Detection of antibodies to Chlamydophila pneumoniae by immunoblotting in patients with cardiovascular diseases.

Chlamydophila pneumoniae, one of the most prevalent human pathogens worldwide, is not only a significant cause of pneumonia, but may also be associated with cardiovascular diseases (CVD) as suggested by multiple studies. A total of 228 sera from CVD patients with hypertension, ischemic heart disease or previous reconstructive vascular surgery were screened for the presence of anti-C. pneumoniae IgG and IgA antibodies by ELISA. Out of 150 positive serum samples, 80 with similar IgG and IgA levels were investigated by immunoblot (IB). IgG antibodies were directed predominantly against the 35 kDa and 39 kDa proteins as well as 50-54 and 56-60 kDa proteins of C. pneumoniae. IgA antibodies reacted most frequently with the 50-54 and 56-60 kDa proteins.

Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria.

Pathogenic bacteria employ many strategies to overcome the host immune system for extended survival and propagation in their hosts. Components of the bacterial outer-membrane play an important role in this process. When invading the host, Gram-negative bacteria often use a strategy, known as phase variation, that involves a reversible change in antigenic determinants, frequently polysaccharides. This means that the genes encoding the outer-membrane antigens undergo reversible changes within repeated simple DNA sequence motifs. The antigenic structure of the bacterial outer-membrane is influenced by the character of the host immune system, as well as by the targets for bacterial invasion. When the selection pressure of the immune system is absent or weak, bacteria can fail to synthesise the outer-membrane antigens, which are not needed at that time. Smooth-to-rough (S-R) mutation, an economical and often irreversible process in some Gram-negative bacteria, involves the gradual shortening of the lipopolysaccharide (LPS) O-chain. Under certain conditions, e.g., propagation in embryonated eggs or cell lines, some bacteria will cease synthesis of the complete LPS O-chain because it is an energy-demanding process. A type of gradual shortening of the LPS O-chain by Coxiella burnetii, traditionally called phase variation, is used in serological tests for the diagnosis of Q fever. This review discusses the role and function of polysaccharides, especially LPS produced by some Gram-negative bacteria, in bacterial survival.

Chlamydia pneumoniae antibodies and markers of inflammation in patients with cardiovascular diseases.

BACKGROUND: Chlamydia pneumoniae is suggested to be associated with cardiovascular diseases. OBJECTIVES: To study the presence of IgG, IgA anti-C. pneumoniae antibodies, interleukin-6 (IL-6), and C-reactive protein (CRP) as markers of previous C. pneumoniae infection and inflammation, in sera of patients with acute myocardial infarction (AIM), hypertension (HT), and coronary heart disease (CHD). METHODS: Determination of these markers by ELISA method. RESULTS: Proportion of samples containing both IgG and IgA antibodies as well as IL-6 was significantly higher in all groups of patients than in a control group. The CRP was significantly higher in patients with AIM and HT, however, in other patients, the proportion of positive samples depended on the chosen cut-off value. CONCLUSIONS: The results obtained indicate the feasibility of following chlamydial antibodies on higher number of serum samples extended to direct detection of C. pneumoniae in blood and vascular tissue (Tab. 2, Ref. 24).

Q fever is absent from New Zealand.

To investigate the presence of Coxiella burnetii in sheep and cattle, the two major ruminant populations of New Zealand, its seroprevalence was determined in aborting cattle and sheepdogs. These groups of animals were chosen because of their accessibility and the fact that they would be good indicators for the presence of the organism. A total of 2181 bovine and 12,556 canine samples were all seronegative. On the basis of these results and previous reports it is argued that New Zealand is free of coxiellosis or Q fever.

Detergent extract from chlamydial elementary bodies used as antigen for enzyme-linked immunosorbent assay.

Soluble antigen (SA) from chlamydial elementary bodies (EBs) was extracted with N-lauroylsarcosine. The extracted SA composed of lipopolysaccharide (LPS) and proteins was compared with EBs using an enzyme-linked immunosorbent assay (ELISA). Patient sera from natural chlamydial infections exhibited ELISA mean absorbance (A(492) and A(405/650)) values 2-5 times higher with SA than with EBs, resulting in a better discrimination between positive and negative human sera.

A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever.

Genetic studies of Coxiella burnetii strains suggested the possibility of differentiating new isolates according to their plasmid DNA content. Virulence and/or clinical manifestations ('chronic' and 'acute' Q fever) had been claimed to correlate with this plasmid typing. A new plasmid, named QpDV, was found to be common to C. burnetii isolates obtained from acute and chronic Q fever. According to the results obtained, plasmid usage for detection and differentiation of respective pathovars of C. burnetii and the correlation between gene specificity and pathovar has to be revised. Closer studies suggested a common origin of C. burnetii plasmids, but also showed some differences characteristic for each plasmid, probably reflecting divergent evolution.

Phase variation of lipopolysaccharide of Coxiella burnetii, strain Priscilla during chick embryo yolk sac passaging.

Changes of lipopolysaccharide (LPS) of Coxiella burnetii strain Priscilla during chick embryo yolk sac passaging were observed by SDS-PAGE and immunoblot analysis. The course of LPS phase variation was similar to that found in other C. burnetti strains, i.e. a conversion of the phase I to the intermediate phase II after 10 passages. The intermediate phase II LPS of Priscilla strain was also detectable by immunoblot analysis using immune serum against Priscilla strain in the 30th passage.

Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.

Ticks are obligate haematophagous acarines that parasitise every class of vertebrate (including man) and have a worldwide distribution. An increasing awareness of tick-borne diseases among clinicians and scientific researchers has led to the recent description of a number of emerging tick-borne bacterial diseases. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 11 tick-borne human bacterial pathogens have been described in Europe. Aetiological diagnosis of tick-transmitted diseases is often difficult and relies on specialised laboratories using very specific tools. Interpretation of laboratory data is very important in order to establish the diagnosis. These guidelines aim to help clinicians and microbiologists in diagnosing infection transmitted by tick bites and to provide the scientific and medical community with a better understanding of these infectious diseases.

Rickettsial diseases and their serological diagnosis.

Rickettsial diseases (typhus and spotted fever group rickettsioses, scrub typhus and Q fever) may pose a serious public health problem, namely when they are non-diagnosed or misdiagnosed. Although rickettsiae can be isolated from or detected in clinical specimens, serological tests still remain an indispensable tool in the diagnosis of rickettsial diseases. The complement fixation test widely used in the past is being replaced by other tests which make differentiation of immunoglobulin classes possible. Of these tests microimmunofluorescence is considered the test of choice followed by the latex agglutination, indirect hemagglutination, immunoperoxidase assay, and enzyme-linked immunosorbent assay. The last one is also suitable for seroepidemiological studies. Immunoblot analysis can be used to confirm the results of other tests. The use of the low-specific and low-sensitive Weil-Felix test should be reserved only for situations in which other serologic tests are not available.

Cross-reactivity between Coxiella burnetii and chlamydiae.

A cross-reactivity among some strains of Coxiella burnetii and chlamydiae with immune rabbit and mouse sera in ELISA and immunoblot analysis was observed. In the latter, the cross-reactivity disappeared after a treatment of C. burnetii or C. psittaci with proteinase K, which indicates that only proteins were involved. The observed cross-reactivity was not influenced by host chick embryo yolk sac proteins. After adsorption of immune rabbit sera with homologous corpuscular antigens the cross-reactivity disappeared. The possibility of influence of such cross-reactivity on serological diagnosis of C. burnetii or chlamydiae infections is discussed.

Immunity in Q fever.

Post-infection and post-vaccination immune mechanisms in Q fever are summarized. Whereas cell-mediated immunity has been found to play a crucial role in developing resistance to Coxiella burnetii infection, data on the role of specific antibodies in Q fever immunity are controversial. The functional state of immunocompetent cells and professional phagocytes seems to be decisive for the persistence of C. burnetii within phagocytic cells and for the control of Q fever at the host level. Defects of cellular immunity, immune complex formation, and immune response modulation by C. burnetii isolates differing in plasmid composition or LPS antigenic structure are implicated as aetiological factors. Immunogenicity and reactogenicity of three possible vaccine candidates (phase I chloroform-methanol treated and untreated corpuscular vaccine, and phase I soluble chemovaccine) for Q fever prophylaxis is discussed, stressing the need for developing suitable models and defined experimental conditions enabling to compare and evaluate the results obtained in different laboratories.

Q fever--still a query and underestimated infectious disease.

Coxiella burnetii (C.b.) is a strictly intracellular, Gram-negative bacterium. It causes Q fever in humans and animals worldwide. The animal Q fever is sometimes designated "coxiellosis". This infection has many different reservoirs including arthropods, birds and mammals. Domestic animals and pets, are the most frequent source of human infections. Q fever may appear basically in two forms, acute and chronic (persistent). The latter form of Q fever in animals is characteristic by shedding C.b. into the environment during parturition or abortion. Human Q fever results usually from inhalation of contaminated aerosols originating mostly from tissue and body fluids of infected animals. Q fever may appear in humans either in an acute form accompanied mainly by fever (pneumonia, flu-like disease, hepatitis) or in a chronic form (mainly endocarditis). Diagnosis of Q fever is based on isolation of the agent in cell culture, its direct detection, namely by PCR, and serology. Detection of high phase II antibodies titers 1-3 weeks after the onset of symptoms and identification of IgM antibodies are indicative to acute infection. High phase I IgG antibody titers >800 as revealed by microimmunofluorescence offer evidence of chronic C.b. infection. For acute Q fever, a two-weeks-treatment with doxycycline is recommended as the first-line therapy. In the case of Q fever endocarditis a long-term combined antibiotic therapy is necessary to prevent relapses. Application of Q fever vaccines containing or prepared from phase I C.b. corpuscles should be considered at least for professionally exposed groups of the population. Infections caused by C.b. are spread worldwide and may pose serious and often underestimated health problems in human but also in veterinary medicine. Though during the last decades substantial progress in investigation of C.b. has been achieved and many data concerning this pathogen has been accumulated, some questions, namely those related to the pathogenesis of the disease, remain open.

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.

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.

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.

Failure of Q fever phase I corpuscular vaccine to influence the persistence and reactivation of Coxiella burnetii infection in mouse and guinea pig tissues.

In mice and guinea pigs infected with Coxiella burnetii, accumulation of large numbers of rickettsiae in the spleen and liver occurring at the early stages of infection was followed by clearing of these organs from the infectious agent but by its persistence in kidneys and reproductive tract at later intervals. The persistence of C. burnetii was not affected when one to six months-infected mice or guinea pigs were immunized with 100 or 500 micrograms of Q fever phase I corpuscular vaccine. Administration of the vaccine also did not substantially influence the reactivation of C. burnetii infection elicited in mice by parturition or developing in mice and guinea pigs upon treatment with cortisone or cyclophosphamide (CPA). Some differences observed between groups of immunized or non-immunized animals were only quantitative in nature. The possibility to disbalance the steady state of persisting C. burnetii in the host tissues by inhibition or stimulation of the immune response is discussed.

Improved method of isolation of Coxiella burnetii proteins.

An improved method of isolation of Coxiella burnetti proteins was developed. It consists of a combination of detergent (sodium dodecyl sulphate (SDS) or sodium deoxycholate (DOC) and hot phenol treatments. The resulting phenol phase (PP) contained either lipopolysaccharide-(LPS) free proteins (DOC extraction) or proteins contaminated with LPS (SDS extraction), while the water phase (WP) contained LPS. Isolated C. burnetii proteins induced in mice and rabbits antibodies reacting in immunoblot analysis with both phase I and II C. burnetii corpuscles. A rabbit serum against C. burnetii prepared by DOC-phenol extraction did not react with purified I C. burnetii LPS in immunoblot analysis.

Coxiella burnetii phase I and II proteins studied by SDS-page.

Coxiella burnetii cells in both phase I and II reveal in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) similar protein profiles with only small differences. C. burnetii protein profile in SDS-PAGE depended on the method of purification of C. burnetii cells from chick embryo yolk sacs. Immune mouse sera against C. burnetii phase I cells recognized in phase I and II cell protein profiles mainly the 61 K and 29 K proteins by the immunoblot method. Hyperimmune mouse and rabbit sera against phase I and II cells reacted in different way with phase I and II cells. Sera against phase I cells recognized in both phase I and II profiles more protein bands than sera against phase II cells. Thus phase I LPS present in phase I cells exerted adjuvant effect on the antibody response in animals immunized with phase I cells.

Some biological properties of an endotoxic lipopolysaccharide from the typhus group rickettsiae.

A lipophilic thermostable lipopolysaccharide (LPS) complex was isolated by phenol extraction from purified suspensions of the typhus group rickettsiae. The LPS complex is antigenic and possesses some endotoxic properties such as toxicity for actinomycin D-treated mice, pyrogenicity for rabbits and guinea pigs, ability to elicit hypothermia in white rats and local Schwartzman reaction and active cutaneous anaphylaxis in rabbits.

Analysis of antibody response and immunoglobulins in sera of rabbits and guinea pigs immunized with Coxiella burnetii.

Rabbit and guinea pig sera and their immunoglobulin fractions (IgM and IgG) were examined by complement-fixation (CF), microagglutination (MA), opsonization-phagocytosis (OP) and serum protection (SP) tests at intervals after immunization with live phase I and phase II Coxiella burnetii suspensions. In general, MA antibodies, but also decreased, earlier than CF antibodies. The anamnestic immune response was higher with lower primary doses. Both phase II and phase I CF and MA antibodies as well as phase I opsonins and protective antibodies were found in either immunoglobulin fraction depending on post-immunization intervals. At later intervals and especially after the second immunizing dose the levels of protective antibodies in whole sera, but mainly in the IgG fractions, showed a better agreement with those of phase I opsonins rather than of phase I CF and MA antibodies.