmids in Human Sera by Nested PCR

Nested PCR assays were used for the direct identification of Coxiella burnetii plasmids in human sera. A total of 81 serum samples from 81 patients with Q fever were tested by nested PCR with four sets of primers. The first set of primers was used to detect the genomic sequences. The second set of primers was used to detect the conserved sequences of the plasmids. Another two sets of primers were used to identify the QpH1 and QpRS plasmids. QpH1 and QpRS plasmid-specific sequences were identified in 40 (49.4%) and 24 (29.6%) of the serum samples, respectively. Both of the QpH1 and QpRS plasmid-specific sequences were detected in 5 (8.6%) of the serum samples but were not found in 12 (20.7%) of the serum samples. Furthermore, all of the 23 acute-phase serum samples were positive for the QpH1 plasmid and negative for the QpRS plasmid. Nested PCR with plasmid-specific primers appears to be a useful method for the direct typing of C. burnetii plasmids in human sera.