RESUMO
Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.
Assuntos
Bordetella pertussis/imunologia , Pentosiltransferases/metabolismo , Toxina Pertussis , Fatores de Virulência de Bordetella/metabolismo , ADP Ribose Transferases , Adjuvantes Imunológicos , Anafilaxia/etiologia , Animais , Antígenos/imunologia , Bordetella pertussis/enzimologia , Bordetella pertussis/genética , Códon , Tolerância a Medicamentos , Histamina/farmacologia , Imunização , Leucocitose/etiologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Ovalbumina/imunologia , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologiaRESUMO
We have demonstrated the safety, immunogenicity, and efficacy of the WC and CMR vaccines in guinea pigs. Vaccination of guinea pigs with either WC or CMR protects animals against challenge with virulent C. burnetii. A total of 2 micrograms of either WC or CMR vaccine was a significant priming dose. A total of 20 micrograms gave complete protection against lethal challenge. Detection of antibodies to phase II cells by microaglutination, after vaccination with either WC or CMR and before lethal challenge, correlated with the ability of guinea pigs to mount a protective immune response. The PD50 values for WC and CMR vaccines, administered as a single dose, were 0.3 and 1.4 micrograms per animal, respectively. In contrast, the PD50 values for the WC and CMR vaccines, administered as two doses, were 0.83 and 0.72 micrograms per animal, respectively. Although the PD50 values for the two vaccines are similar, the CMR vaccine is preferred over the WC vaccine because it induces significantly fewer adverse reactions, and repeat injections can be given. Unvaccinated guinea pigs do not clear infectious microorganisms after challenge infection. Vaccination before challenge infection reduces the infectious load of C. burnetti in the blood and in various organs of the animals. When vaccinated animals were challenge infected and treated with rifampicin, the microorganisms were not eliminated from various organs. However, the combination of vaccination, challenge, and rifampicin treatment is effective in reducing the number of infectious microorganisms in some of these sites. We have demonstrated the safety and immunogenicity of the CMR vaccine in sheep and goats. Animals that were seropositive for one or more antigens developed significant levels of antibodies to alternate antigens, but no adverse reactions were observed at the site of s.c. injection of the CMR vaccine. This demonstrates that seropositive animals can be successfully immunized with this vaccine. These results also indicate that a long-term vaccination program using the CMR vaccine has the potential for producing animals with significant antibody titers to C. burnetii and perhaps lifelong immunity. The goal of a Q fever vaccination program is to produce immunized animals that are able to clear completely the infectious microorganisms. The appropriate vaccination schedule to render adult animals and their offspring "Q fever-free" should now be thoroughly investigated.
Assuntos
Vacinas Bacterianas , Coxiella burnetii/imunologia , Febre Q/prevenção & controle , Animais , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Cabras , Cobaias , Camundongos , Ovinos , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologiaRESUMO
A strain of Rickettsia canada was recovered in 1980 an adult rabbit tick, Haemaphysalis leporispalustris, taken from a black-tailed jack rabbit, Lepus californicus, in Mendocino County, California. In all examined biologic characteristics, this isolate, CA410, is indistinguishable from the prototype, strain 2678, isolated in Ontario, Canada, in 1963. These similarities include serologic and immunologic reactivity in laboratory mice and guinea pigs, cultural characteristics in Vero cells, chick embryo cells and embryonated eggs, low pathogenicity for mice, meadow voles and guinea pigs, unusual resistance to streptomycin, morphology by electron microscopy, and molar percentages of guanine plus cytosine of the deoxyribonucleic acids. Recovery of this second strain in the same species of tick, but far removed in time and place from the origin of the prototype, provides evidence that R. canada is an established, ecologically stable, rickettsia in North America.
Assuntos
Infecções por Rickettsia/transmissão , Rickettsia/isolamento & purificação , Carrapatos/parasitologia , Animais , Arvicolinae , California , Embrião de Galinha , Cobaias , Insetos Vetores/parasitologia , Masculino , Coelhos , Rickettsia/patogenicidade , Rickettsia/ultraestrutura , Infecções por Rickettsia/epidemiologiaRESUMO
The ability of three isolates of serogroup 1 and one isolate of serogroup 4 of Legionnaires' disease bacterium (LDB) to infect and cause fever and death in guinea pigs was studied, as well as their ability to produce plaques in cultured primary chick embryo cells. The serogroup 4 isolate originally was recovered from cord clot and placental tissue from a healthy mother following delivery of a normal child. The effects on LDB of prolonged cultivation on supplemented Mueller-Hinton (MH) agar medium and of subsequent cultivation in yolk sacs of chick embryos were examined. Prolonged cultivation of LDB on MH medium resulted in great loss of ability to produce plaques and to cause fever and death in guinea pigs. Subsequent passage in embryonated eggs of MH-adapted LDB tended to restore ability to produce plaques and to cause infection and illness in guinea pigs. Fatty acid composition profiles of the four strains were similar to each other.
Assuntos
Legionella/patogenicidade , Animais , Embrião de Galinha , Ácidos Graxos/análise , Feminino , Cobaias , Humanos , Legionella/isolamento & purificação , GravidezRESUMO
Rocky Mountain spotted fever (RMSF) or ehrlichiosis was diagnosed in dogs on the basis of specific immunofluorescent testing for each disease. Comparisons between clinical and laboratory findings were made between the 2 diseases. The incidence of RMSF tended to be more seasonal and it affected younger dogs. Purebred dogs appeared to be more susceptible to both diseases. In general, RMSF had a more rapid and severe course of clinical illness than did ehrlichiosis, but acute ehrlichiosis was difficult to differentiate from RMSF. Both diseases were characterized by fever, depression, lymphadenopathy, and signs of neurologic dysfunction; petechial hemorrhages or other signs of hemorrhagic diathesis were evident only in a small proportion of cases. Anemia, leukopenia, and thrombocytopenia were more common in dogs with ehrlichiosis, whereas those with RMSF more often had leukocytosis and thrombocytopenia. Hypoalbuminemia was found in dogs with both diseases, but those with ehrlichiosis usually had concurrent hyperglobulinemia. High serum alkaline phosphatase activity and serum cholesterol concentration, and low serum calcium concentration were more common in dogs with RMSF than with ehrlichiosis. Rising serum titers or positive direct immunofluorescence for Rickettsia rickettsii in skin biopsy specimens were used to confirm RMSF, whereas a single serum titer for Ehrlichia canis enabled detection of ehrlichiosis. In the absence of neurologic deficits and when dogs were treated with tetracycline, dogs with RMSF made a more rapid and consistent recovery than did dogs with ehrlichiosis.
Assuntos
Doenças do Cão/microbiologia , Infecções por Rickettsiaceae/veterinária , Febre Maculosa das Montanhas Rochosas/veterinária , Animais , Diagnóstico Diferencial , Doenças do Cão/diagnóstico , Doenças do Cão/tratamento farmacológico , Cães , Ehrlichia , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/veterinária , Infecções por Rickettsiaceae/sangue , Infecções por Rickettsiaceae/diagnóstico , Infecções por Rickettsiaceae/tratamento farmacológico , Febre Maculosa das Montanhas Rochosas/sangue , Febre Maculosa das Montanhas Rochosas/diagnóstico , Febre Maculosa das Montanhas Rochosas/tratamento farmacológico , Tetraciclina/uso terapêuticoRESUMO
A small inoculum (0.2 microgram) of phase I Coxiella burnetii vaccine given to individuals previously sensitized to CO burnetii elicited a positive skin reaction and a strong IgM phase I antibody response as determined by microagglutination, complement fixation and microimmunofluorescence tests. A similar inoculum administered to nonsensitized individuals did not elitic a skin reaction nor stimulate a recognizable antibody response. Serum from one of these sensitized and skin tested individuals was fractionated by gel filtration methods. The serum and serum fractions were titrated in a mouse seroprotection test using primary chicken embryo cell culture plaque technique as the assay procedure. Results of the mouse seroprotection test indicated that most of the protective activity of the serum was associated with the IgM fraction and that phase I IgM antibody suppressed the growth of C. burnetii in mouse spleen when mixed with the rickettsial suspension prior to inoculation.
Assuntos
Anticorpos Antibacterianos/biossíntese , Vacinas Bacterianas/imunologia , Coxiella/imunologia , Adulto , Coxiella/citologia , Humanos , Imunoglobulina M/biossíntese , Injeções Intradérmicas , Masculino , Febre Q/imunologia , Testes CutâneosAssuntos
Infecções por Rickettsia/epidemiologia , Adolescente , Adulto , Idoso , Testes de Aglutinação , Anticorpos/análise , Antígenos , América Central , Criança , Pré-Escolar , Testes de Fixação de Complemento , Coxiella/imunologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Métodos , Pessoa de Meia-Idade , Febre Q/epidemiologia , Febre Q/imunologia , Rickettsia/imunologia , Infecções por Rickettsia/imunologia , Rickettsia prowazekii/imunologia , Rickettsia rickettsii/imunologia , Rickettsia typhi/imunologia , Tifo Epidêmico Transmitido por Piolhos/epidemiologia , Tifo Epidêmico Transmitido por Piolhos/imunologiaAssuntos
Reservatórios de Doenças , Rickettsia prowazekii , Ovinos , Tifo Epidêmico Transmitido por Piolhos/etiologia , Testes de Aglutinação , Animais , Anticorpos/análise , Encéfalo/microbiologia , Testes de Fixação de Complemento , Feminino , Cobaias , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Fígado/microbiologia , Pulmão/microbiologia , Linfonodos/microbiologia , Microscopia de Fluorescência , Testes de Neutralização , Rickettsia prowazekii/isolamento & purificação , Baço/microbiologia , Fatores de Tempo , Tifo Epidêmico Transmitido por Piolhos/imunologia , Tifo Epidêmico Transmitido por Piolhos/microbiologiaRESUMO
Pertussigen (pertussis toxin (PT] is one of the most effective stimulators of IgE production in mice and rats. Employing flow microfluorimetric analysis (FMF), we showed that PT increases the percentage of blood and spleen lymphocytes with IgE on their surface. The percentage of IgE-bearing cells in the spleen of normal untreated C57Bl/10SCN mice of various ages varied from 2.2 to 12.2%, with an average value of 6.1 +/- 5.4%. In mice treated with 400 ng of PT and 1 mg of chicken egg albumin (EA), the percentage of these cells increased, 14 days after immunization, to an average value of 31.1 +/- 2.2%. Immunization of mice with PT alone increase the percentage of IgE-bearing cells only slightly (13.1 +/- 2.2% of the splenic lymphocytes) while injection of 1 mg of EA alone did not have any detectable action. As little as 6 ng of PT, when given simultaneously with 1 mg of EA, increased the percentage of IgE-bearing lymphocytes. A booster dose of 10 micrograms of EA given on Day 14 induced a further increase in the percentage of these cells even when as little as 0.039 ng of PT had been given at the time of initial immunization. PT was effective when given 4 days before or 5 days after EA. EA was effective when given 4 days before or 4 days after PT, but not 8 days after. The increase in IgE-bearing cells was mainly due to cytophilic binding of IgE to receptors for the epsilon chain of IgE (Fc epsilon) on the surface of lymphocytes rather than to a greater number of IgE-producing cells. This was shown by removing the IgE from Fc epsilon receptors by acid treatment which reduced the percentage of IgE-bearing cells to nearly normal values. The antibodies of IgE class with specificity to EA were increased dramatically, while antibodies with specificity to PT were not detected.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Imunoglobulina E/metabolismo , Linfócitos/metabolismo , Toxina Pertussis , Receptores Fc/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Animais , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Receptores de IgE , Baço/citologiaRESUMO
Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.
Assuntos
Adjuvantes Imunológicos/farmacologia , Bordetella pertussis/imunologia , Toxina Pertussis , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/farmacologia , Adjuvantes Imunológicos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Infecções por Bordetella/imunologia , Infecções por Bordetella/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Hemaglutininas/farmacologia , Imunidade/efeitos dos fármacos , Masculino , Camundongos , Especificidade da Espécie , Vacinas de Produtos Inativados/imunologia , Fatores de Virulência de Bordetella/imunologiaRESUMO
The response of guinea pigs experimentally infected with Coxiella burnetii organisms, the etiologic agents of Q fever, was obtained by the measurement of fever, circulating infectious C. burnetii cells, and anti-C. burnetii antibodies. The detection of antibodies by the enzyme-linked immunosorbent assay (ELISA) and traditional methods against phase I whole cells, phase II whole cells, and phase I lipopolysaccharide (LPS-I) (a virulence marker for phase I cells) antigens in the serum samples of infected animals revealed marked differences between intrastrain phase variants. Animals infected with the phase I Nine Mile strain produced a concomitant increase in temperature, circulating infectious C. burnetii cells, and antibodies against phase II cells, phase I cells, and LPS-I. At 15 weeks, a challenge of phase I-infected animals with viable phase I cells resulted in anamnestic antibody responses to phase I cells and LPS-I but not to phase II cells. Infection of animals with the phase II Nine Mile strain produced antibodies against only phase II cells. The challenge of phase II-infected animals at 15 weeks with viable phase II cells resulted in anamnestic antibody responses to phase I and phase II cells but not to LPS-I. Suppression of anti-phase II responses by the phase I challenge was apparent with only the ELISA, because the immunofluorescence, microagglutination, and complement fixation assays were insensitive to these changes. The sensitivity and specificity of the ELISA with whole-cell and the LPS-I antigens in the detection of phase-specific antibody revealed that avirulent phase II cells induced an immune response to phase I antigenic epitopes. Although the avirulent phase II cells were rapidly cleared by the host immune responses, they were sufficiently infective to induce antibody responses to both phase variants. Thus, in the occurrence of Q fever, any conventional serological technique that uses only phase II antigens may not provide a true incidence of naturally acquired infection with both phase I and II C. burnetii organisms.
Assuntos
Anticorpos Antibacterianos/biossíntese , Coxiella/imunologia , Febre Q/imunologia , Testes de Aglutinação , Animais , Anticorpos Antibacterianos/análise , Antígenos de Bactérias/análise , Testes de Fixação de Complemento , Ensaio de Imunoadsorção Enzimática , Febre , Imunofluorescência , Cobaias , Memória Imunológica , Valor Preditivo dos TestesRESUMO
Q fever vaccines were tested in mixed breed dogs by vaccinating them with formalin-killed Coxiella burnetii whole cells (WC) in either phase I (WCI) or phase II (WCII), or the chloroform: methanol residue (CMR) subunit of phase I cells. Phase I vaccines mixed (1:1) with Freund's incomplete adjuvant (FIA) induced humoral immune responses to phases I and II antigens as measured by microagglutination assay. The CMR vaccine mixed (1:1) with FIA induced greater antigen-specific antibody levels to both phases I and II antigens than the corresponding WCI vaccine. The WCII vaccine induced antibody responses to only phase II antigens. The time course of erythema and induration after skin testing with C. burnetii antigens were suggestive of cell-mediated immunity (CMI). Although granulomas were observed with only WCI and WCII, none of the skin test antigens induced abscesses at the injection site. In contrast, axillary nodes draining the vaccine injection site developed sterile draining abscesses in all dogs by days 19 to 24 for the WCI and CMR, and day 104 for the WCII vaccines. The abscesses had resolved within 30 days after first appearance. Responses to Con A and PHA and recall antigens of lymphocytes from the blood, axillary and mesenteric nodes, and spleen at 222 days after vaccination were variable among dogs. Lymphocytes from various organs responded to one or more of the recall antigens and to both mitogens in the absence or presence of indomethacin. Although these Q fever vaccines induced humoral and CMI, either the antigens or FIA caused sterile draining abscesses.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Vacinas Bacterianas/administração & dosagem , Coxiella burnetii/imunologia , Doenças do Cão/prevenção & controle , Animais , Doenças do Cão/imunologia , Cães , Avaliação de Medicamentos , Feminino , Imunização/veterinária , Masculino , Testes CutâneosRESUMO
Sensitization of mice with 1 mg of bovine serum albumin (BSA) or chicken egg albumin (EA) given intraperitoneally and 300 to 400 ng of pertussigen (pertussis toxin [Ptx]) given intravenously (i.v.) induced a high degree of anaphylactic sensitivity when the mice were challenged i.v. with 1 mg of antigen 14 days later. Regardless of H-2 haplotype, all of the strains tested (CFW, BALB/cJ, DBA/2J, and C3H.SW/SnJ) were susceptible to anaphylaxis. Sensitization of mice by a multiple-dose procedure that has been reported to induce fatal encephalopathy in mice (L. Steinman, A. Weiss, N. Adelman, M. Lim, R. Zuniga, J. Oehlert, E. Hewlett, and S. Falkow, Proc. Natl. Acad. Sci. USA 82, 8733-8736, 1982) (1 mg of BSA on day -1, 100 to 400 ng of Ptx on day zero 1 mg of BSA on day +1, 100 to 400 ng of Ptx on day +2, and 1 mg of BSA on day +6) induced shock in BALB/cJ, DBA/2J, and C3H.SW/SnJ mice, but not in CFW mice. When EA was used instead of BSA, CFW, BALB/cJ, and C3H.SW/SnJ mice did not develop fatal shock, whereas DBA/2J mice did. When dose 3 of antigen (BSA or EA) was postponed to day +21, all mouse strains sensitized by the multiple-dose procedure were found to be susceptible to shock. The fatal shock induced by this procedure, as well as that induced by giving a single sensitizing dose of antigen and Ptx, could be prevented by one to three 1-ml doses of saline given i.v. at the time signs of severe shock appeared. Although only one dose of saline was often sufficient to save the mice, two or three doses were usually needed. Microscopic changes were not found in midsagittal sections of the brains of mice sensitized by either procedure. This was true of mice that died from shock or were saved from shock by injections of saline. From these results, we concluded that the proposed model for encephalopathy induced in mice by Ptx and BSA demonstrates only the well-known anaphylactogenic effect of Ptx or pertussis vaccine. Since there are many other more sensitive methods to detect Ptx, induction of anaphylaxis is not of much value for detection or quantitation of Ptx in pertussis vaccine.
Assuntos
Anafilaxia/imunologia , Encefalopatias/imunologia , Toxina Pertussis , Fatores de Virulência de Bordetella/imunologia , Animais , Volume Sanguíneo , Encefalopatias/patologia , Relação Dose-Resposta Imunológica , Antígenos H-2/imunologia , Camundongos , Camundongos Endogâmicos , Soroalbumina Bovina/imunologia , Fatores de TempoRESUMO
Acute infection with Coxiella burnetii usually results in a self-limited illness, but it can occasionally cause chronic endocarditis or hepatitis. Headache is a common presenting symptom of acute infection with this agent, but specific neurological abnormalities are rare. We report the case of a patient with acute Q fever that caused frank encephalitis. We also review the literature on central nervous system disease attributable to C. burnetii.
Assuntos
Encefalite/diagnóstico , Febre Q/diagnóstico , Adulto , Anticorpos Antibacterianos/análise , Confusão/etiologia , Doxiciclina/uso terapêutico , Encefalite/complicações , Encefalite/tratamento farmacológico , Cefaleia/etiologia , Humanos , Masculino , Febre Q/complicações , Febre Q/tratamento farmacológicoRESUMO
Antigenic fractions of Coxiella burnetii phase variants were identified with an enzyme-linked immunosorbent assay (ELISA). Immune sera from guinea pigs immunized with Formalin-inactivated phase I or phase II whole cells were used to measure the antigenic activity of whole cells and various soluble and particulate preparations. Phase-specific antigens of C. burnetii whole cells and fractions were compared by dose-response curves at different (antigen and antibody) dilutions. Water-soluble extracts prepared by meta-periodate, ether, and phenol extraction of phase I whole cells yielded antigenic fractions which reacted with anti-phase I antibodies. The extraction of phase I whole cells with dimethyl sulfoxide, trichloracetic acid, and Formalin yielded antigenic fractions which detected antibodies in both anti-phase I and -phase II sera. Interestingly, the trichloracetic acid extract of phase I whole cells also contained a component which bound nonimmune immunoglobulin. The sera of animals immunized with whole cells of the phase II Australian QD strain reacted with lipopolysaccharides of the phase I and phase II Nine Mile strains. Therefore, variations in lipopolysaccharide structure among phase variants of C. burnetii were detected as cross-reactions with immune sera from an interspecific strain. Comparisons of immunofluorescence, microagglutination, and the complement fixation assays with the ELISA indicated greater sensitivity and specificity of the ELISA for the measurement of phase-specific antigens and antibodies.
Assuntos
Antígenos de Bactérias/análise , Coxiella/imunologia , Animais , Anticorpos Antibacterianos/análise , Embrião de Galinha , Testes de Fixação de Complemento , Ensaio de Imunoadsorção Enzimática , Formaldeído , Cobaias , Camundongos , Valor Preditivo dos TestesRESUMO
The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Processamento de Proteína Pós-Traducional , Rickettsia/patogenicidade , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Western Blotting , Mapeamento Cromossômico , Eletroforese em Gel de Poliacrilamida , Temperatura Alta/efeitos adversos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Rickettsia/genética , Homologia de Sequência do Ácido NucleicoRESUMO
An enzyme-linked immunosorbent assay (ELISA) was developed to detect immunoglobulin G to Coxiella burnetii phase II. Serum samples from 213 patients who had had Q fever 1 year previously and from 301 blood donors from six localities in Switzerland were tested by ELISA and by indirect fluorescent-antibody (IFA) and complement fixation (CF) tests. The ELISA and the IFA and CF tests detected antibody to C. burnetii in 202 (94.8%), 193 (90.6%), and 166 (77.8%) of the 213 Q fever patients, respectively. With the serum samples from blood donors, the ELISA yielded a higher percentage of positive sera than did the IFA and CF tests. The high specificity of the three tests was confirmed by analyzing paired serum samples from 36 patients suffering from acute pneumonia of viral or bacterial origin. In these cases, the serological results were negative by the three tests, except for three Q-fever cases included as positive control.