INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1987, p. 351-356 Vol. 37, No. 4 0020-7713/87/040351-06$02.00/0 Copyright 0 1987, International Union of Microbiological Societies

Antigenic Heterogeneity Among , Fluoribacter, and Tatlockia Species Analyzed by Crossed Immunoelectrophoresis

MICHAEL T. COLLINS,l* JETTE M. BANGSBORG,2 AND NIELS H@IBY2 Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin 53706’ and State Serum Institute, Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark2

Crossed immunoelectrophoresis (XIE) reference systems were established for Fluoribacter (Legionella) (containing Fluoribacter bozemanae, Fluoribacter dumofii, and Fluoribacter gormanii) and for Tatlockia (Legionella) micdadei. The Fluoribacter and Tatlockia XIE reference systems contained 54 and 72 anode- migrating antigens, respectively. These two systems, together with the previously described polyvalent (serogroups 1 through 6) XIE reference system, were used to study the cross- reactivities of antigens from organisms comprising the three proposed genera in the family Legionellaceae. Antigenic homology was expressed as the matching coefficient (MC), the ratio of the number of cross-reactive antigens to the total number of antigens. The MCs for individual L. pneumophila serogroups when the polyvalent L. pneumophila antibody was used were 0.98 f 0.05, which was significantly higher than the MCs determined by using Fluoribacter or Tatlockia antibodies (0.50 f 0.13) (P < 0.001). The MCs for the three species of Fluoribacter when polyvalent Fluoribacter antibody was used were 0.93 & 0.10, which was also significantly higher than the MCs when heterologous antibodies were used (0.40 f 0.04) (P< 0.001). The MCs for T. micdadei with the two heterologous antibody preparations were similar to each other (0.32 and 0.46) and to all other heterologous MCs among members of the Legionellaceae. The MCs for organisms representing three other families of were 0.16 f 0.04 in all three XIE reference systems and were significantly lower than the MCs among members of the Legionellaceae (P < 0.001). When a priori criteria for MC interpretation established in previous serotaxonomic studies of other bacterial species by XIE were used, our results from studies on the antigenic relationships among Legionella, Fluoribacter, and Tatlockia supported the proposal that there are multiple genera in the family Legionellaceae.

Antigenic analysis of proteins or peptides has been used to lished by Brown et al. (9, 21) and initiated production of study phylogenetic relationships among both procaryotes crossed immunoelectrophoresis reference systems to dem- and eucaryotes (11, 18, 44). In most studies, purified en- onstrate antigenic profiles for Fluoribacter and Tatlockia, as zymes and monospecific polyclonal antisera have been used. well as L. pneumophila (serogroups 1 through 6), as previ- These studies have demonstrated that protein antigens do ously described (2), by using the type cultures available at not cross-react unless they are homologous and isofunc- that time. Using these three crossed immunoelectrophoresis tional and have a common phylogenetic origin (11, 42-44, systems, we quantitated the degrees of antigenic homology 52), that the degree of cross-reaction is detectable until 30 to among the three genera. 40% of the amino acid sequence is substituted (43, 44), and We recognize the controversy concerning legionella no- that there is a linear correlation between immunological menclature, but use the genus designations Fluoribacter and distance and percentage of deoxyribonucleic acid-ribo- Tatlockia because our original hypothesis, upon which this nucleic acid homology down to homology values of 45 to study was founded, was that these organisms are sufficiently 50% (4). antigenically dissimilar to warrant classification as distinct Crossed immunoelectrophoresis techniques are ideal for genera in the family Legionellaceae. simultaneous evaluation of cross-reactions among multiple (40 to 90) protein antigens from different organisms. These techniques have been applied to serotaxonomic studies of MATERIALS AND METHODS (28, 29), (25, 26), injluenzae (47), Bacterial strains. The strains of L. pneumophila sero- (24, 32), Salmonella typhi (16, 17), Staphylococcus aureus groups 1 through 6 used in this study were strains ATCC (46), and Staphylococcus epidermidis (15). 33153, ATCC 33154, ATCC 33155, ATCC 33156, ATCC We previously reported one-way cross-reaction studies in 33216, and ATCC 33215, respectively. The Fluoribacter which a Legionella pneumophila serogroup 1 crossed im- (Legionella) bozemanae serogroup 1, Fluoribacter dumofii, munoelectrophoresis reference system was used (13). Our Fluoribacter gormanii, and Tatlockia (Legionella)micdadei findings were similar to those of Jolly and Kenny (33), who, strains used were strains ATCC 33217, ATCC 33279, ATCC also by using crossed immunoelectrophoresis, showed that 33297, and ATCC 33218, respectively. The , there was a high degree of antigenic homogeneity among L. , and Pseudomonas aeruginosa strains pneumophila serogroups 1 through 4 (27 of 31 antigens in used were clinical isolates from the culture collection of our common) and that only five antigens of Tatlockia micdadei laboratory. cross-reacted with antibodies to L. pneumophila. Conse- Antigens prepared from these organisms and the homolo- quently, in 1981 we adopted the nomenclature validly pub- gous antibodies were designated as follows: Lpl, Lp2, Lp3, Lp4, Lp5, and Lp6 for L. pneumophila serogroups 1through 6, respectively; Lpl-6 for the pool of all six L. pneumophila * Corresponding author. serogroups Fb for F. bozemanae; Fd for F. dumofii; Fg for

351 352 COLLINS ET AL. INT. J. SYST.BACTERIOL.

F. gormanii; Fbdg for the pool of all three Fluoribacter MCs. Matching coefficients (MCs) were used to express species; and Tm for T. micdadei. the degrees of antigenic relatedness among species and Antigen and antibody production and the crossed im- genera in the family Legionellaceae. This numerical measure munoelectrophoresis reference system for Lpl-6 have been of antigenic similarity has been employed in several other described previously (2). serological studies (13, 15, 17, 27, 47). Each MC was Antigen preparations, All organisms were cultivated on calculated as a mean of four independent determinations, buffered charcoal yeast extract agar (GIBCO Diagnostics, and all precipitation line quantitations were done without Madison, Wis.) incubated at 37°C. The organisms were knowledge of the antigen or antibody reagents employed. harvested by gently scraping the agar surface with a sterile The t test was used for pairwise comparison of mean MC bent glass rod, and then the cells were suspended in sterile values. A comprehensive review of the taxonomic applica- distilled water. Antigen preparations were made by sonica- tion of crossed immunoelectrophoresis has recently been tion (three times for 45 s) followed by high-speed centrifu- published (31). gation (20,000 x g for 60 min at 4°C) as described previously Terminology. The terminology guidelines described by (2, 12). The colloidal concentration of each antigen prepara- Chaparas et al. (10) for serotaxonomic studies in which tion was determined by refractometry, using human immu- immunodiffusion and immunoelectrophoresis are used were noglobulin as a standard (12). Concentrations ranged from adhered to. The antibodies which cross-react with antigens 13.3 to 29.3 mg/ml. from different bacterial isolates are those which bind specif- The pooled Lpl-6 reference antigen preparation has been ically to one or more similar epitopes on the antigens. described previously (2). A pooled reference antigen for Antigens carrying such (similar) epitopes were designated Fluoribacter species (Fbdg) was prepared by using equal cross-reactive antigens. amounts (by weight) of all of the individual species antigen preparations. For T. micdadei, the type strain was used RESULTS alone as the reference antigen. Identical antigen preparations The Legionella (Lpl-6) XIE reference system has been were used for both rabbit immunizations and immuno- described previously (2). With this system 58 precipitation electrophoresis. lines were routinely demonstrated. Reference antibody production. Ten adult New Zealand The Fluoribacter (Fbdg) XIE reference system (Fig. 1) White rabbits were used for Fluoribacter reference antibody had 54 precipitates, and the Tatlockia (Tm) XIE reference production, and five rabbits were used for T. micdadei system (Fig. 2) had 72. reference antibody production. At each immunization, 100 Cross-reacting antigens between genera were identified pl of the antigen preparation was homogenized with 100 pl of and quantitated by using three crossed immunoelectropho- incomplete adjuvant and injected intracutaneously at multi- retic techniques (XLIE, XIE-Ab, and XIE) with heterolo- ple sites on the back of the rabbit. The immunization and gous antigen preparations in the first-dimension gel. bleeding schedules were similar to those reported previously All three techniques produced comparable results, but (12, 22). Antisera for each group of rabbits harvested from XIE in which heterologous antigen and antibody prepara- months 5 to 17 were pooled, and the immunoglobulins were tions were used, counting all precipitate lines as cross- purified by using the method of Harboe and Ingild (22). The reactive (Fig. 3), provided the easiest-to-read and most immunoglobulin concentrations were 20.8, 29.3, and 30.4 reproducible results. To confirm that all precipitate lines mg/ml for the Lpl-6, Fbdg, and Tm reference antibodies, produced by XIE represented truly cross-reactive antigens, respectively. a control XLIE gel was run with the same combination of Crossed immunoelectrophoretic techniques. All immuno- heterologous antigen and antibody preparations but with the electrophoretic procedures were performed as described reference antigen, homologous with the antibody, in the previously (2, 12, 13, 29, 48) by using glass plates (5 by 7 intermediate gel. In every instance, 100% of the antigens cm), 1% agarose (Litex LSM, batch 812; Accurate Chemical were absorbed in situ, proving their cross-reactivity (Fig. and Scientific Corp., Westbury, N.Y.), and barbital buffer 3D). XLIE was also used to evaluate cross-reactivity among (pH 8.6; ionic strength, 0.1). First-dimension electrophoresis closely related species, i.e., among L. pneumophila was performed at 10 V/cm until 2 pl of bromothymol serogroups as previously described (2) and among blue-labeled human albumin marker migrated exactly 35 Fluoribacter species (Table 1). mm. Second-dimension electrophoresis was conducted at 2 The MCs for the antigens of Legionella, Fluoribacter, and V/cm for 18 to 20 h. During all electrophoretic procedures a Tatlockia were calculated by using the number of cross- constant temperature of 12°C was maintained by a re- reactive antigens counted by XIE, as shown in Fig. 3, and circulating cooler. are listed in Table 2. The MCs for individual L. pneumophila The following three crossed immunoelectrophoretic tech- serogroups when the polyvalent L. pneumophila antibody niques were used: (i) crossed immunoelectrophoresis with a was used were 0.98 k 0.05, which was significantly higher blank intermediate gel containing saline (100 pl of 0.154 M than the MCs determined with Fluoribacter or Tatlockia NaCI), referred to below as XIE (30); (ii) crossed im- antibodies (0.50 k 0.13) (P< 0.001). The MCs for the three munoelectrophoresis with an intermediate gel containing species of Fluoribacter when polyvalent Fluoribacter anti- antigen preparations at varying concentrations (this tech- body was used were 0.93 * 0.10, which was also signifi- nique has been called crossed-line immunoelectrophoresis), cantly higher than the MCs obtained when heterologous referred to as XLIE (35); (iii) crossed immunoelectro- antibodies were used (0.40 k 0.04) (P< 0.001). The MCs for phoresis with antibody-containing intermediate gels, re- T. micdadei with the two heterologous antibody prepara- ferred to as XIE-Ab (1). tions were similar to each other, 0.32 and 0.46, and to all For all three techniques, a standard amount of antigen other heterologous MCs among members of the Legionel- preparation (60 pg) was placed in the first-dimension well, laceue. The MCs for organisms representing three other and a standard amount of reference antibody (300 1.1) was families of bacteria were all <0.22 in all three XIE reference mixed with the agarose used to pour the second-dimension systems and were significantly lower than the MCs among or back gel (final concentration, 14.3 pl/cm2). members of the Legionellaceae (P < 0.001). VOL.37, 1987 ANTIGENIC HETEROGENEITY 353

FIG. 1. Fluoribacter XIE reference system. A pooled antigen preparation (60 pg) containing equal amounts of F. bozemanae, F. dumofii, and F. gormanii was placed in the first-dimension well (Fbdg Ag). Saline (NaCl) was placed in the intermediate gel. Purified immunoglobulins (300 pl; 14.3 pl/cm2) containing antibodies to the pooled Ffuoribucter antigen were incorporated in the back gel (Fbdg Ab). In the first-dimension electrophoresis the anode was to the right. In the second-dimension electrophoresis the anode was at the top. The horizontal bar shows the migration of a human albumin marker in the first dimension. The gel was stained with Coomassie brilliant blue R. A total of 54 anode-migrating precipitates are numbered.

FIG. 2. Tatfockia XIE reference system. The first-dimension well contained 60 pg of T. micdudei antigen (Tm Ag). Saline (NaCl) was placed ih the intermediate gel. Homologous purified antibodies (Tm Ab) were incorporated in the back gel (300 pl; 14.3 pl/cm2). For technical details see the legend to Fig. 1. A total of 72 anodemigrating precipitates are numbered. 354 COLLINS ET AL. INT. J. SYST.BACTERIOL.

FIG. 3. Crossed immunoelectrophoresis in which heterologous antigens were used in the first dimension. (A through D) A 300-pl amount of L. pneumophila antibody (Lpl-6 Ab) was in the back gel. (A) Control with 60 pg of homologous antigen (Lpl-6 Ag) in the first dimension and saline in the intermediate gel, showing 60 reference system precipitates (peaks). (B)Fluoribacter antigen (60 kg of Fbdg antigen [Fbdg Ag]) in the first dimension and saline in the intermediate gel. The L. pneumophila antibody precipitated 35 antigens. (C) Tutlockia antigen (60 pg of Tm antigen [Tm Ag]) in the first dimension and saline in the intermediate gel. The L, pneumophila antibody precipitated 27 antigens. (D) XLIE control for gel B. This gel contained the same first-dimension antigen (60 pg of Fbdg Ag) and back gel antibody (300 pl of Lpl-6 Ab) as gel B but had 50 pl of Lpl-6 Ag in the intermediate gel. This gel confirined that all Fbdg antigens precipitated by Lpl-6 antibody do, in fact, cross-react with Lpl-6 antigens by the formation of lines of identity (arrows).

DISCUSSION measures of similarities and differences among the taxa studied and have been interpreted as evidence either that all Genetic and biochemical data (3, 5-9, 20, 21, 23, 42, 49, legionellae cluster as members of a single genus (6, 7) or that 50), ubiquinone patterns (34), fatty acid analysis data subgroups within the Legionellaceae occur which are suffi- (3841), peptide profiles (14, 36), and carbohydrate compo- ciently unique to warrant designation as separate genera (9, sitions (19, 51) for members of the family Legionellaceae 19, 21, 36, 50). have been reported and are nicely summarized in a recent Such interpretations are primarily governed by the body of publication by Fox et al. (19). These data are relative data which one chooses to use as a standard. For example, if a family of bacteria with a high degree of phylogenetic TABLE 1. Number of non-cross-reacting antigens among relationship, such as the , is used as a F. bozemanae, F. durnofii, and F. gormanii determined by XLIE standard, then legionellae appear to be a relatively diverse No. of non-cross-reacting antigens group of organisms. On the other hand, comparison of No. of with the following antigen prepn legionellae with families having much broader and deeper Antigen prepn in precipitates first dimension in control in the intermediate gel: phylogenetic groupings, such as the Pseudomonadaceae, XIE gel F. bozemanae F. dumofii F. gormanii makes legionellae appear relatively homogeneous. XIE studies on the antigens of Pseudomonas aeruginosa F. botemunae 40 0 3 5 by H@iby in 1975 (28) revealed a high level of antigenic F. dumufii 36 8 0 5 homogeneity within the species and established methods for F. gormahii 36 8 3 0 reproducibly quantifying the number of cross-reactive anti- VOL. 37, 1987 ANTIGENIC HETEROGENEITY 355

TABLE 2. MCs among Legionella, Fluoribacter, and Tutlocki as species were 0.86 5 0.07 (based on the XLIE data in Table determined by XIE l), and MCs among the genera Legionella, Fluoribacter, and MC with the following polyvalent antibody in the Tatlockia were 0.46 k 0.12. By contrast, MCs for bacteria second dimension: 5 Antigen prepn in representing three other families were 0.16 0.04, similar to previous findings (13). Thus, the antigenic homogeneity first dimension Legionella Fluoribacter Tutlockiri Lpl-6 Fbdg Tm among serogroups of L. pneumophila was significantly higher than that found among Fluoribacter species (P < L. pneurnophila 1.03 * 0.02" 0.58 * 0.08 0.47 * 0.07 0.001). Antigen sharing among Fluoribacter species was serogroup 1 significantly greater than antigen sharing among the three L. pneurnophila 0.96 2 0.02 0.68 * 0.03 0.45 5 0.03 serogroup 2 proposed genera of the Legionellaceae (P < 0.001), and all L. pneurnophila 0.93 +. 0.01 0.58 * 0.05 0.35 2 0.03 strains of legionellae tested had significantly more antigens serogroup 3 in common than they did with representatives of three other L. pneurnophila 0.94 * 0.05 0.67 2 0.03 0.34 k 0.03 families of bacteria (P < 0,001). serogroup 4 Our results illustrate the potential of XIE techniques as L. pneurnophila 0.98 2 0.05 0.59 * 0.02 0.37 * 0.05 serotaxonomic tools, agree with proposed MC cut-off limits, serogroup 5 and support the proposal that there are multiple genera in the L. pneurnophila 1.02 2 0.04 0.64 * 0.09 0.39 * 0.04 family Legionellaceae, which is consistent with reports on serogroup 6 the genetic distance between species of Legionella as deter- mined by studies of 16s ribosomal ribonucleic acid (37) and F. bozernanae 0.41 0.03 1.01 0.07 0.39 2 0.02 * * deoxyribonucleic acid homology (6,7,9,21). In addition, the F. dumofii 0.44 2 0.03 0.92 * 0.04 0.43 * 0.04 F. gorrnanii 0.35 2 0.04 0.85 * 0.08 0.38 k 0.02 relatively low degree of antigen sharing found among L. pneumophila, Fluorihacter sp., and T, micdadei has practi- T. rnicdadei 0.32 * 0.02 0.46 * 0.02 0.99 * 0.04 cal implications for development of polyspecies-specific serodiagnostic tests. Controls E. coli 0.13 * 0.00 0.21 2 0.01 0.19 * 0.01 Pasteurella 0.12 * 0.03 0.19 * 0.01 0.10 * 0.00 ACKNOWLEDGMENTS rnultocida This work was supported in part by a grant from the Research Pseudomonas 0.09 2 0.00 0.13 5 0.01 0.17 ? 0.01 Committee, University of Wisconsin-Madison, and by the Knud aeruginosa HQjgaards Fund. Mean 2 standard deviation based on four determinations. LITERATURE CITED 1. Axelsen, N. H. 1983. Intermediate gel electrophoresis, p. 141- gens between bacterial species by using quantitative im- 149. In N. H. Axelsen (ed.), Handbook of immunoprecipitation- in-gel techniques. Blackwell Scientific Publications, Oxford. munoelectrophoresis techniques. A number of subsequent 2. Bangsborg, J. M., M. T. Collins, and N. H~iby.1986. Antigenic XIE studies on other bacterial species indicated a close homogeneity among Legionella pneurnophila serogroups 1 to 6 agreement between the degree of antigenic homogeneity evaluated by crossed immunoelectrophoresis. Int. J. Syst. Bac- among bacterial species and their taxonomic relatedness as teriol. 36:399404. defined by more traditional methods (12, 16, 17, 26, 28, 32, 3. Barbaree, J. M., A. Sanchez, and G. N. Sanden. 1983. Tolerance 46, 47). For example, of six Neisseria species investigated, of Legionella species to sodium chloride. Curr. Microbiol. all had MCs of 20.79 with N. meningitidis (26). In contrast, 9: 1-15. of 13 Pseudomonas species tested, all had MCs of 10.16 4. Baumann, L., and P. Baumann. 1978. 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BACTERIOL.

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