Extraction of protective components of Rickettsia rickettsii with n-octyl-beta-D-glucopyranoside.

Two Rickettsia rickettsii proteins, with apparent molecular masses of 120 and 155 kilodaltons, have been implicated as important antigens in immunity to Rocky Mountain spotted fever. An n-octyl-beta-D-glucopyranoside-soluble fraction of R. rickettsii was found to be enriched for these proteins along with others. Vaccination of mice with the detergent-soluble fraction protected them against death caused by R. rickettsii and protected guinea pigs from developing Rocky Mountain spotted fever after being challenged with viable R. rickettsii.

Neutralizing activity of monoclonal antibodies to heat-sensitive and heat-resistant epitopes of Rickettsia rickettsii surface proteins.

Antiprotein monoclonal antibodies derived from mice inoculated with Rickettsia rickettsii heated at 56 degrees C for 15 min are of two types: one is type specific for epitopes denatured by moderate temperatures, and the other is specific for epitopes resistant to 100 degrees C for 5 min. The heat-resistant epitopes are found by immunoblotting on multiple polypeptides after solubilization of the rickettsiae at temperatures of 56 degrees C or higher. Most, but not all, antibodies to the heat-sensitive epitopes passively protected mice against two 50% lethal doses of R. rickettsii, whereas none of the antibodies to heat-resistant epitopes did.

Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae.

Analysis of 15 spotted fever group (SFG) and 2 typhus group strains of rickettsiae with a panel of monoclonal antibodies revealed a number of shared and unique epitopes of the 120- and 155-kilodalton surface proteins. All of the SFG strains but neither of the typhus group strains reacted with antibody to the lipopolysaccharidelike antigen of Rickettsia rickettsii; possibly the lipopolysaccharidelike antigen is the common antigen which defines the SFG. North Carolina and Montana strains of R. rickettsii known to differ slightly in virulence for guinea pigs differed in at least one epitope of the 120-kilodalton protein.

Cloned gene of Rickettsia rickettsii surface antigen: candidate vaccine for Rocky Mountain spotted fever.

Two major protective antigens of Rickettsia rickettsii have been previously described. In this study, we cloned the gene encoding one of these antigens into Escherichia coli and tested the effectiveness of the recombinant-made product as a vaccine for Rocky Mountain spotted fever. A clone bank of R strain R. rickettsii DNA was made in E. coli K-12 by using the plasmid vector pBR322. Transformants were screened for their ability to make rickettsial antigens by reactivity with rabbit antibodies to R. rickettsii. One of the transformants, EM24(pGAM21), made a product reactive with two monoclonal antibodies that recognize a 155-kilodalton protein of R. rickettsii. One of the monoclonal antibodies was a member of a class of antibodies that react to heat-sensitive epitopes and protect mice injected with a potentially lethal dose of viable R. rickettsii. The cloned product contained this protective heat-sensitive epitope. In order to obtain enhanced expression, the gene was subcloned downstream of the lactose promoter on the plasmid vector pUC8. A sonic lysate of E. coli harboring the pUC8 subclone was used successfully as a vaccine to protect mice injected with a lethal dose of the viable R. rickettsii.

Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies.

Previously it has been reported that strains of Rickettsia rickettsii that differ greatly in their ability to cause disease in guinea pigs are similar by serological and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. In this study, we used monoclonal antibodies to the virulent R and the relatively avirulent HLP strains to investigate strain differences which might account for the disparate behavior of the strains in guinea pigs. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the R and HLP strains were nearly identical for polypeptides with apparent molecular weights greater than 32 kilodaltons (kDa). All of the monoclonal antibodies to a lipopolysaccharide-like antigen reacted equally well with antigen from both strains by immunoblotting. None of the antibodies to the lipopolysaccharide-like antigen protected mice against challenge with viable rickettsiae. Some antibodies reacted with both 120- and 155-kDa polypeptides of both strains in radioimmune precipitation and immunoblotting tests, and other antibodies reacted only with the homologous strain. The monoclonal antibodies cross-reacted with the heterologous strain in the enzyme-linked immunosorbent assay essentially either completely or not at all. The ability of the monoclonal antibodies to the 120- and 155-kDa polypeptides to protect mice against the two strains was correlated with the ability of the antibodies to react with the antigens in the enzyme-linked immunosorbent assay and radioimmune precipitation or immunoblotting tests. These results demonstrate that R and HLP antigens which appear identical in molecular weight differ in their compositions of antigenic determinants.

Characterization of monoclonal antibodies protecting mice against Rickettsia rickettsii.

Hybridomas producing monoclonal antibodies to Rickettsia rickettsii were prepared from mice to investigate the function of rickettsial antigens. Of the 31 reactive hybridoma lines thus far tested for immunoglobulin subclasses, 11 belonged to the IgG2A subclass, 9 to the IgG2B subclass, and 7 to the IgG3 subclass; four did not react with any of the isotyping sera. Five of the antibodies recognized epitopes present on molecules that were presumed to be polysaccharide and heterogeneous in molecular weight. Twenty monoclonal antibodies reacted with a 170,000-dalton antigen, and six precipitated both the 133,000- and 32,000-dalton polypeptides. Only those antibodies to the 170,000- and 133,000-dalton antigens protected mice from challenge with R. rickettsii. Antibodies to these same antigens were detected in sera from patients convalescing from Rocky Mountain spotted fever. All monoclonal antibodies reacted with antigens apparently located on the rickettsial surface. The protective activity of these antibodies was not correlated with their reactivity in complement fixation, enzyme-linked immunosorbent assay, and immunofluorescence tests.

Biochemical and immunochemical analysis of Rickettsia rickettsii strains of various degrees of virulence.

Six strains of Rickettsia rickettsii from Montana and North Carolina were examined in an effort to identify rickettsial constituents associated with virulence. Fever responses, scrotal reactions, and mortalities of male guinea pigs inoculated intraperitoneally with 1,000 PFU of rickettsial strains revealed that the two Montana patient strains ( Sheila Smith and Norgaard ) and one Montana strain ( Sawtooth female 2) from the wood tick, Dermacentor andersoni, could be placed in the group of highest virulence, the two North Carolina strains (Morgan and Simpson) in the group of lesser virulence, and the Montana strain (HLP) from the rabbit tick, Haemaphysalis leporispalustris , in the group of lowest virulence. The HLP strain was differentiated from the other strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue or with silver. The patient strains could not be differentiated from each other by these procedures. All of the strains apparently had three heat-modifiable proteins. Analysis of proteinase K-digested rickettsial lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the strains had a complex mixture of polysaccharides. These putative polysaccharides probably were not related to the differences in virulence of the strains, since the patterns for all of the strains were identical. At least five antigens (molecular weights of 128,000, 105,000, 84,000, 30,500, and 20,500) were demonstrated by radioimmune precipitation tests employing extracts from radioiodine-labeled rickettsiae and antibodies from infected guinea pigs. With these same sera a minimum of 14 antigens was detected in these strains by an immunoblotting procedure. The apparent molecular weights of several of the HLP antigens differed from those of the presumed corresponding antigens of the other strains. The electrophoretic techniques utilized in this study were not sufficiently sensitive to demonstrate compositional differences in the patient strains which differed in their virulence for guinea pigs.

Evaluation of latex-Rickettsia rickettsii test for Rocky Mountain spotted fever in 11 laboratories.

A latex-Rickettsia rickettsii test for detection of antibodies to Rocky Mountain spotted fever (RMSF) was evaluated during the 1980 RMSF season in 11 laboratories in nine states where the disease is endemic. In a double-blind study, all sera submitted to each laboratory for RMSF testing were also examined by the latex-R. rickettsii test. A portion of each specimen was then sent to the New York State laboratory for testing by latex-R. rickettsii and by the reference microimmunofluorescence test. Results were exchanged at the end of the examination period. At the usual ratio of reactive to nonreactive sera encountered in a diagnostic laboratory on a day-to-day basis, the efficiency of the latex-R. rickettsii test relative to microimmunofluorescence was 96.79% for New York and 93.30% for the collaborating laboratories. Both the latex and microimmunofluorescence tests detected antibodies to RMSF within 7 to 9 days of onset. With the latex-R. rickettsii test--but not necessarily with microimmunofluorescence--a high titer (greater than or equal to 128) on a single serum was diagnostic of active RMSF. Changes in serum titer for patients with multiple sera were similar for both tests. The test detects rickettsial antibodies in patients with active infection, but in most cases it does not detect antibody in patients with past infection. Test reactivity could not be uniquely linked to a particular immunoglobulin class.

Detection of Rickettsia rickettsii antibodies in human sera by crossed immunoelectrophoresis.

To identify Rickettsia rickettsii antigens of immunological importance, we examined sera from patients with serologically confirmed cases of Rocky Mountain spotted fever by crossed immunoelectrophoresis for antibodies to antigens extracted from the R strain of R. rickettsii with the detergent Triton X-100. Sixteen antigens were identified in the detergent extract by crossed immunoelectrophoresis with a hyperimmune rabbit serum raised against whole rickettsiae. When the rabbit antiserum was placed in the reference gel and patient sera were placed in the intermediate gel, antibodies to one or more antigens were detected in 61 of 71 North Carolina sera, all of 7 Oklahoma sera, and 9 of 10 Montana sera obtained from 1 day to 40 years after onset of Rocky Mountain spotted fever. Antibodies to antigens 1 and 16 were found as early as 1 day after onset of illness, and antibody to 16 was found in 20 of 29 sera obtained within the first 7 days of illness. Antibodies to antigens 2 and 3 generally did not appear until the third week of illness but were found in six of seven serum samples collected 4 to 40 years after onset of Rocky Mountain spotted fever. Antibodies to R. rickettsii antigens 1, 7, 8, and 16 were found in sera from patients with illnesses caused by other etiological agents. Four of the Oklahoma and Montana sera from Rocky Mountain spotted fever patients, but none of the North Carolina sera, had antibodies to antigen 12. Sera containing antibodies against antigens 3 and 14 prevented death of mice challenged with two 50% lethal doses of R. rickettsii.

Biological properties of rabbit antibodies to a surface antigen of Rickettsia rickettsii.

Because of the potential significance of external components of the obligate intracellular parasite Rickettsia rickettsii in host-parasite interactions, we have begun the first phase of a study to isolate and characterize surface antigens of this organism. An antiserum to a rickettsial surface component was obtained from rabbits inoculated with immune precipitates prepared by crossed immunoelectrophoresis of Triton X-100 extracts of R. rickettsii strain R. This antiserum (i) protected guinea pigs inoculated with 10,000 guinea pig 50% infectious doses of R. rickettsii against fever, (ii) prevented death of mice challenged with 2 50% lethal doses of R. rickettsii, and (iii) reacted in the microimmunofluorescence test with 9 of 13 spotted fever group serotypes tested. The location of this antigen on the rickettsial surface was demonstrated by immunoelectron microscopy with ferritin-labeled antibodies.

Absorption of Rickettsia rickettsii antibodies by Rickettsia rickettsii antigens in four diagnostic tests.

Immunoabsorption profiles were determined for Rickettsia rickettsii antigens used in four confirmatory tests for detection of antibodies to Rocky Mountain spotted fever. A human serum reactive in the four tests was absorbed with each test antigen and then reexamined by all four tests. The results indicated that the whole organism and complement-fixation antigen absorbed the whole array of antibodies to R. rickettsii, whereas erythrocyte-sensitizing substance coated on latex or erythrocytes did not. With erythrocyte-sensitizing substance coated on latex there was a decrease in titer when tested by the latex, indirect hemagglutination, and complement fixation tests but none when tested by the microimmunofluorescence test. With erythrocyte-sensitizing substance coated on sheep erythrocytes there was a decrease in titer when tested by indirect hemagglutination or complement fixation but none when tested by latex or microimmunofluorescence. These findings confirm that erythrocyte-sensitizing substance contains more than a single antigenic substance and plays a major role in the complement fixation test and a minor role in the microimmunofluorescence test.

Coating of polymeric surfaces for immunoassay by a forced adsorption technique.

A forced adsorption technique for coating polymeric surfaces is described. This technique is simple and allows us to use (i) relatively impure ligands to be coated on latex surface and (ii) the minimum amount of ligands for preparation of large-volume lots of latex reagent. The serologic results are highly reproducible from lot to lot of the reagent, in contrast to the variability found with passively adsorbed reagents. Use of this technique could be extended to coat flat surfaces for solid-phase immunoassays.

Identification of an isolate of Rickettsia canada from California.

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.

Detection of Rocky Mountain spotted fever antibodies by a latex agglutination test.

A latex test for immunodiagnosis of Rocky Mountain spotted fever, using erythrocyte-sensitizing substance from Rickettsia rickettsii adsorbed to latex particles, has been developed. The test was evaluated with a total of 123 single and 118 paired human sera submitted for Rocky Mount spotted fever testing. This test is simple, sensitive, and specific. Its efficiency, relative to the reference microimmunofluorescence test, was 95.1% for single sera and approached 100% for paired sera.

Properties of selected rickettsiae of the spotted fever group.

Eight strains of spotted fever group rickettsiae were studied to gain insight into the extent of variation of their properties. Two standard strains of Rickettsia rickettsii and one strain of Rickettsia conorii were included among the eight for comparison. The molar percentage of guanine plus cytosine for each strain did not differ significantly from that for R. rickettsii, 32.6 +/- 0.7%. Two strains caused extended fever in guinea pigs, one strain caused fever of short duration, and the other strains induced little or no fever. Polyacrylamide gel electrophoresis of the detergent-solubilized rickettsial proteins indicated that the protein content of all strains, except the two strains of R. rickettsii, were different, particularly in the molecular weight range of 40,000 to 60,000. Virulent strains produced large clear plaques in Vero cells monolayers; the strains of low virulence generally produced smaller or more turbid, or both, plaques. On the basis of agglutination reactions with rabbit antisera, the eight strains were placed into five serotypes. These results indicate considerable heterogeneity in properties of spotted fever group rickettsiae in the United States.

Indirect hemagglutination test for detection of antibody to Rickettsia rickettsii in sera from humans and common laboratory animals.

Antibody production in humans and three species of laboratory animals infected with Rickettsia rickettsii was determined with the indirect hemagglutination test. Rabbits, guinea pigs, and mice were inoculated with R. rickettsii and bled at intervals. Antibody which agglutinated both fresh and glutaraldehyde-fixed sheep erythrocytes sensitized with antigen prepared either from purified rickettsiae or from infected yolk sacs was found in rabbit sera at all intervals tested (10 to 59 days postinfection). Antibody which agglutinated fresh but not glutaraldehyde-fixed erythrocytes sensitized with either of the above antigens was detected in guinea pig sera obtained 7, 14, and 28 days postinfection. Antibody was found in mice inoculated with 5.6 x 10(6) plaque-forming units of R. rickettsii but not in mice given 5.6 x 10(2) plaque-forming units. Peak indirect hemagglutination titers occurred in nonvaccinated human Rocky Mountain spotted fever patients about 3 weeks after onset of illness, and antibody was still detectable after 1 year. Both human immunoglobulin G and human immunoglobulin M antibodies agglutinated sensitized cells, but immunoglobulin M antibodies apparently were more efficient. The indirect hemagglutination test is useful for the titration of human, rabbit, guinea pig, and mouse antibodies when the appropriate erythrocytes are used.

A comparison of serologic methods for diagnosis of Rocky Mountain spotted fever.

Sera referred to the North Carolina Division of Health Services for rickettsial serology in 1974 were tested by complement fixation (CF), microimmunofluorescence (micro-IF), microagglutination (MA) and hemagglutination (HA) for antibodies against Rickettsia rickettsii. There was good agreement among micro-IF,MA and HA tests in detecting antibody responses to this agent, but the CF test was definitely less sensitive than the others, even in illnesses with classical clinical manifestations of Rocky Mountain spotted fever (RMSF). Some variables that seemed to influence the CF result were the slow rate of increase in antibody titers, timing of serum collection, early antibiotic treatment and possibly, the particular association of CF antibody response with the IgG immunoliobulin class. Greater use of these newer, but relatively untried, serodiagnostic procedures is recommended infuture studies of RMSF.

New assay of protective activity of Rocky Mountain spotted fever vaccines.

Areas under the fever curves of guinea pigs inoculated with Rocky Mountain spotted fever vaccine over a restricted dose range and infected with a standardized dose of Rickettsia rickettsii varied linearly with log10 dose of vaccine. A calculator was programmed to plot fever curves and calculate the vaccine dose that reduced the fever of infected animals by 50%.

Rhipicephalus sanguineus: vector of a new spotted fever group rickettsia in the United States.

A rickettsia related to but distinct from the spotted fever agent, Rickettsia rickettsii, has been detected in 167 (18.9%) of 884 Rhipicephalus sanguineus taken off dogs in central and northern Mississippi. The organisms could readily be isolated in male meadow voles (Microtus pennsylvanicus), where it produced massive infections in the tissues of tunica vaginalis. It was practically nonpathogenic for male guinea pigs, although inoculation of these animals with infected tunica vaginalis of voles afforded in 30 of 38 instances solid immunity to challenge with virulent R. rickettsii. The Rhipicephalus rickettsia grew well in monolayers of chicken embryo fibroblast, Vero, mouse L, and HeLa cells. Cytopathogenic effects were minimal unless large concentrations of rickettsiae were used as inocula. It also could be established in embryonated hen eggs but only after injection of massive doses of L cell-propagated organisms. Serological tests (complement fixation, microagglutination and/or micro immunofluorescence) indicated that the newly described Rickettsia belongs to the spotted fever group but differs from R. rickettsii, R. akari, and R. conorii. Antigenic differences were also demonstrated by direct fluorescence microscopy as well as by vaccine potency and mouse-toxin neutralization tests.