Mycoplasma arthritidis was demonstrated to incite experimental conjunctivitis and uveitis in Swiss Webster mice which have a known susceptibility to the.
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Mycoplasma arthritidis-Induced Ocular Inflammatory Disease CHARLES E. THIRKILL* AND DALE S. GREGERSON Yale University School of Medicine, Department of Ophthalmology and Visual Sciences, New Haven, Connecticut 06510 Received 26 October 1981/Accepted 29 December 1981
Mycoplasma arthritidis was demonstrated to incite experimental conjunctivitis and uveitis in Swiss Webster mice which have a known susceptibility to the arthritis customarily associated with infection by this mycoplasma. The initial symptom of ocular involvement was conjunctivitis, which appeared as early as 1 day after intravenous injection with viable culture concentrates of M. arthritidis. By day 2, histological analysis showed intraocular localized inflammatory reactions that were confined primarily to the anterior portion of the uvea and produced results which were compatible with those seen in iridocyclitis. Serological assays of the titer and the class of antibodies involved in the early humoral immune response to infection confirmed the predominance of immunoglobulin G (IgG) concentrations over IgM concentrations that was described by others (Cole et al., Infect. Immun. 4:431-440, 1971) and revealed significant titers of the IgG2a and IgG2b subclasses of complement-fixing antibodies. The rapid onset of acute conjunctivitis, together with the early appearance of immunoglobulins of the IgG class, suggests that the M. arthritidis-infected Swiss Webster mice may have experienced an anamnestic response to the mycoplasma antigens. These observations introduce a new animal model for the study of mycoplasma-induced experimental uveitis and conjunctivitis, which are demonstrated here to accompany a disseminated systemic disease process.
Mycoplasma arthritidis has been the subject of many investigations designed to determine the means whereby this microorganism is able to incite localized inflammations in susceptible strains of rats and mice. With the exception of the cataracts produced in neonatal mice after intracerebral inoculations of the suckling mouse cataract spiroplasma (26), the only reports of intraocular lesions that result from infection with mycoplasmas came from studies in which viable organisms (M. pulmonis) were injected directly into the eyes of experimental animals (19, 20, 22). Since M. arthritidis is known to incite extensive joint reactions when introduced intravenously into Swiss Webster mice and since mycoplasmas resembling this organism have been isolated from patients suffering from rheumatoid arthritis in which iridocyclitis is common (2), an interest arose in the possible uveitogenic ability of this mycoplasma. The possible role of mycoplasmas in the induction of the triad of symptoms comprising Reiter's syndrome, especially the conjunctivitis which occasionally extends into iridocyclitis, is of particular relevance to this study (18, 21), particularly since the species of mycoplasma most commonly isolated from such patients, M. hominis, 775
cross-reacts antigenically with the "Campo" strain of M. arthritidis which was formerly designated M. hominis type 2 (7, 11). M. arthritidis incites a polyarthritis in Swiss Webster mice when sufficient numbers of viable particles are introduced through a single tail vein inoculation, whereas killed suspensions only induce a mild, transient immunity to subsequent infection with viable cells (8, 9). Although joint involvement has previously been the major source of interest in studies of
the pathogenic characteristics of M. arthritidis, the related uveitis and conjunctivitis which we have observed to accompany infection with this mycoplasma represent hitherto largely neglected facets of a complex infectious disease. The observations presented here lead us to believe that a form of experimental anterior uveitis and conjunctivitis results in Swiss Webster mice after a single tail vein injection of viable, washed, adjuvant-free M. arthritidis. Therefore, we submit the described procedure as an additional animal model for the study of mycoplasma-induced ocular inflammatory diseases, which in this case results from a generalized infectious disease process. (A preliminary report of these observations
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was presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla., 26 April 1981 to 1 May 1981. MATERIALS AND METHODS Mycoplasma cultivation and enumeration. A viable mycoplasma concentrate of M. arthritidis (strain 158 P1o Pg, kindly supplied by B. C. Cole of the Division of Arthritis, University of Utah College of Medicine, Salt Lake City) was cultivated aerobically at 37°C in Difco PPLO broth and on Difco PPLO agar which contained 10% horse serum, 100 U of penicillin per ml, 0.001% phenol red, 0.5% glucose, and 0.25% L-arginine hydrochloride. A single colony scooped from the surface of a triple-cloned 72-h plate culture was used to inoculate 2-liter broth cultures, which were then incubated at 37°C for 72 h before being harvested by centrifugation at 10,000 x g for 30 min at 4°C. The pooled pellet obtained from each 2-liter culture was washed twice by centrifugation in 100 ml of sterile phosphate-buffered saline, resuspended to a final volume of 2 ml with sterile phosphate-buffered saline, and stored at -20°C for the time required to obtain an approximation of the total viable count. All culture concentrates were standardized and introduced into mice within 1 week of harvest. Culture concentrates of M. arthritidis used for test antigens were homogenized and standardized to 10 mg of protein per ml as previously described (24). Mouse inoculation. Four sets of six female Swiss Webster mice (Charles River Breeding Laboratories, Inc., Wilmington, Mass.), 5 to 6 weeks of age, were used in this study; five test animals and one control animal were in each group. The eyes of all mice were examined to establish that they were free of any gross abnormalities before being used. All mice were test bled from the tail vein 3 days before inoculation to provide preimmune sera for comparisons. On day 0, five test mice were given single tail vein injections of 0.1 ml of the concentrate of a freshly harvested 72-h culture of M. arthritidis containing approximately 106 colony-forming units per ml. The remaining mouse, serving as a negative control, was given 0.1 ml of sterile complete mycoplasma culture medium by the same route. On each subsequent day mice were examined for any gross changes in their physical appearance, and one test mouse was sacrificed by cervical dislocation. Serum samples collected from individual mice in all four groups were labeled and stored at -20°C to be assessed together for overall antibody response to M. arthritidis and for the relative concentration of antibody subgroups involved. The eyes of both the test and the control mice were promptly removed with sterile scissors and placed into a sterile petri dish containing 2 ml of complete sterile PPLO broth. They were cut into two equal longitudinal halves with a sterile razor blade, and the lens, lens capsule, and associated vitreous were immediately transferred to sterile PPLO broth in an attempt to reisolate M. arthritidis from within the eyes of infected mice. Histology. Two halves from different eyes of the same mouse were dropped into 2 ml of 2% glutaraldehyde in phosphate buffer at pH 7.4 for processing into Araldite. The remaining two halves were fixed in 2 ml of 95% ethanol in preparation for embedding in paraffin wax. In the first two sets the negative control mice were killed on day 1 with the first test mouse. In the
INFECT. IMMUN.
second two sets negative controls were killed on day 5 with the last test mouse. All processed eye samples were labeled and stored until the biopsies from the four groups of 6 mice could be assessed as a single set of 20 test mice and 4 control mice. Conjunctivitis was scored by noting the presence of mucoid discharge and the presence of edema and hyperemia of the lids and conjunctiva. Values of 0, 1, or 2 were assigned to scores of none, moderate, or severe, respectively. The total of the individual scores was then divided by the total number of mice. Iridocyclitis was scored on the basis of iris vessel dilation and circumlimbal injection, histological evidence of iris-ciliary body edema, the presence of leukocytic infiltration, the deposition of cells and debris on the corneal endothelium, and the presence of choroidal inflammation. Values of 0, 1, or 2 were assigned to scores of none, moderate, or severe, respectively. The total of the individual scores was then divided by the total number of mice. Serology. The enzyme-linked immunosorbent assay (ELISA), modified from the method described by Voller et al. (27), was used as a means of detecting, titrating, and characterizing the antibody response of mice throughout the 5-day observation period. Absorption readings were made at 405 nm with a Titertek Multiscan (Flow Labs, Inglewood, Calif.). Previous studies have shown that the complement fixation assay does not detect a response before 7 and 4 days postinfection, in mice (6, 8) and rats (4), respectively; hence, that is why we used ELISA. Since the titers of commercially obtained immunoglobulin heavy chain typing sera vary with respect to their reactivity with homologous antigens, each was titrated to ensure uniform potency. Rabbit anti-mouse immunoglobulin heavy chain sera (Litton Bionetics Laboratory Products, Inc., Kensington, Md.) were standardized to exhibit the same color intensity when reacted with a constant concentration of their respective antigens, affinity-purified mouse immunoglobulin subclasses immunoglobulin M (IgM), IgA, IgGl, IgG2a, IgG2b, and IgG3 (Litton).
RESULTS Observation of the four groups of six mice over the 5-day experimental period indicated a consistent alteration in the gross appearance of the test mice which was first apparent 24 h postinoculation. Normal active movement about the cage by the previously bright-eyed, sleek-coated Swiss Webster mice was replaced with an increasing tendency to huddle together with ruffled fur and closed, weepy eyes. By 48 h, a large volume of mucoid discharge was accompanied by edema of the lids and conjunctiva of all infected mice. Rapid resolution of the conjunctivitis began at 96 h, and overt extraocular symptoms continued to decline thereafter. Close examination of the eyes of mice which subsequent histopathological examination revealed to be affected commonly indicated an overall bilateral reddening effect resulting from vascular dilation of the vessels in the iris (Fig. 1). The behavior and appearance of the control mouse in each set remained normal. Histopathology. Alterations in the character of
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intraocular tissues were first observed in day 2 biopsies when indications of edema were apparent within the iris of one of the four test mice in that set (Fig. 2); the remaining mice in this group were asymptomatic. Sections from two of the four test animals in group 3, representing day 3, showed evidence of an ongoing disease process involving both the ciliary process and iris. A cellular infiltrate consisting primarily of polymorphonuclear neutrophils was apparent within the ciliary processes and extended into the iris, which exhibited the extensive edematous changes apparent in Fig. 3. Three of the four test mice in group 4 exhibited ciliary-iris involvement consistent with that already described, whereas the fourth showed the anterior lesions and a localized posterior choroidal abscess with extensive neutrophil infiltration (Fig. 4). Sections cut from the eyes of two of the mice harvested on day 5 showed evidence of anterior inflammation limited to the iris-ciliary body complex, whereas the remaining two test mice in this group were normal. Eyes from the four
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control mice in this study were normal in both gross and microscopic appearance. In all, 9 of the 20 infected test mice were found to have experienced acute bilateral iridocyclitis, confined primarily to anterior uveal tissues, although one also exhibited posterior uveal involvement. Microbiological cultivation. Positive isolations of M. arthritidis from lens-inoculated PPLO broth were obtained from only 2 of the 20 test animals inoculated with this mycoplasma. Tubes inoculated with the lenses of all remaining mice remained sterile after 7 days of incubation. Both positive isolates were obtained from the day 1 groups, 24 h post-inoculation. Serum antibody assessments. The overall antibody response to the antigens of M. arthritidis was detectably elevated by day 1, in comparison to that apparent in the normal controls (Fig. 5). A progressive increase in titer that manifested over the 5-day observation period testified to the presence of an ongoing humoral immune response incited by the disease process. The antibody subgroups involved in this response were dominated by antibodies in the complementfixing subclasses IgG2a and IgG2b. Concentrations of the remaining complement-fixing subclass, IgM, remained comparatively low (Fig. 6). A modest increase in the IgA class was also found.
DISCUSSION Mycoplasmas are responsible for the induction of a variety of infectious diseases in birds and mammals, including respiratory tract, urogenital, joint, and central nervous system involvement. Certain species of mycoplasma have occasionally been isolated from uveitis patients (1, 10, 15-17). The possibility that severe ocular lesions might accompany infection with known arthritogenic species of mycoplasma has been a source of interest to other investigators who have sought to duplicate the symptoms of uveitis in experimental animals through intraocular injections of living culture concentrates (19, 20, 22), although it is well known that the introduction of virtually any proteinaceous compound or microorganism directly into the eye will produce "uveitis." M. arthritidis will incite intraarticular inflammations in both rats and mice, and although many recover completely within 2 months, chronic lesions do persist in some animals and are prone to exacerbation (9). A state of prolonged parasitism is believed to exist in chronically infected rodents in which M. arthritidis FIG. 1. (A) Gross appearance of the normal eye of avoids complete elimination by its ability to survive for lengthy periods sequestered within a control Swiss Webster mouse contrasting with that of (B) a symptomatic test mouse harvested on day 2 in joint spaces and its possession of surface antiwhich the iris vasculature is dilated and contributes to gens that are postulated to closely resemble the observed reddening effect. those of the host (3). An avoidance of immune
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FIG. 2. (A) Normal appearance of the iris-ciliary complex of one of the three asymptomatic test mice harvested on day 2. (B) Iris-ciliary complex of the one symptomatic test mouse harvested on day 2. The edematous iris and the leukocytic infiltration into the ciliary body combine to characterize the predominating type of tissue inflammation observed in these mice. (C) Perivascular infiltration of polymorphonuclear neutrophils in the ciliary body of the mouse harvested on day 2. At this early stage the epithelial layer is largely intact. (D) Extensive infiltration of the ciliary body seen by day 4. The normal architecture of the ciliary body has been lost. Bars in (A) through (D) = 100 jLm.
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surveillance through an overall lack of immunogenicity has been proposed as a survival characteristic which could lead to confusion by the immune response of the host, possibly resulting in autoimmune reactions (14, 25). The results obtained in this study indicate the existence of additional pathogenic characteristics which may be attributed to M. arthritidis, such as the rapid production of severe conjunctivitis followed by the appearance of iridocyclitis within the eyes of mice experiencing a generalized disease. Despite the concurrent increase in the severity of joint involvement in similarly infected groups of Swiss Webster mice, the associated conjunctivitis has, in our experience, undergone complete resolution within 8 days (unpublished data). Histopathologically, the ob-
served intraocular lesions were characterized by edema and polymorphonuclear neutrophil infiltrate that was confined predominantly to the anterior uvea. The fact that both positive isolates of M. arthritidis from lens tissues were obtained from lens biopsies cultured 24 h post-inoculation might argue that a simple bacteremia may be responsible for the success in reisolating M. arthritidis from two of the infected mice. The overall serum antibody response in the test mice indicated the appearance of a prompt antibody rise in titer to the antigens of M. arthritidis that was clearly demonstrable within 24 h of exposure. The early increase in specific antibody content could be interpreted to reflect previous exposure to M. arthritidis, a classical
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DAYS POST-INFECTION FIG. 5. Comparison of ocular inflammation (conjunctivitis and iridocyclitis), arthritis (foot swelling), and anti-M. arthritidis titers during the 5-day period after infection with live organisms. The mean and the standard deviation of the serum antibody response to the antigens of M. arthritidis of a total of 20 infected Swiss Webster mice, as assessed by the ELISA method, are shown (-----). Test mice were sacrificed at 24-h intervals over the 5day observation period. Two negative control mice were sacrificed on day 1, and two more were sacrificed on day 5. Symbols: 0, relative degree of conjuctivitis; *, iridocyclitis; A, arthritis. The degree of joint inflammation was scored as previously described (4). Eye inflammation was scored as described in the text. A 2-fold increase in antibody titers corresponds to a 1.38-fold increase in the absorbance at 405 nm (A405nm).
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DAYS POST-INFECTION FIG. 6. Average content of anti-M. arthritidis-specific antibody subgroups within the infected and control mouse serum samples, as assessed by the ELISA method. Note the relatively high initial content of the complement-fixing subclass IgG2b evident in both the test group and the pairs of controls sacrificed on days 1 and 5. The heavy chain subclasses assayed include y2b (0), y2a (U), y3 (A), yl (A), a (O), and (0). A405nm, Absorbance at 405 nm.
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M. ARTHRITIDIS-INDUCED UVEITIS
anamnestic response resulting from endemic infection with this strain of mice. Since the blood
cytes: induction of cytotoxic lymphocytes and lymphocyte transformation by Mycoplasma arthritidis are under Ir gene control. J. Immunol. 126:922-927. Cole, B. C., L. Golightly-Rowland, and J. R. Ward. 1976. Arthritis in mice induced by Mycoplasma arthritidis. Ann. Rheum. Dis. 35:14-22. Cole, B. C., M. L. Miller, and J. R. Ward. 1967. A comparative study of the virulence of Mycoplasma arthritidis and "Mycoplasma hominis, type 2' strains in rats. Proc. Soc. Exp. Biol. Med. 124:103-107. Cole, B. C., J. R. Ward, L. Golightly-Rowland, and G. A. Trapp. 1971. Chronic proliferative arthritis in mice induced by Mvcoplasma arthritidis. II. Serological responses of the host and effects of vaccines. Infect. Immun. 4:431-440. Cole, B. C., J. R. Ward, R. S. Jones, and J. F. Cahill. 1971. Chronic proliferative arthritis of mice induced by Mycoplasma arthritidis. I. Induction of disease and histopathological characteristics. Infect. Immun. 4:344-355. deGrosz, I. 1962. Significance of mycoplasmas in the pathology of uveitis. Acta Ophthalmol. 40:600-607. Edward, D. G. ff., and E. A. Freundt. 1965. A note on the taxonomic status of strains like 'Campo," hitherto classified as Mycoplasma hominis, type 2. J. Gen. Microbiol. 41:263-265. Gamble, C. N., S. B. Aronson, and F. B. Brescia. 1970. Experimental uveitis. Arch. Ophthalmol. 84:321-330. Gamble, C. N., S. B. Aronson, and F. B. Brescia. 1970. The pathogenesis of recurrent immunologic (Auer) uveitis. Arch. Ophthalmol. 84:331-341. Harwick, H. J., G. M. Kalmanson, M. A. Fox, and L. B. Guze. 1973. Mycoplasma arthritis of the mouse: development of cellular hypersensitivity to normal synovial tissue. Proc. Soc. Exp. Biol. Med. 144:561-563. Hessburg, P. C., L. H. Mattman, C. Barth, and L. T. Dutcheshen. 1969. Aqueous microbiology: the possible role of cell wall deficient bacteria in uveitis. Hernry Ford Hosp. Med. J. 17:177-194. Holland, M. C. 1960. Uveitis and pleuropneumonialike organisms. Ann. N.Y. Acad. Sci. 79:646-649. Holland, M. C., and J. T. Worlton. 1957. Relationship of pleuropneumonia-like organisms and uveitis. Am. J. Ophthalmol. 43:597-606. Kossman, J. C., D. Floret, H. Renaud, N. Skpetjian, and P. Monet. 1980. Reiter's syndrome in children associated with Ureaplasma urelyticum. Pediatrie 35:237-242. Kunishi, M. 1973. Experimental mycoplasma-induced uveitis. Acta Soc. Ophthalmol. Jpn. 77:1204-1217. Kunishi, M. 1974. Experimental uveitis due to Mycoplasma pulmonis. Jpn. J. Ophthalmol. 18:150-160. Oates, J. D., M. J. Whittington, and A. E. Wilkinson. 1959. A note on the results of cultural and serological tests for pleuropneumonialike organisms in Reiter's disease. Br. J. Vener. Dis. 35:184-186. Pavan-Langston, D. 1969. Mycoplasmal anterior and posterior uveitis. Arch. Ophthalmol. 82:245-258. Sabin, A. 1940. Experimental proliferative arthritis in mice produced by filterable pleuropneumonia-like microorganisms. Science 89:228-229. Thirkill, C. E., and G. E. Kenny. 1975. Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis. J. Immunol. 114:1107-1111. Thirkill, C. E., H. G. Muchmore, R. M. Hyde, and L. V. Scott. 1981. Immunologic reactions of rabbit anti-Mycoplasma arthritidis serum with in vitro cultivated rat synovial cells. In Vitro 17:405-411. Tully, J. G., R. R. Whitcomb, D. L. Williamson, and H. F. Clark. 1975. Suckling mouse cataract agent is a helical wall-free prokaryote (Spiroplasma) pathogenic for vertebrates. Nature (London) 259:117-120. Voller, A., D. Bidwell, and A. Bartlett. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections, p. 506-512. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C.
vessels of the uvea are fenestrated, the appearance of circulating immune complexes and their deposition in the anterior uvea could be a mechanism for the rapid onset of iridocyclitis that we have observed. Other investigators have reported a similar acute onset in a model for immune complex-mediated iridocyclitis in which bovine serum albumin is used (12, 13). The rapid onset of ocular inflammation described here may therefore reflect a hypersensitivity reaction in a presensitized host. It is considered possible that undetected foci of arthritogenic mycoplasma may persist in colonies of susceptible mice, a phenomenon which, although not yet shown to be a characteristic of M. arthritidis, has been demonstrated previously by the isolation of arthritogenic mycoplasma from the brains of apparently normal, healthy mice (23). Although the onset of the appearance of antibody in complement-fixing subclasses occurred earlier than that previously reported by others (6, 8), the increased sensitivity of the ELISA method of analysis over that of conventional complement fixation techniques could allow for the differences in reported results. No complement-fixing 19S antibodies were detected in infected mice by Cole et al. (8). Although there are indications that autoimmune reactions may play some part in mycoplasma-induced pathological processes (3, 14, 25) and that genetic influences account for the greater susceptibility of different strains of rodents (5), the exact biological mechanisms involved in mycoplasma-induced arthritis and in the experimental uveitis reported here are not fully understood. ACKNOWLEDGMENTS This work was supported by Public Health Service grants EY-02365, EY-03660, EY-07000, and EY-00785 from the National Eye Institute and unrestricted funds from Connecticut Lions Eye Research Foundation and Research to Prevent Blindness. C.E.T. is a postdoctoral trainee supported by grant EY-07000. We thank Mary Bannon for typing the manuscript.
6. 7.
8.
9.
10. 11.
12. 13. 14.
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16. 17. 18. 19.
20. 21.
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LITERATURE CITED Arm, H. D., R. L. Woolridge, K. Cheng, and I. Chang. 1966. Isolation of mycoplasma organisms (PPLO) from the human eye. Am. J. Ophthalmol. 62:1125-1127. Bartholomew, L. E. 1965. Isolation and characterization of mycoplasmas (PPLO) from patients with rheumatoid arthritis, systemic lupus erythematosus and Reiter's syndrome. Arthritis Rheum. 8:376-388. Cahill, J. F., B. C. Cole, B. B. Wiley, and J. R. Ward. 1971. Role of biological mimicry in the pathogenesis of rat arthritis induced by Mycoplasma arthritidis. Infect. Immun. 3:24-35. Cole, B. C., J. F. Cahill, B. B. Wiley, and J. R. Ward. 1969. Immunological responses of the rat to Mycoplasma arthritidis. J. Bacterial. 98:930-937. Cole, B. C., R. A. Daynes, and J. R. Ward. 1981. Mycoplasma-mediated activation of normal mouse lympho-
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