were otherwise similar (37). We also confirmed that none of

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 28, No. 9 Comparison of Four Mycoplasma arthritidis Strains by Enzyme Immunoassay, Metabolism Inhibition, One- and Two-Dimensional Electrophoresis, and Immunoblotting LEIGH RICE WASHBURN* AND SHANNON HIRSCH Department ofmicrobiology, University of South Dakota School of Medicine, Vermillion, South Dakota Received 27 December 1989/Accepted 10 April 1990 Four Mycoplasma arthritidis strains, two virulent (158plOp9 and 14124plO) and two avirulent (PG6 and H606), were examined for differences in their antigenic compositions. Rabbit antisera prepared against each strain were compared for their reactivities against both homologous and heterologous strains by enzyme-linked immunosorbent assay and metabolism inhibition assay. These tests confirmed a close serologic relationship among the four strains. Only by cross-absorbing each antiserum with intact cells from both homologous and heterologous strains could serologic differences be detected. Interestingly, antigenic variability was minimal among those antigens involved in the metabolism inhibition reaction, suggesting that they may be highly conserved within this species. No differences in protein composition could be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, slight variation was apparent on two-dimensional gels, and antigens possessing strain-specific epitopes were detected by two-dimensional immunoblotting experiments performed with cross-absorbed antisera. The enzyme-linked immunosorbent assay, two-dimensional electrophoresis, and immunoblotting experiments showed that the two virulent strains were closely related to each other, while the avirulent strains were more distantly related to each other and to the other two strains. The relationship of M. arthritidis serologic diversity to virulence remains unclear; however, this study has demonstrated that it may be possible to subdivide M. arthritidis into serogroups on the basis of cross-absorption patterns and to identify those antigens bearing strain-specific epitopes by immunoblotting with cross-absorbed antisera. Mycoplasma arthritidis is a natural arthritogenic agent for rats (9) and is frequently found as an asymptomatic colonizer in rat and mouse colonies (4, 11). Although M. arthritidis has been used extensively in metabolism studies (25, 28), relatively little is known about its membrane and antigenic structures, and even less is known about the basis for strain differentiation. This species is usually considered serologically homogeneous (20), and at present, strains are designated according to the source of the isolate. To our knowledge, no other markers have been identified. However, it has been known for many years that strain differences exist on a number of levels. The most obvious of these, and probably the least understood, is virulence (12, 14, 16), and this was the starting point for most of the earlier comparative studies. Virulence has been associated with slower in vitro growth (12), which may explain the selection of avirulent varieties on laboratory passage (16). It has also been associated with slower clearance from mouse circulation (7) and greater resistance to the action of rabbit metabolism-inhibiting (MI) antibodies (12). On the other hand, both virulent and avirulent strains are resistant to phagocytosis by murine peritoneal macrophages (8), and the avirulent strain H606 can immunize rats against challenge with the virulent strain 158plO (5). We recently showed that two virulent strains, 158plOp9 and 14152pl3, induced higher immunoglobulin M (IgM) and early IgG antibody titers in rats after intravenous inoculation than did two avirulent strains, PG6 and H606, although the antibody responses * Corresponding author were otherwise similar (37). We also confirmed that none of these strains induced MI antibodies in rats (37). These observations support the idea of serologic homogeneity, although in 1965 Tully (32) observed only partial crossreactivity by immunofluorescence between strain PG6 and a rat joint isolate designated JR-3 (32), suggesting that this relationship is not absolute. The purpose of this study was to determine whether four strains of M. arthritidis, two virulent and two avirulent, could be differentiated by antigenic and protein analysis, as has been done for a number of other Mycoplasma species (1, 15, 18, 21, 22, 29, 30). We concentrated on protein antigens, because we and other investigators showed previously that the major membrane antigens of M. arthritidis are protein rather than lipid or carbohydrate in nature (2, 17, 20, 39). MATERIALS AND METHODS Mycoplasmas. The M. arthritidis strains used in this study are designated 158plOp9, 14124plO, PG6, and H606 (6, 10, 12, 13). For simplicity, they are referred to as strains A, B, C, and D, respectively. Strains A and B are highly virulent for rats, while strains C and D are avirulent (12, 37). Mycoplasmas used for one- and two-dimensional electrophoresis, for rabbit injections, and as antigens in enzymelinked immunosorbent assays (ELISAs) were grown in a dialysate medium designated SPCY (soy peptone-caseinyeast extract dialysates), which was modified from Kenny (17) and supplemented with arginine and gamma globulinfree horse serum (34). Mycoplasmas used as antigens in the

2 VOL. 28, 1990 ANTIGENIC VARIABILITY AMONG M. ARTHRITIDIS STRAINS 1975 MI test and for absorption of strain-specific antisera were grown in a modified Edward-type medium supplemented with 7.5% (vol/vol) horse serum (35). Antigens for ELISA and the MI test were prepared as described previously (33, 36). Mycoplasmas for rabbit injections, absorption experiments, and electrophoresis were prepared as follows. After approximately 24 h of incubation at 370C, 1- to 2-liter broth cultures were concentrated by centrifugation at 10,000 x g and suspended in 20 ml of fresh medium containing 15% (wt/vol) sucrose in place of the serum supplement. One-milliliter portions of these concentrated cultures, containing approximately 109 to 1010 CFU/ ml, were stored at -70 C until use. Strain-specific antisera. Female New Zealand White rabbits obtained from a local supplier were immunized with each ofthe four M. arthritidis strains as described previously (38, 39). These rabbits were part of an earlier experiment on the immune response of arthritic rats and rabbits to M. arthritidis membrane antigens (38); therefore, the immunization protocol was actually designed to induce an active infection. Briefly, each rabbit was injected intraarticularly with 1 x 109 CFU in each knee, followed by a second injection of 3 x 109 CFU by the intravenous route 5 weeks later. Rabbits were killed by exsanguination under pentobarbital anesthesia 1 week after the second injection (35). Antisera were divided into equal portions and stored at -20 C. None of the rabbits had detectable antibody against M. arthritidis (measured by ELISA) prior to immunization. Samples of each of the four strain-specific antisera were absorbed with intact cells from the four M. arthritidis strains according to the following procedure. A suspension of mycoplasmas containing approximately 1010 CFU (approximately 1 mg of mycoplasmal protein) (22) was pelleted by centrifugation. The cells were washed and suspended in 0.25 ml of serum and incubated on a rocker overnight at 4 C. The suspension was then centrifuged, and the supernatant antiserum was mixed with a fresh cell pellet, followed by overnight incubation at 4 C. The absorption step was performed a total of three times, so that each antiserum sample was absorbed with approximately 3 mg of mycoplasmal protein. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mycoplasmas were loaded at a concentration of 10,ug of protein onto 3-mm sodium dodecyl sulfate (SDS)-polyacrylamide slab gels (10% T [acrylamide], 2.7% C [N,N'-methylene-bis-acrylamide]) after thorough washing and solubilization in 0.05 M Tris buffer (ph 6.8) containing 2% (wt/vol) SDS, 0.15% (wt/vol) dithioerythritol, and 18% (vol/vol) glycerol. The electrophoretic technique was based on the method of Laemmli (19) and was carried out in a vertical slab gel electrophoresis unit (model SE; Hoefer Scientific Instruments, San Francisco, Calif.) at a constant current of 15 ma per gel in a ph M Tris M glycine buffer containing 0.1% (wt/vol) SDS. Proteins were visualized by silver staining. Low-molecular-weight markers were purchased from Bio-Rad Laboratories (Richmond, Calif.). Two-dimensional electrophoresis. Two-dimensional electrophoresis was performed as described by O'Farrell et al. (24) with slight modifications. The first dimension was performed in a tube gel electrophoresis unit (GT-3; Hoefer). Nonequiibrium ph gradient electrophoresis (NEPHGE) was used rather than isoelectric focusing, because M. arthritidis possesses a large number of basic proteins that do not resolve well by isoelectric focusing (G. L. Budweg and L. R. Washburn, 7th Congr. Int. Org. Mycoplasmology, Badenbei-Wien, Austria, 1988). Thirkill and Kenny observed a similar phenomenon using two-dimensional immunoelectrophoresis (29). For NEPHGE, washed mycoplasmas were solubilized for 10 min in 25,ul of a solution containing 0.5% (wt/vol) SDS, 9.5 M urea, 5% (vol/vol) 2-mercaptoethanol, and 2% (vol/ vol) ampholytes (ph 3 to 10); 25,il of a second solution containing 9.5 M urea, 2% (vol/vol) Triton X-100, 5% (vol/ vol) 2-mercaptoethanol, 1.6% (vol/vol) ampholytes (ph S to 7), and 0.4% (vol/vol) ampholytes ph 3 to 10 was then added. Ampholytes were purchased from Bio-Rad Laboratories. Polyacrylamide tube gels (4% T, 5% C) were prepared as described by Hoefer Scientific Instruments (Hoefer Scientific Instruments Catalog, p , 1986; Hoefer Scientific Instruments) and contained 9.2 M urea, 2% (vol/vol) Triton X-100, 4% (vol/vol) ampholytes (ph S to 7), 1% (vol/vol) ampholytes (ph 3 to 10), 0.07% (vol/vol) N,N,N',N'-tetramethylethylenediamine, and 0.01% (wt/vol) ammonium persulfate. Approximately 0.5 ml of gel solution was loaded into each 1.5-mm electrophoresis tube and overlaid with glassdistilled water. After overnight polymerization at room temperature, the water was completely removed by aspiration, and each tube was loaded with 10,ul of solubilized mycoplasmas containing either 150 (for silver staining) or 200 (for immunoblotting),ug of protein. The sample was overlaid with a solution containing 5% (vol/vol) Triton X-100, 8 M urea, 1.6% ampholytes (ph 5.7), and 0.4% (vol/vol) ampholytes (ph 3 to 10). The cathode (lower chamber) and anode (upper chamber) solutions consisted of 0.02 M NaOH and 0.01 M H3PO4, respectively. Gels were electrophoresed for exactly 4 h at 400 V. After each use, the 1.5-mm glass tubes were washed with acid and coated with Photo-flo 2100 (Eastman Kodak, Rochester, N.Y.). After NEPHGE, gels were immediately removed from the tubes, equilibrated in two 5-min changes of 0.06 M Tris buffer (ph 6.8) containing 2% (wt/vol) SDS and 5% (vol/vol) 2-mercaptoethanol, and stored at -70 C until use. For the second dimension SDS-polyacrylamide gel electrophoresis each tube gel was placed directly into the surface of a 3-mm SDS-polyacrylamide slab gel (7.5% T, 2.7% C). No stacking gel was used. Electrophoresis was carried out at 15 ma per gel as described above. The gel was then either stained with silver or placed in a Transblot apparatus (Bio-Rad) and transferred electrophoretically to nitrocellulose for approximately 18 h at 30 V and then for 1 h at 50 V. Serologic assays. The MI test was performed as modified from Purcell et al. (27) and Washburn (33); antigens consisted of viable cultures of Edward broth-grown mycoplasmas stored at -70 C until use (33). The ELISA was also performed as described previously (36); mycoplasmal antigens consisted of whole-cell lysates prepared from washed organisms grown in SPCY (36). Immunoblotting. Nitrocellulose membranes containing mycoplasmal antigens were blocked for 1 h with phosphatebuffered saline containing 0.05% (vol/vol) Tween 20 (PBS-T) at 37 C and then exposed for 2 h to the specific rabbit antiserum under consideration diluted 1:40 in PBS-T, for 1 h to peroxidase-conjugated anti-rabbit IgG (heavy and light chain specific; Organon Teknika, Cappel Division, Durham, N.C.) diluted 1:1,600 in PBS-T, and finally, for about 20 min to a substrate consisting of 4-chloro-1-naphthol and H202 (3, 31, 38). Blots were given six 5-min rinses with PBS-T between each step.

3 1976 WASHBURN AND HIRSCH TABLE 1. ELISA antibody titers of antisera to four strains against each of the four strains and effect of absorption with intact cells Antiserum Titer of antibody against strain: A B C D Anti-A unabsorbed 5,120 5,120 5,120 2,560 Absorbed with A Absorbed with B Absorbed with C Absorbed with D Anti-B unabsorbed 5,120 2,560 5,120 5,120 Absorbed with A Absorbed with B Absorbed with C Absorbed with D Anti-C unabsorbed 1,280 1,280 5,120 1,280 Absorbed with A , Absorbed with B , Absorbed with C Absorbed with D , Anti-D unabsorbed 5,120 5,120 2,560 5,120 Absorbed with A Absorbed with B Absorbed with C 1,280 5, ,560 Absorbed with D RESULTS ELISA. Each strain-specific antiserum was tested by ELISA against its homologous strain and the three heterologous strains (Table 1). Anti-C showed a fourfold higher titer against strain C than against the three heterologous strains A, B, and D, but none of the other antisera showed more than a twofold difference in titer against heterologous strains. This confirmed previous observations of close serologic identity within this species (20) and indicated the need for a more specific assay. The necessary specificity was achieved by cross-absorbing each strain-specific antiserum with each of the four strains, as described in Materials and Methods. Each absorbed antiserum was then tested against each strain by ELISA (Table 1). Each strain removed nearly all activity against itself from each antiserum. In addition, strains A and B absorbed activity about equally well from anti-a and anti-b, indicating their close serologic relationship. Strains C and D were less effective at absorbing activity against heterologous strains, and of all combinations, strain C was the least effective at absorbing activity from the anti-d. Absorption of the anti-c with strains À and B removed considerable activity not only against strains A and B but also against strain D. Similarly, absorption of anti-d with strains A and B also removed activity against strain C. These data indicate that strains A and B are closely related to each other, while strains C and D may belong to two separate groups. MI assay. The antigens responsible for eliciting MI and growth-inhibiting antibodies are often strain specific in mycoplasmas (15, 18, 21, 26). In view of this and the ELISA results, we expected that antigenic differences would be even more pronounced when tested by the MI assay. This did not prove to be the case. Each of the four (unabsorbed) strain-specific antisera was tested for its ability to inhibit the metabolism of each of the four strains. Titers against heter- TABLE 2. Effect of absorption on MI antibody titers of four strain-specific antisera for their homologous antigens Antiserum Titer of antibody against strain: A B C D Unabsorbed 20,480 10,240 10,240 10,240 Absorbed with A Absorbed with B Absorbed with C Absorbed with D ologous and homologous strains differed by no more than one dilution (data not shown). Cross-absorption provided further evidence for a close, if not identical, relationship among the MI antigens of the four strains. Each absorbed antiserum was tested against its homologous strain (Table 2). Strains C and D were slightly less effective at absorbing MI antibodies from anti-a and anti-b than were strains A and B. In addition, strain D absorbed MI antibodies from anti-c slightly less well than did strains A, B, or C. However, differences in MI titers between homologously and heterologously absorbed antisera were far less dramatic than they were in the ELISA. One- and two-dimensional electrophoresis. In order to determine whether serologic differences observed in the ELISA were accompanied by differences in protein composition, M. arthritidis A, B, C, and D were subjected to one-dimensional SDS-polyacrylamide gel electrophoresis. Protein separation profiles were essentially identical (Fig. 1). For increased sensitivity in detecting minor differences, each strain was further subjected to two-dimensional electrophoresis (Fig. 2). Protein profiles were still very similar for all four strains; however, minor variations could be seen. A peptide labeled a was seen in strains A and B but not in strain A B C D J. CLIN. MICROBIOL. k D a 97 2 ta 66.2 _ L,, FIG. 1. One-dimensional electrophoretic analysis of total cellular protein from four M. arthritidis strains. Lane A, 158plOp9 (strain A); lane B, 14124plO (strain B); lane C, PG6 (strain C); lane D, H606 (strain D). Molecular size markers are given on the right in kilodaltons. Proteins were stained with silver. No differences in protein composition were detected among these strains by this method.

4 VOL. 28, 1990 J A' c ik,.q.1 O.,f 4, 4 I ANTIGENIC VARIABILITY AMONG M. ARTHRITIDIS r STRAINS 1977.*.. ' a.0m r, 's -*a o Ji 4..4e ao 4 *b.*-s gik. bt, p. t _:4a. B op d X w&.. r t;ô- W/-" O t e ;'a e.* 4b.1,*e FIG. 2. Two-dimensional electrophoretic analysis of total cellular protein from four M. arthritidis strains. (A) 158plOp9 (strain A); (B) 14124plO (strain B); (C) PG6 (strain C); (D) H606 (strain D). Proteins were stained with silver. Circled and lettered peptides appeared to show some specificity. a is unique to strains 158plOp9 and 14124plO; b is seen only in strain PG6; w, x, and y are seen only in strain H606, while z is seen in all strains except H606. C or D, and one labeled b appeared only in strain C. Strain D was the most diverse; sets of peptides labeled w, x, and y appeared in strain D but not in the other three strains, while the peptide labeled z appeared in strains A, B, and C, but not in strain D. Immunoblotting from two-dimensional gels. In order to identify antigens containing strain-specific epitopes, we performed a series of two-dimensional immunoblotting experiments with absorbed antisera. M. arthritidis antigens separated by two-dimensional electrophoresis were blotted onto nitrocellulose membranes as described in Materials and Methods and were exposed to absorbed and unabsorbed strain-specific antisera. Each antiserum was tested against its homologous strain only; results with strains A, C, and D are presented in Fig. 3, 4, and 5, respectively. Results with strain B were identical to those with strain A and are not shown. Any antigens appearing on blots that were exposed to absorbed antisera possessed epitopes that were not shared by the absorbing strain. Strains A and B showed nearly complete cross-reactivity by this technique. Both absorbed most of the activity against A and B from anti-a and anti-b (Fig. 3; results with anti-b not shown), and both strains absorbed antibodies to the same antigens from anti-c and anti-d (Fig. 4 and 5). The collection of antigens labeled 1 and 2 possessed the greatest degree of strain specificity. Although these antigens were identical in strains A and B (Fig. 3), their counterparts in db a * strains C and D were different from A and B and from each other (Fig. 4 and 5). Strain C possessed the fewest unique epitopes. Strains A and B absorbed activity to most strain C epitopes from anti-c (Fig. 4), with the exception of antigen 1, the antigen 2 complex, and the acidic ladder labeled 9 and 10. Strain C shared fewer epitopes with strain D than it did with strains A and B. Differences between C and D appeared in antigens 1, 2, 5, 7, 9, and 10 and four unnumbered, slightly more acidic, lower-molecular-weight antigens (Fig. 4 and 5). Strain D had the greatest number of unique epitopes, which was in agreement with the ELISA results. These included antigen 1; the antigen 2 complex; antigens 3, 4, and 5; the antigen 6 triplet; antigens 7, 8, and 10; and the set of four very low-molecular-weight antigens labeled 11. Many of these strain-specific antigens contained both unique and shared epitopes. For instance, antibodies against peptide 8 were absorbed from anti-a antiserum by strain D (Fig. 3D) but not vice versa (Fig. SA), suggesting that the strain D version had both common and unique epitopes, while the strain A version had only the common ones. Similarly, antigens 3, 4, and 5 of strains A, B, and D possessed both common and strain-specific epitopes, but the corresponding antigens in strain C possessed only the common epitopes, because all three heterologous strains completely absorbed antibodies against them from anti-c antiserum. n Fi q, X

5 1978 WASHBURN AND HIRSCH J. CLIN. MICROBIOL. A c u~~~~~~~.._ts_ ?7' 1 40V0, 3ç '') DISCUSSION We confirmed that strain-specific rabbit antisera against four strains of M. arthritidis cross-reacted extensively by ELISA and MI. However, we also showed that serologic differences did exist and could be demonstrated by crossabsorption. When cross-absorbed antisera were tested by ELISA against homologous and heterologous strains, nearly complete cross-reactivity between two virulent strains A and B (158plOp9 and 14124plO, respectively) was revealed, while two avirulent strains, C and D (PG6 and H606, respectively), were more distantly related to each other and to the other two strains. Of these, strain D was the most distinct. Earlier we showed that a third virulent strain, 14152pl3, was also nearly identical to strain A (L. R. Washburn, and G. L. Budweg, 7th Congr. Int. Org. Mycoplasmology, Baden-bei- B D 2< O ( 1i 7 4) 5\ FIG. 3. Two-dimensional immunoblots of strain 158plOp9 (strain A) exposed to homologous unabsorbed rabbit antiserum (U) and homologous antiserum that was absorbed with intact cells from strains 158plOp9 (A), 14124plO (B), PG6 (C), and H606 (D). Antigens that are circled and numbered contained epitopes that were not shared by all four strains. Wien, Austria, 1988). If this relationship holds true for additional virulent strains, this could prove to be a powerful tool for the study and identification of virulence factors. Serologic differences were not reflected by protein profiles on one-dimensional polyacrylamide gels, and only minor differences were seen by two-dimensional electrophoresis. However, immunoblotting of two-dimensional gels with cross-absorbed antisera confirmed the relationships among the four strains detected by ELISA and identified several peptides containing strain-specific epitopes. The most highly strain-specific antigens were the high-molecular-weight peptides labeled 1 and 2, followed by peptides 3, 4, 5, and 7 in the middle-molecular-weight range (Fig. 3 through 5). Most of these were not completely strain specific but possessed epitopes that were also shared by one or more of the heterologous strains. It is interesting that none of these strain-specific antigens corresponded to the minor protein spot differences seen on silver-stained two-dimensional gels, suggesting that the latter either were not surface oriented or were not important antigens. It is by no means certain that all of the strain-specific epitopes identified in this study were, in fact, surface antigens. However, in preparing our strain-specific antisera, we induced an intra-articular infection with viable mycoplasmas (38), so that the antibody response should have been directed more toward surface than cytoplasmic components.

6 VOL. 28, 1990 ANTIGENIC VARIABILITY AMONG M. ARTHRITIDIS STRAINS A c U u 1o 2.'N 4r 0 *0.,. 4w 9.- gof - *; ~ ~~ 4 The ability of intact mycoplasmas to remove almost all activity from homologous antisera suggested that this was the case. That these cells were not absorbing anticytoplasmic activity was suggested by another set of experiments, in which we prepared rabbit antisera by immunizing with sonically disrupted mycoplasmas. These antisera presumably contained considerable antibody against both soluble and membrane antigens, and we were unable to successfully absorb ELISA activity from them with intact cells, regardless of how much antigen we used (G. L. Budweg and L. R. Washburn, unpublished data). However, a third set of antisera prepared once again against viable mycoplasmas provided results essentially identical to those for the first set (Washburn and Budweg, 7th Cong. Int. Org. Mycoplasmology, 1988). Therefore, we believe that most, although per- f B DO 7 ~1 2Q(~~1 9 e FIG. 4. Two-dimensional immunoblots of strain PG6 (strain C) exposed to homologous unabsorbed rabbit antiserum (U) and homologous antiserum that was absorbed with intact cells from strains 158plOp9 (A), 14124plO (B), PG6 (C), and H606 (D). Antigens that are circled and numbered contained epitopes that were not shared by all four strains. haps not all, of the strain-specific antigens demonstrated by cross-absorption with intact cells were surface oriented. In 1970, Golightly-Rowland et al. (12) reported that avirulent strains were more sensitive to rabbit MI antibodies than were virulent strains; however, the present study does not confirm this. The reason for the discrepancy is not clear, since we used four of the same strains and a similar assay system as were used in the earlier investigation. MI antigens are located on the surface of the M. arthritidis cell (39), and these antigens are strain specific for a number of Mycoplasma species (15, 18, 21, 26). In view of the results of earlier studies, we were surprised to find only minimal differences among the MI antigens from these four M. arthritidis strains. This close relationship has now been confirmed by cross-absorption with a total of eight strains (L. R. Washburn, unpublished data). This condition is not unique to M. arthritidis; for example, the MI antigens of Mycoplasma arginini are also reported to be homogeneous (1) İn 1965, Tully reported that two M. arthritidis isolates from different sources failed to completely cross-react by immunofluorescence (32). To our knowledge, this is the first time since then that distinct antigenic differences have been reported among different strains of M. arthritidis. This work provides a basis on which to study the relationship between virulence and the antigenic makeup of M. arthritidis and may

7 1980 WASHBURN AND HIRSCH J. CLIN. MICROBIOL. QQi9< ,5C 65i' 4 A c u.1 il> 2 * 8e) /'Av &. 3 (< u Z (Z,4 --" O * F 4K 6*..( 7 ;# also allow a systematic grouping of strains within this species. ACKNOWLEDGMENTS This work was supported, in part, by grants from the University of South Dakota School of Medicine Parson's Endowment Fund, the University of South Dakota General Research Fund, and Public Health Service grant R01 AR36946 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. We thank Richard Duman for skilled assistance in the preparation of media and reagents. LITERATURE CITED 1. Alexander, A. G., and G. E. Kenny Characterization of the strain-specific and common surface antigens of Mycoplasma arginini. Infect. Immun. 29: B D il FIG. 5. Two-dimensional immunoblots of strain H606 (strain D) exposed to homologous unabsorbed rabbit antiserum (U) and homologous antiserum that was absorbed with intact cells from strains 158plOp9 (A), 14124plO (B), PG6 (C), and H606 (D). Antigens that are circled and numbered contained epitopes that were not shared by all four strains. 2. Bartholomew, L. E., E. Wheeler, and F. R. Nelson Heat-sensitive mycoplasma antigens. J. Lab. Clin. Med. 72: Batteiger, B., W. J. Newhall V, and R. B. Jones The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55: Cassell, G. H., J. R. Lindsey, H. J. Baker, and J. K. Davis Mycoplasmal and rickettsial diseases, p In H. J. Baker, J. R. Lindsey, and S. H. Weisbroth (ed.), The laboratory rat, vol. I, biology and diseases. Academic Press, Inc., New York. 5. Cole, B. C., J. F. Cahill, B. B. Wiley, and J. R. Ward Immunological responses of the rat to Mycoplasma arthritidis. J. Bacteriol. 98: Cole, B. C., M. L. Miller, and J. R. Ward A comparative study on the virulence of Mycoplasma arthritidis and "Mycoplasma hominis type 2" strains in rats. Proc. Soc. Exp. Biol. Med. 124: Cole, B. C., and J. R. Ward Fate of intravenously injected Mycoplasma arthritidis in rodents and effect of vaccines. Infect. Immun. 7: Cole, B. C., and J. R. Ward Interaction of Mycoplasma arthritidis and other mycoplasmas with murine peritoneal macrophages. Infect. Immun. 7: Cole, B. C., and J. R. Ward Mycoplasmas as arthritogenic agents, p In J. G. Tully and R. F. Whitcomb (ed.), The mycoplasmas, vol. II, human and animal mycoplasmas. Academic Press, Inc., New York.

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