Antigenic classification of Rickettsia felis by using monoclonal and polyclonal antibodies

Rickettsia felis is a flea-transmitted rickettsia. There is a discrepancy between its reported phylogenic and phenotypic identifications. Following the first report of R. felis, it was considered by tests with serologic reagents to be closely related to another recognized flea-transmitted rickettia, R. typhi. Subsequently, it appeared to be more closely related to spotted fever group (SFG) rickettsiae by genetic analysis. In the present work, R. felis was studied by microimmunofluorescence (MIF) serologic typing and with monoclonal antibodies (MAbs). Mouse polyclonal antisera to R. felis cross-reacted only with SFG rickettsiae. A neighbor-joining analysis based on MIF indicated that R. felis is actually related to SFG rickettsiae antigenically, clustering with R. australis, R. akari, and R. montanensis. A panel of 21 MAbs was raised against a 120-kDa protein antigen or a 17-kDa polypeptide of R. felis. They cross-reacted with most members of the SFG rickettsiae but not with R. prowazekii, R. typhi, or R. canadensis of the typhus group (TG) rickettsiae. Sixty-four MAbs previously generated to seven other ricketttsial species were tested with R. felis. Three MAbs reacted with the 120-kDa antigen and were generated by R. africae, R. conorii, and R. akari, respectively. They exhibited cross-reactivities with R. felis. All our data show that R. felis harbors the antigenic profile of an SFG rickettsia.     

Serological classification by monoclonal antibodies of Rickettsia tsutsugamushi isolated in Korea

Antigenic types of 113 strains of Rickettsia tsutsugamushi isolated from Korean patients were analyzed by using murine polyclonal and monoclonal antibodies. The isolates can be classified in six groups according to their reaction to a panel of monoclonal antibodies. Nine isolates of group I were identified as the Gilliam serotype, and 13 isolates of groups II and III were identified as the Karp serotype. There were two groups that were considered to be a mixture of groups I and II or groups I and III, respectively. The remaining 88 strains of group IV had a unique antigenic determinant that was not present in the prototype strains (Karp, Kato, Gilliam), in addition to sharing common antigens with the prototype strains. Therefore these strains, which are more prevalent in Korea, seem to belong to a new serotype closely related to the Karp serotype.     

Sensitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies Against Typhus Rickettsiae, Rickettsia prowazekii and Rickettsia typhi

An enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of rickettsial antibodies in human and animal sera. Two preparations of soluble typhus-group antigens were obtained from Rickettsia typhi and Rickettsia prowazekii by ether extraction: a standard antigen from infected yolk sacs (YS antigen) and one free of yolk sac contaminants from Renografin-purified rickettsiae (PR antigen). Rabbit, mouse, and guinea pig sera were obtained by immunization with viable purified R. typhi or R. prowazekii. Human sera were obtained from individuals who had recovered from laboratory infections with either typhus rickettsia months or years previously. Goat-derived anti-immunoglobulins were conjugated to alkaline phosphatase with glutaraldehyde. Although the PR and YS antigens gave equivalent antibody titers in the complement fixation test, the PR antigen was clearly superior in the ELISA. With this antigen, the titration curves of all antisera were linear over a wider range of serum concentrations and the titers were higher than with the YS antigen. With YS and PR antigens, ELISA titers were higher than those obtained by complement fixation by one and two orders of magnitude, respectively. In human sera, immunoglobulin G and immunoglobulin M antibodies were demonstrated by their respective anti-immunoglobulins and by differential susceptibility to ethanethiol. ELISA titers showed some type specificity, whereas none was observed in complement fixation tests. The ELISA is highly sensitive, reproducible, and easily adaptable to the various requirements of clinical and research laboratories.     

Detection of Neisseria gonorrhoeae by dot-enzyme immunoassay using monoclonal antibodies

A highly sensitive and specific dot-enzyme immunoassay for the detection of Neisseria gonorrhoeae was developed using a pool of monoclonal antibodies (MAbs). The MAbs were obtained following immunization of mice with lithium acetate extracted outer membrane (OM) preparations. Western immunoblotting experiments demonstrated that MAbs NG26 and NG38, both IgG2a, reacted with lipopolysaccharides (LPS) and with the major OM protein, P1, respectively, MAb NG28, an IgG3, did not react in Western immunoblotting, MAbs NG28 and NG38 failed to react with OM treated with proteolytic enzymes or with semi-purified preparation of LPS. MAb NG26 reacted with the same LPS preparation. Binding radioimmunoassay with live bacteria showed that all the MAbs adsorbed to cell surface-exposed antigenic determinants. The limit of detection of the dot-enzyme immunoassay was between 1 and 4 x 10(4) cfu per dot. Using a panel of 177 strains of N. gonorrhoeae, MAbs NG28 and NG38 recognized only P1A and P1B strains respectively. MAb NG26 reacted with P1A, P1B and non-typable strains. These MAbs did not react with other Neisseria species or other bacterial species. Using this pool, the dot-enzyme immunoassay had a sensitivity of 93.2% and a specificity of 100%.     

Production of monoclonal antibodies against Rickettsia massiliae and their use in antigenic and epidemiological studies.

Rickettsiae are gram-negative, obligate intracellular bacteria which have historically been divided into three groups: the typhus group, the scrub typhus group, and the spotted fever group (SFG). Recently, several new SFG rickettsiae have been characterized, and most of these species are associated with ticks and have, as yet, no known pathogenicity toward humans. Rickettsia massiliae, which is widely distributed in Europe and Africa, is one such rickettsia. In order to investigate the antigenic relationships between R. massiliae and other rickettsial species and to develop a more convenient methodology for identifying R. massiliae, we produced monoclonal antibodies against the type strain (Mtu1T) of R. massiliae by fusing immunized splenocytes with SP2/0-Ag14 myeloma cells. A panel of 16 representatives were selected from the 163 positive hybridomas identified on initial screening, and their secreted monoclonal antibodies were further characterized. The reactivities of these 16 monoclonal antibodies with a large panel of rickettsial species were assessed by the microimmunofluorescence assay. All species of the SFG rickettsiae reacted with the monoclonal antibodies directed against epitopes on lipopolysaccharide, which is the common antigen among the SFG rickettsiae. Some closely related species of the SFG, such as Bar29, "R. aeschlimanni," and R. rhipicephali, showed strong cross-reactivities with the monoclonal antibodies directed against epitopes on the two major high-molecular-mass heat-labile proteins (106 and 120 kDa). In addition, species-specific monoclonal antibodies demonstrated that R. massiliae is antigenically different from other rickettsial species. Moreover, these species-specific monoclonal antibodies were successfully used for identifying R. massiliae in the ticks collected from southern France, and are therefore potentially useful tools in the identification and investigation of R. massiliae in ticks in large-scale field work.     

Production and characterization of monoclonal antibodies to Rickettsia rickettsii

Five mouse ascitic fluids (MAFs) containing monoclonal antibody to Rickettsia rickettsii were produced from three original fusions by murine hybridoma technology. The five MAFs were fractionated and purified; each contained monoclonal antibody of the immunoglobulin G2a subclass. Each monoclonal antibody-containing MAF was titrated by indirect immunofluorescence against three R. rickettsii isolates from humans and four other spotted fever group rickettsiae. Each MAF was also titrated in the complement fixation, latex agglutination, microagglutination, and indirect hemagglutination tests. Two of the MAFs were examined for their ability to prevent fever and rickettsemia in susceptible guinea pigs after a 1:100 dilution of each was mixed with viable R. rickettsii, and all five MAFs were titrated in the mouse toxicity phenomenon assay. All MAFs had high indirect immunofluorescence titers to the three strains of R. rickettsii (1:200,000 to 1:800,000), reduced indirect immunofluorescence titers to R. montana, and were nonreactive with R. akari, R. sibirica, and R. conorii. Each MAF was able to fix complement in the presence of spotted fever group antigen reagent and agglutinate a suspension of purified R. rickettsii, and each was negative in both the latex agglutination and the indirect hemagglutination tests. The two MAFs which were tested proved to be capable of preventing rickettsemia and death in guinea pigs, and each MAF was able to prevent death in mice at dilutions ranging from 1:40 to 1:80.     

Analysis of antigenic characteristics of Rickettsia tsutsugamushi Boryong strain and antigenic heterogeneity of Rickettsia tsutsugamushi using monoclonal antibodies.

Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.     

用16S rRNA探针定量测定普氏立克次体在细胞内的繁殖量

目的以普氏立克次体为研究对象，建立一种定量测定胞内微生物繁殖的方法。方法从立克次体感染细胞提取总ＲＮＡ为样品或用硫氰酸胍裂解感染细胞，以裂解物为样品，用３２Ｐ标记的１６ＳｒＲＮＡ特异探针做ＲＮＡ酶保护分析。根据探针分子结合数，计算出检测到的靶序列分子数，反映胞内微生物的繁殖量。结果从０．５μｌ直接裂解物可检测到３．９４×１０４个１６ＳｒＲＮＡ分子；从６ｐｇ的总ＲＮＡ中可检测到５．８２×１０４个１６ＳｒＲＮＡ分子。用本法试测立克次体在宿主细胞内的生长曲线，只表现出迟缓期及对数生长期，而无稳定期及衰退期。结论用特异性探针通过ＲＮＡ酶保护分析法可以定量测定胞内微生物在宿主细胞内的繁殖量。由本法测定到的普氏立克次体生长曲线表明难以沿用细菌的生长曲线来描述胞内微生物在宿主细胞内的生长过程     

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.     

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.     

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.     

Peculiarities of Rickettsia prowazekii in the cell culture as revealed by cryoultramicrotomy

Ultrastructure of Rickettsia prowazekii has been followed in L-929 cells 4 days post-infection (p.i.) by cryoultramicrotomy. Groups of rickettsiae were present in the cytoplasm outside of vacuoles forming microcolonies. The size of rickettsiae amounted to 400 X 700 nm, the average thickness of the cell wall was 5 nm, that of periplasmic space and cytoplasmic membrane 14 and 6 nm, respectively. Within intracytoplasmic colonies the rickettsiae were tightly packed and their cell walls were closely adjacent to each other. No halo or capsule-like coating around them was detected. No ultrastructural details were observed in the light translucent spaces between cells. Marginal rickettsiae of the microcolonies were often in close contact with the host cell mitochondria.     

Multiplication of Rickettsia prowazekii in cotton rat macrophage cultures

The susceptibility of cotton rat macrophages to Rickettsia (R.) prowazekii, the percentage of the affected cells, and the intensity of damage to individual cells by rickettsiae were found to be much higher than those in guinea pig macrophages infected under similar conditions. At the same time, cotton rat macrophages proved to be more resistant to the effect of rickettsiae than guinea pig macrophages. Some common features of infection in cell culture and in animals have been observed. It is suggested that the outcome of interaction of rickettsiae with macrophages of one or another animal species may be important in generating acute or persistent infection.     

Cytokine sensitivity and methylation of lysine in Rickettsia prowazekii EVir and interferon-resistant R. prowazekii strains.

Modified Rickettsia prowazekii strains have been derived from the avirulent Madrid E strain by passage in the lungs of white mice (strain EVir) or by selection for resistance to gamma interferon (IFN-gamma) (strains 427-19 and 87-17) or alpha/beta interferon (IFN-alpha/beta) (strains 83-2P, 60P, 103-2P, and 110-1P). Compared with the Madrid E strain, strain EVir has increased virulence (N. M. Balayeva and V. N. Nikolskaya, J. Hyg. Epidemiol. Microbiol. Immunol. 17:11-20, 1973) and a different lysine methylation profile in its surface protein antigen (A. V. Rodionov, M. E. Eremeeva, and N. M. Balayeva, Acta Virol. 35:557-565, 1991). The other six strains differ from the Madrid E strain in their resistance to IFN and their ability to grow well in untreated macrophagelike RAW264.7 cells. In the present study, to determine which properties are shared by these strains, we examined R. prowazekii EVir for the following: (i) the sensitivity of its growth in L929 cells to the cytokines IFN-alpha/beta, IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma plus TNF-alpha; (ii) the ability to grow in untreated RAW264.7 cells; and (iii) the ability to induce interferon in L929 cell cultures; we also evaluated strains 83-2P and 87-17 for lysine methylation. Multiplication of strain EVir in growing L929 cells was not markedly inhibited by either IFN-alpha/beta or IFN-gamma. In X-irradiated L929 cells, growth of strain EVir was slightly inhibited (11%) by TNF-alpha alone, somewhat inhibited (38%) by IFN-gamma alone, and markedly inhibited (87%) by IFN-gamma plus TNF-alpha. Nitrite production was induced in X-irradiated, strain EVir-infected L929 cell cultures treated with TNF-alpha alone or IFN-gamma alone; however, more nitrite was produced in infected cultures treated with IFN-gamma plus TNF-alpha. Nitrite production, the dramatic inhibitory effect of IFN-gamma plus TNF-alpha, and the modest inhibitory effect of IFN-gamma on the growth of strain EVir in X-irradiated L929 cells were all alleviated by the addition of the nitric oxide synthase inhibitor NG-methyl-L-arginine. Strain EVir grew very well in untreated macrophagelike RAW264.7 cells and appeared defective in the ability to induce IFN in L929 cell cultures. All strains grown in L929 cells in the presence of radiolabeled lysine had similar percentages of their radioactivity as methylated lysines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.     

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.     

A century of typhus, lice and Rickettsia

At the beginning of the 20th century, it was discovered at the Pasteur Institute in Tunis that epidemic typhus is transmitted by the human body louse. The complete genome sequence of its causative agent, Rickettsia prowazekii, was determined at Uppsala University in Sweden at the end of the century. In this mini-review, we discuss insights gained from the genome sequence of this fascinating and deadly organism.