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Journal of Clinical Microbiology, April 1998, p. 887-896, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Taxonomic Relationships among Spotted Fever Group Rickettsiae as Revealed by Antigenic Analysis with Monoclonal Antibodies

Wenbin Xu and Didier Raoult*

Unité des Rickettsies, CNRS UPRES-A 6020, Faculté de Médecine, Université de la Mediterranée, 13385 Marseille Cédex 5, France

Received 17 November 1997/Returned for modification 16 December 1997/Accepted 14 January 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

The spotted fever group (SFG) is made up of more than 20 different rickettsial species and strains. Study of the taxonomic relationships among the group has been attempted by phenotypic, genotypic, and phylogenetic analyses. In this study, we determined taxonomic relationships among the SFG rickettsiae by comparative analysis of immunogenic epitopes reactive against a panel of monoclonal antibodies. A total of 98 monoclonal antibodies, which were directed against epitopes on the major immunodominant proteins or on the lipopolysaccharide-like antigens of strains of Rickettsia africae, Rickettsia conorii, Rickettsia massiliae, Rickettsia akari, Rickettsia sibirica, and Rickettsia slovaca, were used in the study. The distribution and expression of the epitopes among 29 SFG rickettsiae and Rickettsia bellii were assessed by determination of reaction titers in a microimmunofluorescence assay. The results were scored as numerical taxonomic data, and cluster analysis was used to construct a dendrogram. The architecture of this dendrogram was consistent with previous taxonomic studies, and the implications of this and other findings are discussed.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

Spotted fever group (SFG) rickettsiae are obligate intracellular, gram-negative bacteria which maintain a life cycle in mammalian cells or arthropods (65). Over 20 different globally distributed species (12, 62) have now been described, and new species continue to be recognized in different geographical regions (7, 9, 13, 38).

The strict intracellular lifestyle of the rickettsiae dictates their fastidious nature in vitro, and thus they cannot be characterized by the physiological and biochemical methods usually applied to axenically cultivatible bacteria (63, 64). Furthermore, production of the amount of cell biomass prerequisite for other phenotypic and genotypic characterization methods is impractical (34, 52). Thus, current taxonomic studies of rickettsiae have been based on the comparative analyses of their gene sequences, following their amplification by PCR. To date, these phylogenetic studies have been based on comparisons of sequences of the 16S rRNA-encoding gene (49, 56), the citrate synthase-encoding gene (gltA) (51), or the rickettsia outer membrane protein A (rOmpA)-encoding gene (ompA) (24, 50). Theoretically, phylogenetic relationships among the rickettsiae derived from sequence comparisons of antigenic protein-encoding genes, such as ompA, should be compatible with taxonomies derived from direct comparison of antigenic differences and similarities between the species.

Historically, the differentiation of rickettsial species has been carried out on the basis of serological analysis, and several methods have been described (16, 42, 43, 54). In 1978, Philip et al. developed the microimmunofluorescence (micro-IF) assay, in which the reciprocal reaction titers of antimouse polyclonal sera were determined to serologically type different rickettsial species (41). This technique permitted the concurrent comparison of a number of new isolates with reference strains through calculation of the specificity differences (41). Furthermore, antisera were produced by an immunization protocol of short duration, which reduced the titer of antibodies against the group-reactive lipopolysaccharide (LPS)-like antigens of the rickettsiae. This method remains regarded as the "gold standard" for identifying new SFG rickettsiae (9, 13, 69).

Rickettsiae express both LPS-like and protein antigens. The LPS-like antigens are group specific and thus cannot be used to differentiate among the SFG rickettsiae (3, 36, 59, 67, 68). Antigenic differences among species can, however, be elucidated by comparison of their protein composition. Comparison of protein profiles following sodium dodecyl sulfate-polyacrylamide gel electrophoresis has demonstrated marked differences among the high-molecular-mass proteins (3, 8), and the two immunodominant proteins, designated rOmpA and rOmpB (27, 28), lie within this range. These proteins both induce a strong humoral response in the immunized animals and appear to exist on almost all SFG rickettsial species (1, 27, 36, 68). The characterization of monoclonal antibodies against three different SFG rickettsial species (Rickettsia africae, R. conorii, and R. massiliae) demonstrated that their rOmpA and rOmpB proteins were more immunodominant than either the LPS-like or other protein antigens (66-68). Furthermore, these two proteins were found to express a large number of different specific immunogenic epitopes, as demonstrated by their different distributions among the SFG rickettsiae (66-68). None of these immunogenic epitopes were expressed on the typhus group rickettsiae. Further analyses of the distribution of these epitopes revealed that SFG rickettsiae which shared close phylogenetic relationships also shared more common epitopes with each other than were shared between more distantly related species, suggesting that evolutionary homology was reflected in phenotypic similarity (66, 68).

In this study, in an attempt to assess the taxonomic relationships among the SFG rickettsiae by using immunogenic criteria, we comprehensively investigated the distribution and expression of a number of epitopes recognized by a large panel of monoclonal antibodies raised against 6 SFG rickettsial species among 29 members of the SFG rickettsiae by using a micro-IF assay. Results were used to measure antigenic variation between strains and to infer taxonomic relationships from this variation.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

Rickettsial strains. Details of the 29 SFG rickettsiae and Rickettsia bellii used in this study are presented in Table 1.

                              
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  		TABLE 1.   SFG rickettsiae used in the study

Preparation of rickettsial antigens. All rickettsia strains were cultivated on L929 cell monolayers (ATCC CCL 1 NCTC clone 929) at 32°C supplemented with Earle's minimal essential medium (Eurobio, Les Ulis, France) containing 4% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Ltd., Paisley, Scotland) and 2 mM L-glutamine (GIBCO BRL) (66). Heavily infected cells, as monitored by Gimenez staining (29), were harvested with sterile glass beads and stored in aliquots at -80°C. These unpurified infected L929 cells were used as antigens in the micro-IF assay.

Monoclonal antibodies. A total of 98 monoclonal antibodies were used in this study. The monoclonal antibodies designated with the prefixes AF, RC, MA, AK, RS, and SV were raised against R. africae Z9-Hu, R. conorii Seven, R. massiliae Mtu1, Rickettsia akari Kaplan, Rickettsia sibirica 232, and Rickettsia slovaca 13-B, respectively. The monoclonal antibodies against R. akari Kaplan, R. sibirica Netsvetaev, and R. slovaca 13-B were kindly provided by D. H. Walker. The production and characterization of monoclonal antibodies against R. africae, R. conorii, and R. massiliae, through the fusing of immunized splenocytes with SP2/O myelomas, have been described in our previous studies (66-68).

Among the 83 monoclonal antibodies produced against R. africae, R. conorii, and R. massiliae, 76 (91.6%) were directed against the two major immunodominant proteins (21 against the rOmpA protein and 55 against the rOmpB protein), 6 (7.2%) were directed against the LPS-like antigen, and 1 (MA1-D2), which was directed against R. massiliae (67), could not be identified. The five anti-R. akari monoclonal antibodies are directed against the outer membrane protein (rOmp). The specificities of 10 monoclonal antibodies against R. sibirica and R. slovaca were not determined.

In this study, hybridoma culture supernatants were collected as the sources of monoclonal antibodies, with the exception of those raised against R. akari, R. sibirica, and R. slovaca. Cultures with high antibody concentration were obtained as follows. Hybridomas were grown in hybridoma medium (Seromed, Berlin, Germany) supplemented with 20% FBS (GIBCO BRL) at 37°C with 5% CO2. When the cell density of hybridomas reached saturation (approximately 5 × 105 to 5 × 106 cells per ml), cells were removed from the culture media by centrifugation at 400 × g for 5 min. Five percent of the cells from the pellets were resuspended in the centrifugation supernatant and were reinoculated as described above. Hybridomas were allowed to grow to saturation until death, and their culture supernatant was collected by centrifugation at 800 × g for 10 min at 4°C and then stored in aliquots at -20°C until required.

Micro-IF assay. Infected L929 cells were used as antigens and were aliquoted into each well of the 24-well microscope slides with a pen nib as follows. Four different rickettsia-infected L929 cells were applied to different positions on one well. Eight wells in the same line were pointed with the same 4 rickettsiae so that one slide contained eight spots of 12 different rickettsiae.

After air drying, the antigens on slides were fixed in acetone for 20 min at room temperature. Slides were either used immediately or were stored hermetically sealed at -20°C until required. The micro-IF assay was carried out as described previously (41, 66). Briefly, each slide was overlaid with twofold-diluted hybridoma culture supernatant at concentrations ranging from 1:4 to 1:512 and then was incubated in a humidified chamber at 37°C for 30 min. After three 3-min washes in phosphate-buffered saline, the slides were air dried and then overlaid with the dichlorotriazinyl amino fluorescein-conjugated goat anti-mouse immunoglobulin G and immunoglobulin M (heavy and light chains; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) diluted 1:200 in phosphate-buffered saline containing 0.2% Evans blue (BioMérieux, Marcy l'Etoile, France). The slides were incubated and washed as described above. Dried slides were mounted with Fluoprep (BioMérieux) and examined with a Zeiss epifluorescent microscope (Axioskop 20, Carl Zeiss, Göttingen, Germany) at a ×400 magnification. The endpoint value of highest dilution at which the organisms of certain rickettsial species could still be observed was recorded as the reaction titer. If the reaction titer of the monoclonal antibodies was higher than 1:512, a micro-IF assay with twofold dilutions of hybridoma supernatant from 1:512 was performed until the endpoints were obtained.

Numerical taxonomic analysis. The specific reaction titer of each monoclonal antibody with each SFG rickettsia was scored in terms of the level of expression of a specific epitope. Thus, strong expression was correlated to a higher reaction titer, and weaker or no expression was inferred from a lower reaction titer or a negative result. The score of each epitope expression in each rickettsia was designated as being (log2 T - 1), where T is the reaction titer of the rickettsia with the corresponding monoclonal antibody. Accordingly, the reaction titers of 1:4, 1:8, 1:16, etc., were scored as 1, 2, 3, etc., respectively, and a negative result is scored as 0. All rickettsiae tested therefore accumulated 98 different scores which reflected the different expression of all epitopes. Jaccard coefficients (SJ) for each rickettsial pair were obtained based on their reactivities as follows: SJ = a / (a + b), where a was the number of positive matches and b was the number of negative matches (53, 68). These scores and SJ similarity were used to construct a matrix, and then a dendrogram was constructed from the matrix by the unweighted pair group method with arithmetic mean (UPGMA) available in the PC-TAXAN software package (Sea Grant College, University of Maryland, College Park) according to the manufacturer's instructions. The SJ similarity was used as a measure of the taxonomic relationships between the SFG rickettsiae in a dendrogram tree (53).

A phylogenetic tree inferred from sequence alignment of SFG rickettsial ompA, as described previously (24, 44, 50), was compared with the dendrogram we obtained.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Reactivities of monoclonal antibodies. The reactivities of all 98 monoclonal antibodies with the 29 SFG rickettsiae and R. bellii are presented in Table 2.

                              
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  		TABLE 2.   Expression of immunogenic epitopes of R. africae, R. conorii, R. massiliae, R. akari, R. sibirica, and R. slovaca among 29 SFG rickettsiae and R. bellii

As expected, monoclonal antibodies reactive with the LPS-like antigens of R. conorii and R. massiliae cross-reacted with all SFG rickettsial strains but not R. bellii (67, 68). The anti-R. sibirica monoclonal antibody RS4-C4 and anti-R. slovaca monoclonal antibodies SV2-C21 and SV2-C22 reacted in the same manner and are therefore also likely to be directed against LPS, which constitutes the SFG-specific antigen (3, 36, 67, 68). Because all anti-LPS monoclonal antibodies yield the same reactivity pattern against the SFG rickettsia, we do not know if each was directed against a different group-specific epitope or if all are reacting with a single immunodominant epitope. One monoclonal antibody raised against R. massiliae (MA1-D2) also reacted with the non-SFG rickettsial species, including R. bellii, R. prowazekii, R. typhi, and R. canada (67), confirming that immunogenic rickettsial epitopes are shared beyond the group level.

Previous studies have demonstrated that none of the monoclonal antibodies directed against the major outer membrane proteins of R. africae, R. conorii, R. massiliae, and R. rickettsii reacted with Rickettsia helvetica, R. akari, and Rickettsia australis (3, 66-68). Because the remaining anti-R. sibirica and anti-R. slovaca monoclonal antibodies also failed to react against these species, they too are likely to be directed against epitopes on the immunogenic outer membrane proteins (Table 2). The major outer membrane proteins of R. africae, R. conorii, R. sibirica, and R. slovaca possessed epitopes which were shared by all SFG rickettsial species studied, except R. helvetica, R. akari, and R. australis. However, the major outer membrane protein epitopes of R. massiliae had a far more limited distribution, existing on only six other species. Furthermore, of the five monoclonal antibodies raised against R. akari, only two cross-reacted with other species, and these two reacted only with R. australis (Table 2).

The monoclonal antibodies raised against the major outer membrane proteins of R. africae, R. conorii, R. sibirica, and R. slovaca yielded 55 different cross-reactivity patterns with the other SFG rickettsiae, and we considered this to indicate that 55 different epitopes were being recognized. Of the 13 raised against R. massiliae, 6 yielded unique profiles, whereas two profiles were shared by two monoclonal antibodies, and one profile was shared by 3 monoclonal antibodies (Table 2). Again, shared profiles may signify that the different monoclonal antibodies were directed against the same epitope. Of the five monoclonal antibodies raised against R. akari, three were species specific and two reacted only with R. akari and R. australis. As before, we could not assume that these monoclonal antibodies recognized more than two epitopes.

Different expression of the immunogenic epitopes. The levels of expression of the reactive epitopes among the SFG rickettsiae were investigated by determining their reaction titer against each monoclonal antibody in the micro-IF assay. The monoclonal antibodies directed against the LPS-like antigen yielded the same reaction titers among all of the SFG rickettsiae (Table 2), indicating that the LPS-like antigens were not only distributed widely but were also expressed to an equal degree among the SFG rickettsiae. Conversely, for the monoclonal antibodies directed against the major outer membrane proteins, highly variable reaction titers were observed, indicating that some epitopes, although present on numerous species, were immunogically expressed to markedly different degrees. The basis of these differences is not clear, although the differences may result from variation in the epitope itself or from different levels of hindrance exerted by neighboring structures.

Western immunoblotting of electrophoretically resolved proteins has previously been used to confirm that all anti-protein monoclonal antibodies are directed against either rOmpA or rOmpB protein epitopes (1, 2, 36, 66-68). However, comparison of the reactivities of anti-rOmpA and anti-rOmpB monoclonal antibodies demonstrates general differences between them (1, 3, 66-68). The SFG rickettsia rOmpA protein expressed strain-specific epitopes, whereas the rOmpB protein expressed species- and subgroup-specific epitopes (Table 2) (67, 68). Furthermore, the epitopes recognized on the rOmpB protein tended to be expressed by more of the SFG rickettsial species than those on the rOmpA (68). However, when making such generalizations, it must be remembered that the epitopes we detected may represent only a tiny sample of those which exist, and thus they may not be truly representative of the antigenicity of the protein as a whole.

Numerical analysis of data. Pairwise comparisons of SFG rickettsial reactivity with the large monoclonal antibody panel and derived SJ similarity values based on epitope expression are presented in Table 3. The two R. massiliae strains tested exhibited indistinguishable expression of all of the epitopes and therefore shared 100% SJ similarity. Previous studies have also demonstrated that these two isolates were antigenically and genotypically indistinguishable (6, 49, 67). R. conorii Manuel differed from both the Kenya tick typhus rickettsia and the Seven strains at only one epitope, and thus each pair was scored as sharing 97.6% SJ similarities. Some of the other rickettsia pairs, such as R. conorii Seven-Kenya tick typhus rickettsiae, R. conorii Indian tick typhus rickettsia-Kenya tick typhus rickettsiae, R. conorii M-1-Kenya tick typhus rickettsiae, R. conorii M-1-Moroccan, R. africae Z9-Hu-Ethiopian, R. sibirica-BJ90, and BJ90-"R. mongolotimonae," also showed very high levels of SJ similarities. Similarities between most of the SFG rickettsiae and R. helvetica, R. akari, and R. australis were derived solely from anti-LPS monoclonal antibodies, and thus SJ values were very low (Table 3). R. bellii reacted weakly with only one monoclonal antibody raised from R. massiliae (MA1-D2) and therefore was scored as sharing virtually no antigenic similarity with other SFG rickettsiae (Tables 2 and 3).

                              
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  		TABLE 3.   Relationships of SFG rickettsial pairs revealed by expression of immunogenic epitopesa

Taxonomic relationships among SFG rickettsiae. In order to weight each epitope equally, only one representative of any monoclonal antibodies sharing a reactivity pattern was included in the analysis. Thus the results of 20 of 23 R. africae, 29 of 40 R. conorii, 9 of 13 R. massiliae, 4 of 4 R. sibirica, 3 of 3 R. slovaca, and 2 of 5 R. akari monoclonal antibodies were used for dendrogram construction. A dendrogram including the 29 SFG rickettsiae and R. bellii was inferred by cluster analysis of SJ similarities (Fig. 1A). A phylogenetic tree derived from ompA gene sequence alignment (24, 44, 50) is also presented in comparison with the dendrogram (Fig. 1B). The overall architectures of the two reconstructions were consistent with one another.


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  		FIG. 1.   Taxonomic dendrogram of the 29 SFG rickettsiae and R. bellii obtained by using the unweighted pair group method with arithmetic mean inferring SJ similarity based on different distribution and expression of the immunogenic epitopes on R. africae Z9-Hu, R. conorii Seven, R. massiliae Mtu1, R. akari Kaplan, R. sibirica Netsvetaev, and R. slovaca 13-B (A) and phylogenetic tree of the SFG rickettsiae derived from sequence alignment of the ompA gene (B). The scale bar (lower right) represents a 1.5% difference in nucleotide sequences. Bootstrap values are indicated at the nodes of the phylogenetic tree.

R. conorii is probably the older, but also most often isolated and most geographically distributed species of the SFG rickettsia (61, 68). In our study, strains of R. conorii, particularly the Indian tick typhus rickettsia, Kenya tick typhus rickettsia, M-1, and Seven strains, all showed very similar expression of epitopes. The Astrakhan fever rickettsia and the Israeli tick typhus rickettsia also expressed most of the R. conorii epitopes (68), concurring with their inclusion in an R. conorii subgroup (R. conorii complex) as proposed by genotypic comparative methods (20, 24, 60). Isolates of R. conorii from different geographical regions as well as from both humans and ticks, have been shown to exhibit the extremely-well-conserved macrorestriction patterns (48). When ompA gene sequences of strains of R. conorii were compared, they also exhibited high similarities to each other (24, 47). Indeed, the gltA sequences of four strains (M-1, Indian tick typhus rickettsia, Moroccan, and Seven) were identical (51). Thus, the evolutionary homology of R. conorii as demonstrated by these studies is reflected in the high levels of antigenic similarity revealed by our work (Tables 2 and 3) (68).

Within the R. conorii complex, the Astrakhan fever rickettsia and Israeli tick typhus rickettsia have been demonstrated to share antigenic and genotypic similarities with strains of R. conorii sensu stricto, although variation has also been noted (Tables 2 and 3) (20, 30, 37, 47, 60, 68). Therefore, it was not unexpected that in our study, the Astrakhan fever rickettsia and Israeli tick typhus rickettsia were clustered with the R. conorii strains, albeit at a lower level of similarity than that observed intraspecies (Fig. 1). Israeli tick typhus rickettsiae have been shown to have a protein profile distinguishable from those of the other SFG rickettsiae, including R. conorii (19 [data not shown]). At the genetic level, the Israeli tick typhus rickettsia ompA gene has been estimated to contain 15 rOmpA repeat unit-encoding regions, whereas those of the R. conorii Seven and Moroccan strains contain only 10 and 6 repeat unit-encoding regions, respectively (26, 28, 60), and these differences may account for antigenic variation on the rOmpA protein. Because the Israeli tick typhus rickettsia demonstrates marked antigenic and genotypic variation from R. conorii, it should perhaps be classified as a new species. Similarly, antigenic differences between Astrakhan fever rickettsia and R. conorii may also result from variation in the number of repeating regions encoded by their ompA genes (60). However, variation in the number of the ompA repeating unit is not the sole basis of antigenic variation within the R. conorii complex. Although the ompA genes of Astrakhan fever rickettsia and Israeli tick typhus rickettsia both encode the 15 repeating unit (60), marked variation in anti-rOmpA monoclonal antibody reactivity was observed (Table 2). The rOmpA protein epitope variation must therefore also result from variation within individual repeating unit sequences and/or variation with nonrepeating unit sequences at the 5' and 3' ends of the ompA gene (4, 26, 28). Comparison of sequence similarities for these nonrepeating unit regions indicates that about 26 base substitutions have occurred between Astrakhan fever rickettsia and Israeli tick typhus rickettsia (24).

R. africae has recently been identified as a new species of SFG rickettsia (35), being more akin, antigenically and phylogenetically, to Rickettsia parkeri than to R. conorii, which also exists in Africa (21, 24, 25, 49-51, 66, 68). However, subsequently, the ompA and 16S rRNA gene sequences of strain S, a new isolate from Armenia, were compared with an R. africae strain, and these two strains were found to share very high similarities (24, 49). The phylogenetic position of R. africae, inferred from comparison of alignments of these sequences, was found to be markedly nearer strain S than R. parkeri (24, 50, 51, 66). In our study, strain S and R. parkeri possessed almost all of the R. africae rOmpA and rOmpB protein epitopes, and their expression of epitopes shared with other SFG rickettsiae was also very similar (Tables 2 and 3). On the dendrogram, these three SFG rickettsiae clustered together with an SJ similarity of 83.6% (Fig. 1A).

The taxonomic interrelationships of "R. mongolotimonae" and strain BJ90, two new SFG isolates from China, have previously been studied by polyphasic methods (21, 49-51, 69), on the basis of which, strain BJ90 has been proposed as a strain of R. sibirica, whereas "R. mongolotimonae" has been proposed as a novel species (69). In this study, the reactivity pattern of BJ90 differed from that of R. sibirica with only three monoclonal antibodies. Similarly, "R. mongolotimonae" showed almost identical epitope expression to both R. sibirica and strain BJ90, and thus these three rickettsiae clustered tightly together at a high level of SJ similarity (Table 3 and Fig. 1A). These results question the true taxonomic nature of the relationship between "R. mongolotimonae" and R. sibirica, and there is some justification for the two species being unified; certainly, the expression of their immunogenic epitopes is very similar, far more so than that, for example, of members of R. conorii sensu stricto. Furthermore, comparison of the 16S rRNA genes of the two species has demonstrated them to be identical (48).

"R. slovaca" was found to cluster more loosely with the subgroup defined above (Fig. 1). Previous genotypic analysis, based on comparative macrorestriction analysis, suggested that "R. slovaca" was most closely related to Thai tick typhus rickettsia (48), but in both gltA- and ompA-inferred phylogenetic analysis, "R. slovaca" lay on a monophyletic branch, demonstrating no specific relationship to other SFG rickettsiae (48, 51).

R. honei, a pathogenic SFG rickettsia isolated from Flinders Island in Australia (7), has been demonstrated to be antigenically and genotypically different from R. australis, a species which occurs in the same geographical region (7, 55). In our study, R. honei shared antigenic similarity with R. australis only on the SFG group-specific LPS-like antigen, instead exhibiting a reactivity pattern similar to that of the Thai tick typhus rickettsia (Tables 2 and 3 and Fig. 1). The proximity of these two species has also been demonstrated by phylogenetic studies after sequence alignment of the ompA, gltA, and 16S rRNA gene fragments (47). R. rickettsii also clusters, albeit more loosely, with these species (Fig. 1) (7). The immunogenicity of R. rickettsii has been well studied previously by Anacker and colleagues through production of monoclonal antibodies (2, 3). The rOmpA and rOmpB proteins were first demonstrated to express different specific epitopes in their studies. Antigenic heterogeneity among R. rickettsii strains has also been proved with these monoclonal antibodies (2). When other SFG rickettsiae were tested against these monoclonal antibodies, it was observed that these two major surface proteins expressed a number of epitopes which were not expressed among the other SFG rickettsia species but which were distributed widely within the R. rickettsii strains (3). Similarly, in this study, R. rickettsii was found to express only a few common protein epitopes with R. africae, R. conorii, R. sibirica, and "R. slovaca." Moreover, among these common epitopes, most of them were also shared with R. honei and Thai tick typhus rickettsia (Table 2).

Strain Bar29, a newly identified isolate from Catalonia in Spain, has been demonstrated to exhibit antigenic and genotypic characteristics very similar to those of R. massiliae (9, 24, 47-51, 67). These two SFG rickettsiae, both isolated from the Mediterranean regions, also showed similar expression of epitopes in our study (Table 2). Rickettsia aeschlimannii shared several R. massiliae protein epitopes and thus clustered in the group formed by Rickettsia montana and Bar29 (Table 2 and Fig. 1). R. montana exhibited distribution and expression of the immunogenic epitopes similar to those of R. aeschlimannii and were grouped together (Fig. 1A). These four SFG rickettsiae also formed a subgroup, the R. massiliae subgroup (8, 13, 24, 48-51, 67). However, Rickettsia rhipicephali, which has previously been shown to share a close phylogenetic relationship with this subgroup (24, 48-51), did not cluster in the subgroup in our study (Fig. 1A).

R. akari, R. australis, and R. helvetica have been demonstrated to be antigenically and genotypically very different from the other SFG rickettsiae (24, 27, 28, 47-51, 56, 67, 68). Phylogenetic analysis, inferred from comparison of the gltA and 16S rRNA genes, clearly demonstrated that these three species lay apart from the other SFG rickettsiae (24, 49-51, 56). The species could not be included in ompA-based phylogenetic assessments, because PCR amplifications with conserved primer pairs failed (21, 45, 47). In our study, these three species showed marked antigenic divergence from the other SFG rickettsiae, sharing only the group-specific LPS-like antigens with the other species. R. akari epitopes either were species specific or were shared with R. australis only (Table 2). Although all three formed a cluster in the dendrogram in which R. bellii was also included (Fig. 1A), this grouping is likely to be artifactual, serving only to demonstrate their distance from the other SFG rickettsiae, and not to indicate any specific relationship between the species. The group of R. akari, and R. australis has been suggested as being unique (51, 56); indeed, they may perhaps best be considered as being outside the SFG rickettsiae within the genus Rickettsia (56).

The taxonomic relationships inferred among the SFG rickettsiae in the study were generally consistent with the findings of previous phylogenetic studies, in particular with that derived from ompA sequence alignment (24, 48-51). However, differences between the two dendrograms were observed (Fig. 1), some of which may have resulted from shortfalls in our approach. Because we used monoclonal antibodies raised against only six SFG species, our taxonomic reconstruction did not assign equal "weight" to all strains; an optimal analysis would have included an equal number of monoclonal antibodies raised against all strains. Such an approach would, however, entail an enormous amount of work and is clearly not practical. Despite this drawback, the general applicability and accuracy of this antigenic approach to SFG taxonomy have been demonstrated in this study, and the method presently represents the most practical way to study taxonomic relationships on the basis of phenotypic characteristics.

    ACKNOWLEDGMENTS

We are grateful to Hervé Tissot-Dupont for help in data analysis and Richard J. Birtles for critically reviewing the manuscript.

    FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies, Faculté de Médecine, 27 Blvd. Jean Moulin, 13385 Marseille Cédex 5, France. Phone: (33) 4 91 32 43 75. Fax: (33) 4 91 83 03 90. E-mail: Didier.Raoult{at}medecine.univ-mrs.fr.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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Journal of Clinical Microbiology, April 1998, p. 887-896, Vol. 36, No. 4
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