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Virulence of four Coxiella burnettii phase I strains associated with acute (Nine Mile and Luga strains) and chronic (S and Priscilla strains) forms of Q fever, respectively, for outbred guinea pigs and inbred BALB/c mice was compared. In guinea pigs exposed to infectious aerosol, virulence of the strains Nine Mile, Luga and S was similar, because they all caused lethal infection and high febrile reaction (FR). By contrast, exposure to similar doses of the strain Priscilla resulted in the non-lethal infection manifested only by low, dispersed and protracted FR during a 20 day-period of observation. Lower virulence of the strain Priscilla than of the strains Nine Mile and S was observed also in guinea pigs infected intraperitoneally (ip). In mice, comparable degree of multiplication in the spleen on day 6 post ip infection was observed with the strains Nine Mile, Luga and S, but not with the strain Priscilla, namely when lower infectious doses were used. Whereas the highest doses (10(7) EID50) of the strains Nine Mile and S were lethal for mice during the second week post infection (p.i.), the strains Luga and Priscilla did not kill mice with 10-times lower (10(6) EID50) doses. At the same time agglutination antibody response detected 4 weeks p.i. was very similar in guinea pigs with all strains under study and in mice it did not differ markedly, reflecting the infectious dose used.

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In the years 1984-1988 we observed, besides the routine examinations of history, clinical state, and laboratory tests, also the presence of antibodies to Coxiella burnetii antigens by means of microagglutination reaction (MAR). The examinations were performed in our citizens returning to Czecho-Slovakia from a long termed business stay in the developing countries. Antibodies to C. burnetii antigen 2 have been detected entirely in 91 serum samples, out of which 48 cases following their stay in Libya, 42 cases following their stay in Iraq, and 1 in Syria. Diagnostically significant antibody titres (64) were detected in 44 serum samples, in which the serological positivity was proved by complement fixation test (CFT) examination. Clinical symptoms in the history, responding to the acute form of "Q" fever, occurred in 44 serologically positive cases, 37 of which developed high antibody titres, which is considered to be a significantly high incidence. The results imply the necessity to become more concerned by the incidence of "Q" fever, its diagnostics and therapy, eventually prevention in our citizens working in a more exacting climate of some developing countries. (Tab. 1, Ref. 15.).

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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.

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Prevention of rickettsial infections is aimed at individual control and epidemic measures (especially in epidemic typhus), vector and rodent control, milk pasteurization (in Q fever), chemoprophylaxis and immunoprophylaxis. In vector and rodent control, the main obstacle is the rise in resistance to insecticides and rodenticides. For this reason in vector control, apart from insecticides, enhancement of the natural immunity acquired by animals in response to tick infestation and vaccination with concealed tick antigens as well as the use of hormones, chemosterilants and genetic manipulation can also be considered. For short-term high-risk exposure, doxycycline may be an effective prophylaxis of illness but may not prevent infection with scrub typhus or spotted fever group rickettsiae. At present, for specific prevention by vaccination, only Q fever vaccines are available for common use. However, development of subunit vaccines, namely immunogenic rickettsial proteins, cloned and expressed in Escherichia coli, seems to be very promising.

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Cellular immunity against phase I and II Coxiella burnetii antigens was evaluated by the lymphocyte blast transformation test in a) Q fever convalescents; b) persons immunized with a chemovaccine against Q fever; and c) control persons. The first two groups consisted of workers in a ricketsiology department. Of the 9 convalescents, all (100%) were positive against phase I antigen and 4 (44.4%) against phase II antigen. Of the 22 vaccinees, 77.3% were positive against phase I antigen and all were negative against phase II antigen. All 10 controls were negative against both antigens.

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Endocarditis is a rather frequent complication of Q fever caused by Coxiella burnetii. We examined the ability of phase I (virulent) or phase II (avirulent) C. burnetii to coagulate blood in the presence of human blood mononuclear cells in vitro. After incubation for 4 h, virulent phase I C. burnetii was an effective stimulant for mononuclear cells. Since this interaction is a potent trigger of blood coagulation through the extrinsic pathway, it could be responsible for the local deposition of fibrin on the surface of infected valves and the development of large vegetations in cases of endocarditis complicating Q fever.

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Electrophoresis in polyacrylamide gel gradients in the presence of sodium dodecyl sulphate revealed at least 18 identical protein bands in eight strains of Coxiella burnetii. By staining with Coomassie Brilliant Blue R-250 it was possible to visualize clearly at least 40 proteins. The protein pattern showed the greatest variability in the region from 27 to 18.5 kD. Strains S and Priscilla differed from the other strains also in proteins smaller than 18.5 kD. The lipopolysaccharide pattern obtained by the same technique consisted of from 6 to 12 various bands in the region from 23.5 to 11.8 kD. The protein and lipopolysaccharide patterns of four strains isolated from ticks did not significantly differ from those of strains Henzerling and L-35 isolated from men.

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Optimal conditions of extraction (time and temperature) by trichloroacetic acid of soluble antigen from phase I Coxiella burnetii (TCAE), possessing protective properties and used as a chemovaccine against Q fever in men, were studied. Extracts prepared under various conditions were analysed for their polysaccharide, protein and phosphorus contents. Forty-five min of extraction at 0 degrees C were sufficient to obtain a soluble antigen reacting in immunodiffusion with hyperimmune rabbit antiserum. The polysaccharide contents decreased with prolonged extraction at 0 degrees C. At higher extraction temperatures (37 and 100 degrees C), the polysaccharide contents increased while that of proteins decreased. TCAE prepared at 100 degrees C gave no positive immunodiffusion reaction.

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We compared the sensitivity of indirect hemagglutination and indirect hemolysis tests by detection of antibodies in sera of man, rabbits and sheep after infection with R. slovaca, R. sibirica and C. burnetii. We found that the titres of antibodies in indirect hemolysis are 4 times higher than in indirect hemagglutination namely with the sera of man, rabbits and sheep. This method can be recommended for diagnoses of rickettsiae of Spotted fever group.

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Two hundred forty-nine cases of Q fever were documented at the laboratories of the Centro Nacional de Microbiología, Virología e Inmunología Sanitarias (CNMVIS) during the 5-year period 1981-1985. Two hundred thirty-four cases corresponded to acute infections, mostly sporadic but including two epidemics. The clinical presentation was respiratory in 74% of the cases and febrile in 18%. Fifteen cases, all but one of which were endocarditis, were categorized as chronic. The cases studied were referred from almost every region of Spain. The clinical and epidemiologic analyses and the number of cases reported permit only an approximation of the true incidence and characteristics of Q fever in Spain.

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Different concentrations of chemically-treated (by potassium periodate oxidation or mild acid hydrolysis) purified phase I Coxiella burnetii (C.b.) corpuscles and natural (untreated) purified phase I and phase II C.b. corpuscles were compared by ELISA for detection of both phase I (directed to antigen 1) and phase II (directed to antigen 2) antibodies in human Q fever convalescent sera. As to the absorbance values the most sensitive was the antigen obtained by mild acid hydrolysis (0.1 mol/l HCl of phase I corpuscles for 30 min at 100 degrees C) followed by phase I corpuscular antigen (treated with 0.01 mol/l potassium periodate for 4 hr at 45 degrees C). The natural phase I (the 3rd egg passage) and phase II (the 162th egg passage) C.b. corpuscles gave lower absorbance with some sera not even distinguishing the 6th egg passage (EP6 or EP3 in phase I) from EP 162 or from negative controls.

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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.

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Common isolation procedures on chick embryos and laboratory animals are not of great importance for routine diagnosis of rickettsioses. Detection of rickettsiae in skin lesions by immunofluorescence technique allows early diagnosis of Rocky Mountain spotted fever (RMSF). Broad spectrum of methods is at disposal for serological diagnosis of rickettsial diseases. Their choice is determined by laboratory equipment, professionality of laboratory staff, economy and simplicity of the given test. Though complement-fixation and microagglutination tests held their position and will certainly be used in future, the use of indirect immunofluorescence test is recommended for its sensitivity and simplicity. Latex agglutination test is valuable especially in the diagnosis of acute rickettsial infections. Recently introduced ELISA method is expected to fulfil the highest requirements as to sensitivity in differentiation of rickettsioses within the known classification groups. The efforts to obtain efficient antirickettsial vaccines have been limited to preparation of the vaccines against RMSF and Q fever. As to the latter, elaboration of chemovaccine and preparation of chloroform-methanol-treated phase I C. burnetii suspension of decreased reactogenity seem promising in field trials.

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Antibodies were detected to C oxiella burnetii, Rickettsia slovaca and Chlamydia psittaci in the blood of small terrestrial mammals, larger wild and domestic animals and humans in the southern part of the Protected Landscape Area of the Sumava region between 1977 and 1982. The data obtained have contributed to the safe development of livestock raising in this region.

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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.

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The skin test (ST) with Q fever chemovaccine revealed more positive reactors than the serological examination by microagglutination (MA) test among humans, who had suffered from Q fever one to eleven years ago or who had been vaccinated with Q fever chemovaccine from three months to four years ago. When examining the sera harvested either at skin-testing or two weeks thereafter by MA test, seroconversion or rise in antibody titres were found to both main Coxiella burnetii (C. b.) antigens. The rise or appearance of antibody response was similar in Q fever convalescents and vaccinees, in the latter occurring more often in ST positive than in ST negative individuals. Results of ST at different postvaccination (p.v.) intervals corresponded well to those of lymphocyte transformation (LT) test, but not to those of inhibition of leukocyte migration ( ILM ) test.

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Two strains of Chlamydia were isolated in McCoy cell cultures and hens' yolk sacs from urethral scrapings of men suffering from "nonspecific" urethritis. Their identification as Chlamydia trachomatis was based on cytoplasmic inclusions staining with iodine and on indirect immunofluorescence with anti-LGV serum. Both tests were performed in McCoy cells.

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Lipopolysaccharides (LPSs) isolated from Coxiella burnetii cells in phase I and pure phase II behaved as antigens reacting in serological tests (passive haemolysis, passive haemolysis inhibition, complement-fixation, immunoprecipitation) with sera of rabbits immunized with C. burnetii strains in the corresponding phase. No cross-reactions were observed between LPSs isolated from C. burnetii in phase I and phase II, respectively.

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