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Although some viruses and bacteria can induce chronic arthritis, the mycoplasmas as a group are the most common etiologic agent of naturally occurring chronic joint inflammation (12). Mycoplasmas are well known arthritogenic agents in humans, cattle, sheep, goats, swine, rodents and poultry. Mycoplasma-induced arthritis of poultry under field conditions is almost entirely caused by Mycoplasma synoviae infection (18). M. synoviae infection most frequently occurs as a subclinical upper respiratory infection but may become systemic, resulting in arthritis, termed commonly infectious synovitis. Infectious synovitis is an acute to chronic disease of chickens and turkeys, involving primarily the synovial membranes of joints and tendon sheaths and producing an exudative synovitis, tendovaginitis, or bursitis (22). Following M. synoviae egg transmission, infectious synovitis has been observed in 6-day-old chickens. In flocks with clinical synovitis, morbidity varies from 2 to 75%, with 5 to 15% morbidity being most usual (22). The leg joints are most consistently involved, particularly the tibiotarsal-tarsometatarsal joints (hock joints) and tarsometatarso-phalangeal joints. Some of the arthritic and systemic forms of M. synoviae disease of chickens resemble an immune complex disease (12, 18). Whether autoimmune or other immunologic mechanisms are involved in the pathogenesis is not clear (12). In experimentally induced M. synoviae arthritis in chickens, granular immunoglobulin G (IgG) deposits along with vasculitis and glomerulonephritis have been observed (19-21). Rheumatoid factors have been reported in both natural and experimental mycoplasmal infections (26, 37), but they have been seen in uninfected birds as well (12). Cold agglutinins may appear in M. synoviae-infected turkeys (32), but the significance of this is unclear.

Resistance to lesions induced by M. synoviae is bursal lymphocyte dependent (20, 23, 36), while thymus-dependent lymphocytes may be needed for the development of macroscopic synovial lesions (19, 20, 23). While B and particularly T lymphocytes seem to play an important role in the pathogenesis of M. synoviae-induced arthritis, there is no available information about the involvement of lymphocyte subpopulations. Moreover, data about cellular immunity in M. synoviae infection in chickens are lacking, and the cell-mediated response has been demonstrated only by leukocyte migration inhibition and skin testing (12, 35). A local antibody response to M. synoviae in synovial fluid from chickens has been reported (3) but without evidence of which proteins are targets of the antibody response. M. synoviae synthesizes several major membrane antigens which undergo phase-variable expression associated with hemagglutination (HA) and hemadsorption (HAD) to chicken erythrocytes (4, 24, 25). M. synoviae (type strain WVU 1853) putative hemagglutinins MSPA (50 kDa) and the coexpressed MSPB (45 to 47 kDa) have recently been described (24). Because a single gene from a multigene family encodes M. synoviae hemagglutinin, MSPA and MSPB proteins are products of posttranslational cleavage (25). Adhesins play a crucial role in the initial stage of infection with pathogenic Mycoplasma species (28); this is probably also the case with M. synoviae. In M. synoviae AAY-4 isolated from a chicken in Slovenia (15), we identified groups of phase-variable surface membrane proteins with molecular masses from 45 to 80 kDa associated with HA and HAD (4, 6). A number of isogenic lineages with HA-positive (HA⁺) and HA-negative (HA) phenotypes were established. Proteins relevant to the HA⁺ phenotype were defined with monoclonal antibodies (MAbs) raised against M. synoviae AAY-4 hemagglutinin (4, 6).

The present study was undertaken to investigate the influence of the hemadherent phenotype of M. synoviae in experimentally induced arthritis. Our previous pilot experiments showed that inoculation of chicken hock joints with 10⁶ to 10⁸ CFU of M. synoviae induced infectious synovitis in the majority of inoculated birds and that cyclosporin A (CsA) could reduce clinical signs of synovitis if it was injected into the infected joint. Frey's broth medium used to grow M. synoviae (22) or heat-inactivated broth cultures (2 × 10⁸ CFU; heated at 60°C for 30 min) did not induce synovitis even if they were inoculated into hock joints three times at 2- or 3-week intervals. For further studies, groups of 4- to 5-week-old broiler-type chickens from mycoplasma-free flocks were used. Before each experiment they were examined for mycoplasma infection by culture of tracheas and choanal clefts on Frey's agar and broth media and for antibodies to M. synoviae, M. gallisepticum, and M. iowae by rapid serum agglutination, HA-inhibition (HI), indirect immunoperoxidase assay (IIPA), and immunoblotting as described previously (3, 5, 22). All chickens tested negative before the experiments, and uninoculated chickens (negative controls) also remained negative at the end of each experiment.

M. synoviae cultures used in experiments. In this study in three experiments HA⁺ or HA cultures of a low-passage (<10 passages) arthrogenic M. synoviae strain, AAY-4, were used. To obtain HA⁺ and HA clones, AAY-4 colonies (derived from a single colony) were examined for HAD of chicken erythrocytes. Individual HAD-positive (HAD⁺) and HAD-negative (HAD) colonies were grown in broth to expand isogenic lineages, and aliquots of washed pelleted cells were assayed for HA titers to confirm their HA⁺ or HA phenotype (22, 30). In experiments 1, 2, and 3, HA⁺ clones designated P1 (3 × 10⁷ CFU; HA titer, 2 × 10⁴), P2 (2.8 × 10⁷ CFU; HA titer, 4 × 10⁴), and P3 (1.1 × 10⁶ CFU; HA titer, 4 × 10⁴), respectively, were used. In experiments 1 and 2, HA clones N1 (3 × 10⁷ CFU; hemagglutinin titer, <10²) and N2 (3.1 × 10⁷ CFU; hemagglutinin titer, <10²) were used. Groups of 10 chickens were inoculated in the hock joint cavity with 0.2 ml of broth cultures in the log phase of growth. The design of experiments 1, 2, and 3 is shown in Table 1. Following inoculation with M. synoviae, joint lesions (swelling, edema) were observed daily and evaluated macroscopically. Lesions were scored from 0 to 3 (a score of 2 or higher was counted as positive) as described elsewhere for experimentally induced M. synoviae synovitis of chickens (23). Synovial fluid was collected at necropsy 2 or 3 weeks post inoculation (p.i.) by syringe. From the majority of swollen hock joints a small sample of synovial fluid was taken during an acute phase when the first signs of inflammation became apparent. Synovial fluid samples (20 µl) were seeded onto Frey's agar plates. M. synoviae colonies were identified by the direct immunofluorescence test (2), and the number of colonies was counted.

Higher frequency of synovitis induced by HA⁺ than by HA M. synoviae cultures. In experiment 1, chickens inoculated with the HA⁺ culture developed signs of acute synovitis (lameness and apparent swelling around the affected hock joint) from 7 to 10 days p.i. Nine of ten chickens developed synovitis, while in the group inoculated with the same number (3 × 10⁷) of CFU of the HA culture only one chicken developed mild swelling (score 2) of the inoculated hock joint. The mean joint lesion scores were 2.8 and 0.5 for groups of birds inoculated with HA⁺ and HA clones, respectively. The difference between the two groups in the frequency of clinical signs of acute synovitis is highly significant (P = 0.001; chi-square test [FREQ, Statistical package SAS/STAT, SAS 96]). M. synoviae isolation from synovial fluid 2 weeks p.i. did not correlate with signs of synovitis (Table 1, experiment 1). Articular lesions were not correlated with recovery of mycoplasmas, and this has also been reported previously (23).

Differences between proteins of M. synoviae HA⁺ and HA clones. An interesting finding was that two HA⁺ clones caused a significantly higher frequency (P = 0.001) of acute synovitis than did the same number of viable cells of two HA clones derived from the same M. synoviae population. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the whole-cell proteins of HA⁺ and HA clones of the AAY-4 strain was done, and proteins were stained with Coomassie blue (1, 5). It was apparent that HA⁺ clones (including clones P1, P2, and P3 used to induce synovitis) synthesized proteins of 48 to 50, 53, and 67 to 70 kDa more abundantly than the corresponding HA clones (including clones N1 and N2). Further analyses using Triton X-114 partitioning to obtain integral membrane proteins of M. synoviae (1) and immunoblotting with the PhastSystem (Pharmacia LKB) (5) indicated that the 48- to 50-kDa proteins defined by MAb 125 (17) are homologues of the MSPB proteins in M. synoviae WVU 1853 (17, 24). Furthermore, the 53-kDa protein defined by MAb 3E10/C9 (which inhibits binding of M. synoviae to erythrocytes) is homologous to MSPA hemagglutinin (4, 6, 24). The N-terminal amino acid sequences determined for the proteins defined by MAb 3E10/C9 in the AAY-4 HA⁺ clone (KF9) indicate the cleavage site in M. synoviae hemagglutinin which cleaves into MSPB and MSPA (6, 25). The 67- to 70-kDa proteins have the capability to hemagglutinate chicken erythrocytes and seem to be size and antigenic variants of M. synoviae hemagglutinin (6). HA⁺ and HAD⁺ phenotypes of the AAY-4 strain are thus associated with the expression of proteins detected by MAbs 125 and 3E10/C9. HA and HAD clones lack the 53-kDa protein defined by MAb 3E10/C9 and synthesize truncated (27- to 30-kDa) proteins recognized by MAb 125. About 1% of the HAD⁺ population reverts in vitro to an HAD subpopulation, and the reversion from HAD to HAD⁺ occurs at similar rate (4, 6). M. synoviae attachment to erythrocytes (via sialic acid receptors) may be not exactly the same as its attachment to the cells on the surface of the hock joint cavity, but HA⁺ clones are more capable of inducing acute synovitis than are HA clones. We found this to be the case also for HA⁺ and HA clones of the type strain WVU 1853 of M. synoviae (unknown, high level of passages; obtained from Janet M. Bradbury, University of Liverpool). Cells of an HA⁺ clone (2 × 10⁷ CFU; HA titer, 2 × 10⁴) induced acute infectious synovitis in 50% of inoculated hock joints (n = 8), while a similar number of cells of an HA clone (2.2 × 10⁷ CFU; HA titer, <10²) did not induce synovitis. It has been previously reported that in strain WVU 1853, the hemadherent population expresses MSPA and MSPB, which are not expressed in the nonhemadherent population (24, 25). Our data also indicate a lack of MSPA-like protein in HA clones of WVU 1853. However, in HA clones, MSPB defined by MAb125 appears in a truncated form of 27 to 30 kDa, which might be equivalent to the 25- to 30-kDa MSPC protein which shares antigenic determinants with MSPB (6, 24, 25). It is possible that other proteins besides hemagglutinins are also involved in the induction of acute synovitis with M. synoviae, but this remains to be investigated.

Suppression of synovitis by CsA. CsA is a well-known immunosuppressor which, bound to its receptor (cyclophilin), prevents synthesis of interleukin-2 by blocking a late stage of the signalling pathway initiated by the T-cell receptor (33). In experimentally induced M. synoviae synovitis in chickens, T lymphocytes are necessary for development of macroscopic lesions (23). We attempted to suppress M. synoviae-induced synovitis with CsA. When hock joints were inoculated with M. synoviae, the left joints were also inoculated with 1 mg of CsA. Joints of half of the birds in experiment 2 and 10 birds in experiment 3 (groups A and C) were inoculated with 1 mg of CsA three times at 2-day intervals (Table 1). In experiment 2, in chickens inoculated with the HA⁺ culture, the frequency of synovitis was reduced from 70% in the CsA-untreated joints to 10% in the CsA-treated joints, and there was no swelling of hock joints treated with CsA three times. In addition, CsA completely prevented synovitis induced by the HA culture which induced mild swelling of a hock joint of only one chicken (Table 1). In experiment 3 with M. synoviae HA⁺ clone P3 (10⁶ CFU), CsA given in multiple doses (three doses of 1 mg) completely prevented acute hock joint swelling (7 to 14 days p.i.), which otherwise appeared in 90% of control joints which were not treated with CsA. Altogether, CsA treatment significantly suppressed the induction of acute joint swelling with M. synoviae (P = 0.001). In addition, CsA reduced the mean lesion score of joints from 2.60 (induced with HA⁺ cultures) to 0.25 (Table 1, experiments 2 and 3). Inoculation with CsA only did not have any adverse effect on joints. Moreover, inoculation of the vehicle used to dissolve CsA did not induce any effect on synovitis or local antibody response (Table 1). The mechanism of CsA activity in suppressing M. synoviae-induced synovitis is at present unclear. It may be due in part to a mycoplasmastatic effect which we observed in vitro and has been also reported for M. pneumoniae (29). On the other hand, CsA activity in vivo was not mycoplasmacidal since it did not significantly reduce isolation rates and the number of CFU of M. synoviae from joints (Table 1). At least a part of CsA suppressor activity seems to be associated with the influence of CsA on immune response cells rather than with its mycoplasmastatic activity since CsA also prevented synovitis induced with M. synoviae membranes (data not shown).

Examination of synovial fluid. Marked edema and large amounts of synovial fluid of the inflamed joint cavity are characteristic of M. synoviae infectious synovitis (22, 23). In experiment 2, an HA⁺ culture caused synovitis lesions in which (3 weeks p.i.) the mean volume of synovial fluid was 0.43 ml, while with the HA culture the corresponding value was 0.15 ml. As shown in Table 1, the clinical signs of synovitis as well as the volume of synovial fluid were suppressed in the CsA-treated joints. The mean volume of synovial fluid was approximately three to four times lower than the corresponding values from joints inoculated with HA⁺ cultures but not treated with CsA. On the other hand, large differences in the volume of synovial fluid within the same groupi.e., in experiment 2 in group A, exudate volumes in joints inoculated with the HA⁺ culture varied from 0.2 to 0.75 mlindicate that synovial exudate volume may not provide an accurate assessment of the degree of joint inflammation. However, volumes of synovial fluid collected at 3 weeks p.i. probably were not comparable with those during the acute stage of synovitis (e.g., 7 to 10 days p.i.) since at 3 weeks p.i. the joint inflammation could be regarded as an early chronic stage.

Antibodies to M. synoviae. It has been suggested that specific antibodies may inhibit M. synoviae replication in the joints (23). We observed that in experimental infections of chickens with M. synoviae strains, inoculation into the upper respiratory tract might fail to induce a detectable systemic antibody response, while intrajoint inoculation with the same strains and numbers of organisms consistently induced a vigorous antibody response (data not shown). In this study all chickens inoculated with M. synoviae developed specific antibodies detectable in serum by rapid plate agglutination, IIPA, and immunoblotting. Immunoblotting using the PhastSystem (Pharmacia LKB) and M. synoviae AAY-4 HA⁺ and HA clones were conducted as described elsewhere (5). Briefly, following transfer of M. synoviae proteins onto an Immobilon P membrane (Millipore), the membrane was blocked in phosphate-buffered saline (PBS) (pH 7.4) containing 0.5% Tween 20 (0.5% PBS-T) for 30 min and cut into strips of appropriate width. The strips were incubated with chicken sera diluted at least 1:100 for 45 to 60 min. Following three consecutive washings for 15 min in 0.05% PBS-T, strips were reacted for 45 min with MAbs (to chicken IgA, IgG, or IgM) conjugated with horseradish peroxidase (5, 14). Following washing in 0.05% PBS-T (twice for 15 min) and PBS (15 min), reacting proteins were visualized by incubation in a solution containing 3,3'-diaminobenzidine (0.25 mg/ml; Sigma) in 0.1 M Tris (pH 7.5) and 0.03% (vol/vol) H₂O₂. Examination of sera from chickens in experiment 2 (group A) showed that when acute synovitis became apparent (7 to 10 days p.i.), IgA, IgM, and IgG antibodies to several M. synoviae proteins were present. Examination of serum samples obtained 2 and 3 weeks p.i. indicated a tendency toward disappearance of specific IgA and IgM antibodies, while the titers of specific IgG antibodies in the IIPA with M. synoviae AAY-4 increased. In immunoblots, specific IgG antibodies recognized at least 10 M. synoviae proteins, with molecular masses from <25 to >200 kDa. They reacted with the greatest intensity with 48- to 50-kDa proteins of an HA⁺ clone of strain AAY-4. Antibodies recognized the MSPB protein (48 to 50 kDa) defined by MAb 125. In the IIPA those sera at low dilutions (1:2 to 1:8) blocked binding of MAb 125 to M. synoviae colonies. Sera obtained from the same birds before inoculation of M. synoviae into joints did not show this activity.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Immunoblots of M. synoviae Triton X-114-extracted proteins with IgG antibodies from synovial fluids from hock joints of chickens following intra-articular infection with M. synoviae AAY-4 HA+ clone culture (experiment 2, group A, right joints). (A) M. synoviae AAY-4 HA+ clone (lanes a) or HA clone (lanes b) immunostained with MAb 125, which defines MSPB proteins (lanes 1), and synovial fluid samples (diluted 1:200) taken from joints 8 days p.i. (lanes 2 to 4). Reactions were stopped when staining of MSPB proteins appeared. The arrow indicates MSPB proteins of 48/50 kDa of HA+ clone. (B) Same antigens described for panel A (same lanes) immunostained with rabbit antiserum (diluted 1:1,000) against HA+ culture of AAY-4 strain (lanes 1) and synovial fluid samples (diluted 1:500) from the same joints as described for panel A, but 3 weeks p.i. (lanes 2 to 4). Bar markers show proteins MSPA (A), MSPB (B), and the truncated 27- to 30-kDa MSPB, which predominates in HA culture (C). At the right side the molecular masses of proteins (in kilodaltons) are indicated.

Association of T lymphocytes with synovitis. Several studies have suggested that T lymphocytes are associated with the pathogenic mechanisms of M. synoviae-induced arthritis in chickens (20, 23). We conducted a few analyses of subpopulations of chicken CD4- and CD8-positive T lymphocytes, of cells expressing major histocompatibility complex II homologue molecules, of B cells expressing IgM, and also of T cells expressing CD3 and T-cell receptor 1 (TCR1) () or TCR2 (). MAbs recognizing these molecular markers were kindly provided by M. Cooper and N. Le Douarin and have been previously used in studies of others (7-10, 16).