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Detection of Coxiella burnetii in market chicken eggs and mayonnaise.

We tried to detect C. burnetii in market chicken eggs and mayonnaise by nested PCR assay. The PCR target was the com 1 gene of C. burnetii. The positive rate for egg and mayonnaise samples was 4.2% and 17.6%, respectively. Direct sequence of some of the positive egg samples shows mutations whereas no mutation was found in the positive mayonnaise samples. The number of molecules of the Q fever agent is estimated at 10(4) to 10(6) per egg, according to our quantitative PCR test.

Coxiella burnetii↗

[Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever].

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.

Agglutination Tests↗

[Coxiella burnetii antigens--inducers of tumor necrosis factor and interleukin-1].

The cultivation of mouse peritoneal macrophages in the presence of antigenic preparations obtained from C.burnetii was accompanied by the appearance of phagocytes and considerable amounts of tumor necrosis factor and interleukin 1 in the culture medium. The production of cytokines depended on the doses of preparations used as inducers. The special treatment of C.burnetii antigen, selectively removing its phospholipid components, led to a sharp drop in its capacity for stimulating the production of cytokines.

Animals↗

Detection of antibodies against spotted fever group Rickettsia (SFGR), typhus group Rickettsia (TGR), and Coxiella burnetii in human febrile patients in the Philippines.

A total of 157 sera from febrile patients in the Philippine General Hospital in Manila, Luzon, and the Northern Samar Provincial Hospital, the Philippines, were used. Serum antibodies against spotted fever group Rickettsia (SFGR) and typhus group Rickettsia (TGR) were detected by indirect immunofluorescence test. Antibody positive rates were 1.3% for SFGR (Rickettsia japonica) and 2.5% for TGR (R. typhus), respectively. Rickettsial antibodies in humans in the Philippines were found for the first time. These results underscore the need for further epidemiological study of clinical rickettsioses in the Philippines.

Antibodies, Bacterial↗

[Development of the technology of the purification of Coxiella burnetii from components of culture medium].

The technology of the purification of C.burnetii from the yolk-sac components of the cultivation medium, including differential centrifugation, membrane filtration, enzymatic hydrolysis, the standardization of the preparation thus obtained and its stabilization by the method of lyophilization, was developed. Antigens, prepared in accordance with this technology, were sufficiently active and highly specific. The possibility of their use in different serological tests (the complement fixation test and the indirect fluorescent analysis) was studied. In addition, these antigens were found to have good capacity for sorption on the solid-phase carrier (plates) and to be suitable for use in such highly sensitive test as the enzyme immunoassay.

Animals↗

[The morphological changes in the body of guinea pigs in response to the administration of different Coxiella burnetii antigens].

In this work the evaluation of macro- and micro-changes in the tissues of guinea pigs to the injection of inactivated antigens of C. burnetii is presented. The injection of these antigens in a dose of 50 micrograms induced only an insignificant reaction at the site of injection; in the brain of the animals, as well as in all examined internal organs, no visible pathological changes were present. During the first two weeks after the injection of the antigens signs of irritation of the reticuloendothelial system and hyperplasia of lymphoid tissue in T- and B-dependent zones were observed in the regional lymph nodes of the experimental animals. These changes became moderate by day 28.

Animals↗

[An experimental study of the immunogenic and protective properties of Coxiella burnetii strain M-44 and a live enteral vaccine against Q fever].

High effectiveness of C.burnetii strain M-44 was confirmed in experimental studies on subcutaneously immunized guinea pigs ¿correction of pig] after their intratracheal infection with the virulent culture of the infective agent. The study revealed that the dose necessary for the enteral immunization of guinea pigs and monkeys was 10(4.8) -- 10(5.0) times less than that needed for the oral immunization of the animals. Live enteral vaccine obtained on the basis of this strain possessed sufficiently pronounced immunogenic and protective properties, protecting 75% of immunized monkeys from subcutaneous infection with C.burnetii virulent culture.

Animals↗

Electron microscopy of Coxiella burnetii in tissue culture. Induction of cell types as products of developmental cycle.

An in vitro ultrastructural study was carried out on tissue cultures (J774, murine macrophage-like tumour cell line, and BHK-21, baby hamster kidney cell line) persistently infected with C. burnetii to investigate whether the events of cellular differentiation could be visualized. At a given stage of the developmental cycle, a proportion of the cells within the affected phagolysosomes clearly underwent cellular differentiation. The cells initially showed asymmetrical septation, the primary stage of cellular differentiation, and ended with the formation of the differentiated product, a precursor to the small cell. The results verified our initial observation that the events occurring during growth in a phagolysosome represent stages of a complex developmental cycle consisting not only of i) vegetative growth by typical transverse binary fission, but also ii) cellular differentiation.

Animals↗

Unusual causes of reactive arthritis: Leptospira and Coxiella burnetii.

Reactive arthritis is a well-defined clinical syndrome occurring after various infections, although most cases are usually associated with Chlamydiae and gastrointestinal pathogens. Its immunologic background has been extensively studied, as has its relationship with HLA-B27. We describe two cases of reactive arthritis arising after infections with two pathogens not so far related to the occurrence of reactive arthritis: one patient exhibited migratory oligoarthritis shortly after the course of acute Q fever, and another patient developed monoarthritis during recovery from leptospirosis. Arthritis was transient and did not exhibit a chronic course in either patient. We further discuss the context of pathophysiology of the arthritis in these patients, with an emphasis on the immunomodulatory properties of these two pathogens.

Adult↗

[Prevalence of antibodies against Coxiella burnetii in 2 geographical zones of Tuscany].

Q Fever, caused by Coxiella burneti, was first identified as a separate syndrome in 1937 in Australia. Usually it takes the form of an acute atypic lung pneumonia disease, but subclinic or non-typical forms are also known. Q Fever is a zoonoses. The most important source of infection is represented by sheep; the transmission to man occurs by vectors (ticks), or more often by carriers as aerosol, non-pasteurized milk and dairy products. In Italy the first outbreaks of Q Fever date back to the end of World War II and lated until the beginning of the sixties. Since the seventies only a few sporadic cases have been notified, suggesting a low endemic situation. In Tuscany Q Fever presented the same epidemiological characteristics as in Italy. The causes of this particular epidemiological evolution are not clear. We suspect that it might be partly an illusion, caused by an inaccurate evaluation of the real number of cases, due to both the high frequency of atipic clinic forms and the lack of attention of doctors. On the other hand, a reduction in the circulation of C. burneti in the environment could be explained by a limitation of infection to man caused by changed sheep-breeding systems and a consequence of socio-economic evolution. To evaluate the actual reduction in the incidence of Q Fever in general, it is necessary: 1 - to make a careful research of Q Fever cases in man 2 - to evaluate the frequency of C. burneti antibodies in man and sheep. In this light, the Authors carried out a seroepidemiological study in individuals living in two different geographic areas of Tuscany. A total of 130 serum samples were tested. Half of them were collected from people professionally in contact with ovines, the other half from urban people presumably not in contact with animals. The antibody response to C. burneti was tested by indirect immunofluorescence test (IIT), a method known to be sensitive, specific, practical and economic. Antibodies titers indicating previous infection (greater than 1:20) were more frequently found in subjects belonging to the rural group (49.1%) than in those belonging to the urban group (6.1%). The seropositive subjects presented a negative clinic anamnesis. These results suggest a persistent circulation of C. burneti in man, linked to contacts with sheep. Also there appear, however, to be a good number of unnotified or subclinic Q Fever forms. Young people appear to be slightly less frequently infected, which suggest that the infection may be in fact becoming rarer.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

[Direct demonstration of Coxiella burnetii from air samples on slides coated with kraton 1107].

Inactivated Coxiella burnetti (strain Henzerling) was introduced into a sealed chamber in aerosol form and recovered on Kraton 1107-coated slides with the aid of slit samplers. Following auramine staining, the coxiellae could be demonstrated directly by microscopy. Under the experimental conditions chosen, the limits of demonstrability were initial Coxiella contents of 2.26 X 10(3) in 14 L or 3.6 X 10(5) in 1 m3 of air.

Aerosols↗

[In vivo and in vitro growth of the TK-1 strain of Coxiella burnetii].

All A/J, BALB/c and C57BL/6 murine strains were resistant against the intraperitoneal infection with TK-1 strain, but an enhancement of susceptibility of mice were revealed by the administration of cyclophosphamide (CPA) in BALB/c and A/J strains. CPA-treated BALB/c mice allowed an increase of TK-1 strain up to 1.7 x 10(6) coxiellar particles/mg spleen. But athymic nude mice of BALB/c strain showed only a slight increase of coxiellar particles in spleen. The resident peritoneal macrophages from BALB/c and A/J, but C57BL/6, showed proliferation of the TK-1 strain in the large infected cell population, and a part of the infected macrophages allowed TK-1 strain to survive. On the other hand, the elicited peritoneal macrophages from resistant C57BL/6 showed the largest infected cell population, number of intracellular coxiellar particles, and following decrease of TK-1 strain in later stage of infection. These in vitro infectivity of TK-1 strain seemed to relate to the in vivo infectivity in mice, and indicated existence of macrophage subpopulation, in which destruction or proliferation of TK-1 strain occurred.

Animals↗

[The host reaction to the administration of different components of Coxiella burnetii].

After the intraperitoneal injection of corpuscles of C. burnetii antigen (Ag), phospholipid (PL), and sediment obtained after the extraction of PL from Ag with chloroform-methanol (CM) slight leukocytic reaction developed in the peritoneum on day 1, and on day 2 it could be observed in the liver and in the spleen. Ag induced the most pronounced morphological changes. In the spleen they were manifested by the activation of T- and B-dependent zones of white pulp from day 2 and by the pronounced hyperplasia of reticular cells and macrophages, leading to splenomegaly, by days 7-14. Simultaneously lymphoid-macrophagal granulomas and hepatomegaly developed in the liver. By days 7-14 the foci of necrosis in the liver were caused by the thrombosis of portal veins and were not registered after the injection of PL and CM (and earlier also in experiments with Ag in doses of 0.1-0.3 mg).

Animals↗

Predominant immunoglobulin A response to phase II antigen of Coxiella burnetii in acute Q fever.

Diagnosis of acute Q fever is usually confirmed by serology, on the basis of anti-phase II antigen immunoglobulin M (IgM) titers of >/=1:50 and IgG titers of >/=1:200. Phase I antibodies, especially IgG and IgA, are predominant in chronic forms of the disease. However, between January 1982 and June 1998, we observed anti-phase II antigen IgA titers of >/=1:200 as the sole or main antibody response in 10 of 1,034 (0.96%) patients with acute Q fever for whom information was available. In order to determine whether specific epidemiological or clinical factors were associated with these serological profiles, we conducted a retrospective case-control study that included completion of a standardized questionnaire, which was given to 40 matched controls who also suffered from acute Q fever. The mean age of patients with elevated phase II IgA titers was significantly higher than that usually observed for patients with acute Q fever (P = 0.026); the patients were also more likely than controls to live in rural areas (P = 0.026) and to have increased levels of transaminase in blood (P = 0.03). Elevated IgA titers are usually associated with chronic Q fever and are directed mainly at phase I antigens. Although the significance of our findings is unexplained, we herein emphasize the fact that IgA antibodies are not specific for chronic forms of Q fever and that they may occasionally be observed in patients with acute disease. Moreover, as such antibody profiles may not be determined by most laboratories, which test only for total antibody titers to phase I and II antigens, the three isotype-specific Ig titers should be determined as the first step in diagnosing Q fever.

Acute Disease↗

Molecular detection of Coxiella burnetii in the sera of patients with Q fever endocarditis or vascular infection.

In the absence of a specific diagnosis based on serology, chronic Q fever is inevitably fatal. However, diagnosis is often delayed because the test is not widely available. To shorten the diagnostic delay, we adapted a nested-PCR assay with serum as a template and the LightCycler as a thermal cycler, termed LCN-PCR. We retrospectively and prospectively applied this method to samples from 48 patients diagnosed with Q fever endocarditis or vascular infection and to samples from 100 controls with endocarditis caused by other microorganisms. We also prospectively applied this technique to samples from 30 patients treated for a Q fever endocarditis and to samples from 13 patients with a convalescent acute Q fever with ambiguous immunoglobulin G (IgG) phase I titer. LCN-PCR had a specificity of 100%. It was positive only in samples from patients with evolutive Q fever, as none of the samples from patients with a treated chronic Q fever or with a convalescent acute Q fever presented positive results. When performed prospectively on recently stored sera, the sensitivity of LCN-PCR is 64% (7 of 11 samples; P = 0.004), but the efficiency of LCN-PCR was dramatically altered by the storage of specimens at -20 degrees C. High IgG phase I titers decreased the sensitivity of LCN-PCR. A significant difference was observed among LCN-PCR results for sera with IgG phase I titers of > or =1:25,600 compared to sera with IgG phase I titers of <1:25,600 (0 of 15 samples versus 13 of 33 samples; P = 0.004). In patient samples with titers below 1:25,600 tested prospectively, sensitivity was 100% (7 of 7). The LCN-PCR assay may be helpful in establishing an early diagnosis of chronic Q fever.

Adult↗

[Contamination in areas of Slovakia with strains of Coxiella burnetii imported from abroad].

Following the upsurge of Q fever epidemic in the village Jedl'ové Kostol'any, Nitra district, Slovakia, in April 1993, the possibility of the penetration of coxiellae into free nature was investigated for the next two years. Micromammals and their ectoparasites, primarily ticks, were employed as indicators of the presence of the infection in nature. The infestation of mammals and their ectoparasites was followed in J. Kostol'any on the farm where the goats were housed as well as in its close proximity. The infestation of ticks with coxiellae was further followed in four localities of the Rimavská Sobota district, where the goats after the eradication of Q fever outbreak in J. Kostol'any were transferred for breeding. The presence of coxiellae was not proved in these investigated localities. It is suggested that the pathogenicity of imported C. burnetii strains through Bulgarian goats was decreased under local environmental conditions including the ability to circulate freely in nature or these strains found inconvenient conditions for maintaining their viability.

Animals↗

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)

Acute Disease↗