Experimental forms of infection and serological analysis of the antigenic structure of Rickettsia canada

Experimental forms of Rickettsia canada infection were characterized and serological analysis of the antigenic structure of R. canada was carried out. According to its pathogenicity for experimental animals, R. canada can be characterized as a poorly virulent species of rickettsiae, similar to R. prowazekii (for guinea pigs). The complement-fixing, haemagglutinating and agglutinating antigens of R. canada are fairly similar to those of the typhus group rickettsiae. The region of antigenic activity common to or identical in R. canada and the typhus group rickettsiae, is larger in R. canada than in the typhus group rickettsiae. R. canada has common antigens with Proteus OX19. R. canada has active toxic substances similar to those of R. prowazekii which, however, are detectable only with sera of Brill's disease convalescents. The position of R. canada in the taxonomy of rickettsiae is discussed.     

Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.     

Plaque Assay of Rickettsiae in a Mammalian Cell Line

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.     

Characteristics of recombinant fragments of the protective antigen SPA of epidemic typhus pathogens

Fragments of a gene for species-specific protective antigen SPA of Rickettsia prowazekii earlier cloned in lambda gt11 were recloned into the in-frame expression vector pQE30. Polypeptides encoded by these fragments were shown to be synthesized in Escherichia coli with a yield of up to 100 micrograms/ml of culture and to be accumulated in the cells as inclusion bodies. The partially purified antigens were used in enzyme immunoassay with the sera of humans convalescing from epidemic typhus, tick-borne rickettsioses, and other infectious diseases. One of two recombinant proteins was shown to react in immunoblotting and ELISA with homologous, but not with heterologous, sera. The immunoreactivities in ELISA of the recombinant antigens and heat-denatured SPA proved to be similar, but substantially lower than that of the native SPA. These data as well as the data of other investigators show that serodiagnostics of epidemic typhus using recombinant antigens remains a problem.     

Detection of antibodies to Rickketsia prowazekii by using the antigen neutralization test]

The authors studied a possibility of using the antigen neutralization test with dry immunoglobulin typhus erythrocytic diagnostic agent for the purpose of detection of Rickettsia prowazeki antibodies. Blood sera of 315 healthy persons, 24 patients with sporadic typhus, and 18 laboratory animals immunized with R. sibirica and R. burneti, as well as with Proteus OX19 were examined. The results obtained pointed to the high specificity and sensitivity of the given serological test. A possibility of its use for antibody detection both in the typhus patients and in persons who sustained this infection in the past was demonstrated. In difference from the complement fixation test it permits to study anticomplementary sera.     

Characterization of recombinant fragments of the protective antigen SPA of epidemic typhus agent

Fragments of a gene for species-specific protective antigen SPA ofRickettsia prowazekii earlier cloned in λgt11 were recloned into the in-frame expression vector pQE30. Polypeptides encoded by these fragments were shown to be synthesized inEscherichia coli with a yield of up to 100 μg/ml of culture and to be accumulated in the cells as inclusion bodies. The partially purified antigens were used in enzyme immunoassay with the sera of humans convalescing from epidemic typhus, tick-borne rickettsioses, and other infectious diseases. One of two recombinant proteins was shown to react in immunoblotting and ELISA with homologous, but not with heterologous, sera. The immunoreactivities in ELISA of the recombinant antigens and heat-denatured SPA proved to be similar, but substantially lower than that of the native SPA. These data as well as the data of other investigators show that serodiagnostics of epidemic typhus using recombinant antigens remains a problem.     

Murine and epidemic typhus rickettsiae: how close is their relationship?

Typhus fever has occurred globally as epidemic and endemic disorders. In 1910, Brill reported a typhus-like illness which Zinsser and others determined to be recurrent epidemic typhus fever. Maxcy, in 1926, proposed rodents and fleas as reservoir and vector, respectively, of endemic typhus, which Dyer confirmed in 1930. Animals experimentally infected with epidemic typhus (Rickettsia prowazeki) are immune to murine typhus (Rickettsia typhi) and vice versa. Similar solid cross-immunity exists for humans. The two diseases are clinically similar in pathologic and serologic reactions. Human epidemic typhus presumably involved a man-louse-man cycle without an animal reservoir. This concept is now questioned. Antibodies to R. prowazeki have been reported in livestock in Africa, rats in Manila, and from flying squirrels and humans in the United States. R. prowazeki was recovered from blood specimens of goats, sheep, from ixodid ticks, louse, and flea-ectoparasites of flying squirrels, and tissues of flying squirrels. More than 20 cases of squirrel-related acute epidemic typhus have been reported in the United States. R. prowazeki has not been recovered from human cases. Chemical studies of R. prowazeki and R. typhi show genetic similarities but differences in genome size and degree of hybridization suggest that interconversions between the two agents do not occur rapidly in nature. It is proposed that, with time, their relatedness will become even closer.     

Separation of antigens from Sarcocystis species using chromatofocusing

Crude antigen preparations from bradyzoites of Sarcocystis species exhibit a high degree of cross-reactivity with antisera against heterologous Sarcocystis species, preventing the development of a species-specific immunological test for sarcocystiosis. In this study, we fractionated bradyzoite-derived protein extracts from Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, and Sarcocystis muris by chromatofocusing and obtained distinct protein elution profiles for each species. We then examined the isolated protein fractions for antigenicity with homologous and heterologous reference sera in an enzyme-linked immunosorbent assay. Whereas some antigenic fractions of bradyzoite proteins had equally high reactivity with the homologous and heterologous sera, the reactivity of other fractions was 3-38 times higher with homologous serum than with heterologous sera. Mice immunized with less cross-reactive protein fractions of S. gigantea and S. muris bradyzoites produced a specific immune serum. Thus, it is possible to isolate species-specific antigens from crude mixtures of bradyzoite-derived Sarcocystis antigens for development of species-specific immunological tests for sarcocystiosis.     

Experimental infection of domestic animals with R. prowazeki and R. canada]

The authors infected lambs with R. prowazeki and R. canada to ascertain their possible role in the natural infection of the animals. The lambs were infected subcutaneously with increasing doses; rickettsiemia was recorded with the aid of tests on guinea pigs and Ixodidae and Argasidae ticks fed on the lambs. Dynamics of antibody formation was ascertained in the infected animals in the agglutination reaction and in the complement fixation test. The antigenic affinity of R. canada and rickettsia of the typhoid group and the presence of common antigenic determinants with the Proteus OX19 was confirmed. The absence of any clinical manifestations, the character of antibody formation, impossibility of inducing the generalized infection and of the isolation of the causative agent from the blood pointed to the low susceptibility of lambs to R. prowazeki and R. canada; thus a possibility of circulation of the causative agents of typhius among the domestic animals scarcely probable.     

Serological demonstration of the detection of tick-borne rickettsial disease in Astrakhan Province]

Starting from 1978, noncontagious febrile diseases of unclear etiology, accompanied by pronounced headache, roseolous-papular eruptions, prolonged convalescence period, are registered in May-September in Astrakhan Province. These diseases can be effectively treated with chrolamphenicol. In 11 out of 12 sera obtained from such patients the complement fixation test with the antigens of rickettsiae causing tick-borne spotted fever, epidemic typhus, as well as Coxiella burnetii antigen, revealed the presence of antibodies (in 8 sera) only to the antigens of rickettsiae causing tick-borne spotted fever (R. akari, R. conorii, R. sibirica), or the titers of antibodies to these antigens were greater (1 serum), equal and lower (2 sera) in comparison with those of the antigens of rickettsiae causing epidemic typhus. The dynamics and values of antibody titers in 7 patients with the antigens of three rickettsial species of the tick-transmitted biotype indicated that the disease was related to tick-borne spotted fever.     

Species-specific BALB/c mouse antibodies to rickettsiae studied by Western blotting

Abstract BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.     

Staining of rickettsial antigens for microagglutination reaction.

Staining of antigens of R. prowazeki, R. canada and R. conori with hematoxylin-Harris for use in MAR was developed. Optimal results were obtained when rickettsial antigens were stained at 4 degrees C for 18 hours. Comparison of MAR with CFR has shown that MAR using stained rickettsial antigens is specific and as sensitive as CFR.     

Antigenic and cross-protection studies on two turbot scuticociliate isolates

The protection induced in turbot by inactivated vaccines containing either of two isolates (I(1) and C(1)) of the scuticociliate parasite Philasterides dicentrarchi, which causes important mortalities in turbot cultures, was evaluated in the present study. The results obtained after challenging the fish with the two isolates show that vaccination protected fish only against the homologous isolate, but did not confer cross-protection. The two isolates constitute two serotypes, as shown in the immobilization tests with mouse and turbot anti-I(1) and anti-C(1) antisera, in which only the homologous antisera immobilized the ciliates. ELISA assays, using total antigen free of proteases (TAWP), cytosolic antigens (CYA), ciliar antigens (CA) or membrane protein fraction (MPF), were also carried out. Differences in the levels of antibodies produced in mouse against the homologous and heterologous antigens were observed; these differences were significantly different when the antigen preparations used in the ELISA were TAWP, CYA or CA. Nevertheless, ELISA assays using turbot sera against TAWP did not show significant differences in the levels of antibodies against the homologous and heterologous antigens. Antigenic cross-reactivity was also detected in the Western blot assays, as well as significant differences in the patterns of antigenic recognition in the two isolates - in both reduced and non-reduced TAWP antigens, but which was noteworthy when mouse antisera were used. The results obtained in the present study demonstrate for the first time the existence of serotypes of the ciliate parasite of turbot Philasterides dicentrarchi that display clear antigenic differences, which must be taken into consideration in the future development of a vaccine against scuticociliatosis.     

Serological evidence of typhus group rickettsia in a homeless population in Houston, Texas

We tested sera from 176 homeless people in Houston for antibodies against typhus group rickettsiae (TGR). Sera from 19 homeless people were reactive to TGR antigens by ELISA and IFA. Two people had antibodies against Rickettsia prowazekii (epidemic typhus) and the remaining 17 had antibodies against Rickettsia typhi (murine typhus).     

Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), Hope E. Hopps, Michael F. Barile, and Barbara C. Bernheim. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90:1387-1404. 1965.-In an effort to make a valid comparison of the ultrastructure of several intracellular parasites, selected agents were propagated under identical conditions in a single type of tissue culture cell; such infected preparations were processed for examination by electron microscopy by use of a standardized procedure for fixation and embedding. The organisms studied were: the Breinl and E strains of epidemic typhus, Rickettsia prowazeki; the Bitterroot strain of R. rickettsii; the Karp strain of R. tsutsugamushi (R. orientalis); R. sennetsu; the P-4 strain of ornithosis virus; and the HEp-2 strain of Mycoplasma hominis type I. Each of the rickettsial species examined had a cell wall and a plasma membrane, and contained ribosomes and deoxyribonucleic acid (DNA) in a ground substance. However, certain differences were noted. Both strains of R. prowazeki contained numerous intracytoplasmic electron-lucent spherical structures (4 to 10 mmu), not previously described. R. sennetsu, unlike the other rickettsiae, was not free in the host cytoplasm but was always enclosed in a vacuole. R. rickettsii was observed intranuclearly and in digestive organelles of the host cell as well as in the cytoplasm. Cells infected with ornithosis virus contained several forms representing the stages in its life cycle. The "initial bodies," made up of ribosomes and DNA strands, were morphologically similar to the rickettsiae. In cultures infected with M. hominis, most of the cells became large and multinucleate. Although the Mycoplasma organisms were readily cultivated from these cultures, only a few could be found in the electron microscope preparations. These organisms were extracellular and lacked a cell wall, being bound only by a unit membrane. Again, the internal components were ribosomes and DNA strands. Under the uniform preparative conditions employed here, the three groups of organisms were morphologically distinguishable from one another.     

Isolation of a New Soluble Antigen from the Yeast Phase of Histoplasma capsulatum

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.     

Seroepidemiology of spotted fever group and typhus group rickettsioses in humans, South Korea

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1: 40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill-Zinsser disease was prevalent in Korea.     

Effect of thimerosal on the whole yeast phase antigen of Histoplasma capsulatum

Summary Thimerosal (merthiolate) and formalin treated whole-cell yeast phase antigens ofHistoplasma capsulatum were prepared and their reactivities with sera from cases of histoplasmosis, blastomycosis and coccidioidomycosis were compared. Thimerosal treated antigens often gave complement fixation titers with heterologous sera 2 to 8 fold lower than the titers obtained with formalin treated antigens. However, with certain anti-histoplasmosis sera, thimerosal killed antigens had less reactivity with homologous antisera also. In virtually all cases an equal or higher specificity ratio was obtained with thimerosal killed antigens. The effects of thimerosal and formalin were independent, indicating different sites of reactivity of these reagents. Uptake of thimerosal at several concentrations suggested two types of reactions with live yeast phase cells. Analyses of the cellular fractions for thimerosal showed it was present only in the soluble fractions from which it was readily removed by dialysis. Cellular fractions killed with thimerosal retained several of the same physical and antigenic characteristics of those fractions isolated from frozen and thawed cells.