In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens

veterinary microbiology ELSEVIER

Veterinary

Microbiology

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In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens Sharon Levisohn a, Renate Rosengarten b, David Yogev bq* aDivision of Avian Diseases, Kimron Veterinary Institute, Bet Dagan 502.50, Israel b Department of Membrane and Ultrastracture Research, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel Received 7 September

1994, accepted

17 January

1995

Abstract The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs lE5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gullisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67aphenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection. Keywords: Mycoplasma gallisepticum; Surface variable proteins; In vivo antigenic Chronic Respiratory Disease

0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10378-1135(95)00039-9

variation;

Immune evasion;

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1. Introduction Mycoplasma gallisepticum is generally recognized as the most significant avian mycoplasma pathogen and has been widely studied since first described by Nelson more than 60 years ago (Yoder, 1991) , Infection with M. gallisepticum may be manifested as an overt respiratory disease (Chronic Respiratory Disease in chickens or Infectious Sinusitis in turkeys), but under other conditions, infection occurs without clear clinical signs in the host. The variability of M. gallisepticum infection has been variously attributed to antigenic and genetic heterogeneity among strains and/or host-associated or environmental factors including secondary infections (Jordan, 1979). Another feature of M. gallisepticum infection, shared by many pathogenic mycoplasmas, is that infection persists in spite of the presence of high levels of humoral antibodies ( Yoder, 199 1) . Thus the potential for disease remains and in the case of broiler-breeders, perpetuation of the infection by transmission to the progeny. Control of mycoplasmosis, and in particular M. gallisepticum infection, is a keystone of a modern intensive poultry industry. The economic impact is measured not only by the losses due to disease, treatment and inefficiency of production due to sub-clinical infection, but also in expense of the diagnostic procedures needed to determine the mycoplasma status of breeding stock (Kleven, 1990). Since M. gallisepticum, like other organisms of the genus Mycoplasma (Razin, 1992), lack a cell wall, the single membrane surrounding the cell represents a unique microbial barrier in which all host-cell interactions, including encounters with the immune system, take place. It has become apparent in the last few years that mycoplasmas possess genetic systems allowing them to rapidly alter surface antigens and to switch from one antigenic coat to another which apparently facilitates escape from the host immune response. Evidence of such in vitro antigenic variation has been reported for several pathogenic mycoplasma species, including M. gallisepticum (Behrens et al., 1994; BenEina et al., 1994; Garcia et al., 1994; Markham et al., 1993; Panangala et al., 1993; Rosengarten and Wise, 1990; Rosengarten et al., 1994; Theiss et al., 1993; Watson et al., 1988; Yogev et al., 1991; Yogev et al., 1994). The impressive capability of diversifying the antigenic character of the mycoplasma cell surface may also play an important role in “fine tuning” the specific interaction between mycoplasma adhesins and host cell surface receptors, thus affecting adherence, tissue tropism, invasiveness and avoidance of the immune response. Understanding of these processes will aid in control of mycoplasmosis. Understanding of the antigenic variation phenomenon and the ability to monitor this process in vivo are also crucial for mycoplasma control programs in which the diagnostic tests must be such that they can cope with a wide spectrum of antigen presentations. The possibility of a non-expression state of a given epitope must also be taken into consideration. Talkington et al. ( 1989) presented evidence for antigenic variation of the V-l protein of M. pulmonis in mice, thereby demonstrating that generation of variant phenotypes also occurs in vivo. Marked differences in the expression of surface antigens detected by monoclonal antibodies has been reported by Olson et al. ( 1991) for sequential isolates of M. * Corresponding author. Tel: 972 2 758 176, Fax: 972 2 757 413.

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hominis in a patient with chronic septic arthritis and observed also in rats infected by M. arthritidis (Droesse et al., 1994). BenEina et al. ( 1993, 1994) have suggested that in vivo antigenic variation of certain proteins may also occur in M. gallisepticum whereas others (Garcia and Kleven, 1994) indicate that certain epitopes may serve as stable markers for recognition of specific strains. In this study we followed the expression of major surface proteins of M. gallisepticum in individual chickens during the course of experimental respiratory infection. In particular we have used two specific monoclonal antibodies ( lE5 and 12D8) to focus on two distinct surface proteins which have been previously shown to undergo high-frequency phenotypic switching in vitro (Garcia et al., 1994; Yogev et al., 1994).

2. Materials and methods 2.1. Mycoplasma

strains and growth conditions

M. gallisepticum strain R, a widely used pathogenic strain, has been previously used in our laboratory for experimental infection (Levisohn and Dykstra, 1986) and molecular studies (Yogev et al., 1988; Yogev et al., 1994). The strain was obtained from Dr. S.H. Kleven (University of Georgia, Athens, GA) and was used at the 9th passage level. M. gaElisepticum R/E is a clonal variant of R strain with the phenotype PvpA+p67a-, as recently described by Yogev et al. ( 1994). Notably, this phenotype appears to be stable in vitro. Further properties of variant R/E are shown in Fig. 4A, lane 1 of the above indicated publication. Cultivation of mycoplasmas for infection was carried out in Frey’s standard medium (Anonymous, 1989). Isolation of mycoplasmas from the trachea of experimental chickens was carried out by standard methods in which the swab was inoculated into Frey’s medium or, when indicated, streaked directly onto solid mycoplasma medium. Isolates were identified by direct immunofluorescent staining. Mycoplasmas for western blot analysis were grown under standard conditions in modified Edward medium (Razin and Rottem, 1976). 2.2. Experimental

infection

Mycoplasma-free Leghorn-type chicks were obtained from a commercial breeder flock at day 1 and maintained in positive pressure Horsfal-Bauer isolation cells. At two weeks the birds were divided into experimental groups and inoculated by fine-spray aerosol as previously described (Levisohn and Dykstra, 1986). Overnight cultures of M. gallisepticum R and R/E used for infection contained about 2 X 10’ and 8 X lo6 cfu/ml respectively. Samples of the inoculum cultures were saved for comparative studies. Birds were bled at day 8 post infection (PI) and thereafter at approximate weekly intervals. Isolation of mycoplasma was carried out at 3 weeks PI and thereafter at indicated times. Birds were numbered and samples tested on an individual basis. No difference in clinical signs or in temporal response in standard serological tests was observed between groups of birds infected with M. gallisepticum R or R/E, both of which showed transitory respiratory signs

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and morbidity. A non-infected group was maintained for control and was negative for serological tests and isolation until the end of the experiment at 8 weeks PI. 2.3. Chicken antiserum Preparation of the anti-M. gallisepticum chicken serum used as a reference has been previously described (Yogev et al., 1994). In short, experimental infection of chickens with M. gallisepticum strain R was carried out by fine spray aerosol, as described above. Morbidity and characteristic clinical respiratory signs were observed from l-3 weeks PI, with development of antibodies detectable by serological tests using commercial antigens (Rapid Slide Agglutination, Intervet, Boxmeer, The Netherlands; ELISA Pro-Flok kit, Kirkegaard and Perry Laboratories, Gaithersburg, MD). Hemagglutination Inhibition was tested by standard USDA recommended methods. Activity in the Growth Inhibition (GI) test was only marginally positive. During the convalescent stage of infection (6 weeks PI), the birds were injected intramuscularly with the same inoculum strain. Two weeks after injection, high activity was detected also in the GI test. Serum samples from 5 birds were pooled and filter-sterilized. No reactions were found with M. synouiae in any of the above serological tests, or with heterologous mycoplasmas or mycoplasma media components in western immunoblotting. In the present temporal study, experimentally infected chickens were bled at indicated times, and the antisera from the individual birds tested in standard serological tests indicated above. Sera were maintained at - 20°C or, after filter sterilization, at 4°C until used for western blotting. 2.4. Monoclonal

antibodies (mAbs)

mAb lE5: The preparation and characteristics of mAb lE5 have been described previously (Rosengarten et al., 1994). The protein detected by mAb lE5 has been designated PvpA (Yogev et al., 1994). mAb 1208: This mAb was kindly provided by Dr. S.H. Kleven (University of Georgia, Athens GA). The properties of mAb 12D8 have been previously described (Garcia et al., 1994). In our studies it detects a protein of the size 67 kDa in M. gallisepticum strain R, which has been designated p67a and recently identified as a phase-variable membrane lipoprotein (Yogev et al., 1994). mAb A3: This mAb was obtained from Dr. Gy. Czifra (Swedish National Veterinary Institute, Uppsala, Sweden) and described by Czifra et al. ( 1993). mAb A3 detects a protein of the size 67kDa in M. gallisepticum strain R, thus comigrating in SDS-PAGE with the protein detected by mAb 12D8. The proteins can be readily distinguished by consecutive immunostaining. This protein has been designated p67b.

2.5. SDS-PAGE and western immunoblotting For sodium dodecyl sulfate-polyacrylarnide gel electrophoresis (SDS-PAGE) and westem blotting, equivalent amounts of organisms from mid-logarithmic-phase cultures were harvested by centrifugation for 5 min at 12000 X g. Pellets were resuspended in SDS-PAGE

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sample buffer (2% SDS, 5% [v/v] 2-mercaptoethanol, 10% [v/v] glycerol, 62.5 mM Tris, pH 6.8,3% urea) and heated at 100°C for 5 min. Protein samples were separated by SDSPAGE according to the method of Laemmli (1970) as previously described in detail (Rosengarten et al., 1994). After electrophoresis, proteins were transferred electrophoretically to nitrocellulose membrane filters as described by Towbin et al. ( 1979). Blots were blocked for 1 h at room temperature with TS buffer ( 10 mM Tris, 150 mM NaCl, pH 7.4) containing 3% bovine serum albumin and then incubated overnight at 4°C with the primary antibodies diluted 1:50 (mAb lE5) or 1:250 (mAb 12D8, mAb A3 and chicken sera) in PBS (phosphate-buffered saline, pH 7.2) containing 10% (v/v) fetal calf serum. After three washes in TS buffer, blots were incubated for at least 2 h at room temperature in peroxidase-conjugated goat antiserum to mouse IgM (p chain) or mouse IgG, heavy and light chains (Jackson Immuno Research Laboratories, West Grove, PA) or to chicken IgG, heavy and light chains (BioMakor, Rehovot, Israel), all diluted 1: 1000 in PBS containing 10% ( v/v) fetal calf serum. For detection, enzyme substrate 4-chloro- 1-naphthol ( Sigma, St. Louis, MO) was used. 2.6. Colony immunoblotting The expression of PvpA and p67a within M. gallisepticum populations was detected in colony immunoblots as recently described (Rosengarten et al., 1994; Yogev et al., 1994). Colony immunoblots were immunostained (without blocking) with mAbs lE5 and 12D8 as described above for western irnnnmoblots. To distinguish among clonal variants expressing PvpA or p67a only, and those coexpressing PvpA and p67a in the same colony blot, immunostaining with mAbs lE5 and 12D8 was performed consecutively using enzyme substrates o-dianisidine (Sigma, St. Louis, MO), which gives a brown color for detection of PvpA, and 4-chloro- 1-naphthol, giving a blue color for detection of p67a.

3. Results In order to study in vivo variation of antigen expression in M. gallisepticum, an experimental respiratory infection was carried out in chickens using the pathogenic strain R as an aerosol inoculum. Total cell proteins of the inoculum strain R (Fig. 1, lane 1) and of M. gallisepticum reisolates from three individual infected chickens (Fig. 1, lanes 2-4) were analyzed by western blot. Panel a shows the results that were obtained by consecutive immunostaining with mAb lE5,12D8 and A3, which permits detection of the three corresponding proteins (PvpA, p67a, and p67b) (see Materials and Methods). Panel b shows the results obtained by staining with reference anti-M. gallisepticum chicken serum (described in Materials and Methods). The data show that the most prominent surface antigen of strain R is p67a which is strongly recognized by both mAb 12D8 (panel a, lane 1) and the chicken serum (panel b, lane 1) . Interestingly, however, the reisolates from infected chickens recovered 3 weeks (lanes 2 and 3) or 8 weeks (lane 4) PI exhibit a p67a-negative phenotype, with a constant and weak level of expression of the corn&rating p67b protein, detected by mAb A3. Additional M. gallisepticum specific proteins undergoing variation in expression were observed by use of the reference chicken

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PvpA-e-

-

Fig. 1. In vivo antigenic variability of M. galliseptieum strain R. Panels a and b: western immunoblot analysis of total cell proteins of strain R in~~urn (Lane 1) and of three reisolates recovered 3 weeks (lanes 2 and 3) or 8 weeks (lane 4) post inoculation (PI) from three chickens aerosol-inoculated with strain R. Blots were immunost&red consecutively with mAbs lE5, 12D8 and A3 (panel a) or with reference anti-M. gallisepticum chicken antiserum (panel b). The 55 kDa PvpA recognized by mAb 1E5 and two corn&rating 67 kDa proteins (p67a and p67b), recognized by mAbs 12D8 and A3 respectively, are indicated. Other immunogenic proteins (p46, p4’7, ~72, ~75, ~79) varying in expression are also indicated. Each lane represents proteins from equivalent amounts of bro~-~~ organisms.

antiserum (Fig. 1, panel b). For example, while p67a switches from ON (panel b, lane 1) to OFF in expression state (panel b, lanes 2-4), other proteins (~46, ~47, ~72, ~75 and p79) are turned ON in the various reisolates (panel b, lanes 2-4). Notably, PvpA is weakly recognized by this serum in the inoculum strain and in all 3 reisolates. To further examine in vivo surface variation of the relevant epitopes, the colony immunoblot technique using two mAbs distinguishable by substrate reaction color differences was applied. A marked difference was seen with respect to the phenotypic expression of p67a and PvpA between the M. gal~~septi~umR strain inoculum and M. gallisepticum isolated from the chicken 8 days PI (Fig. 2). I~unost~ning of the inoculum culture with mAb anti-PvpA ( lE5) showed variation with respect to expression of the epitope, with the majority of the colonies exhibiting a PvpA f phenotype (panel a). Restaining of the same colony blot with mAb anti-p67a ( 12D8) indicated that the predominant phenotype of this culture was PvpA+p67a+ (panel b) although all 4 possible phenotypes (PvpA’p67a+, PvpA+p67a-, PvpA-p67a+ and PvpA-p67a-) could be distinguish~. Interestingly, 8 days after infection the predominant phenotype of the inoculum R strain was completely replaced by a PvpA+p67a- phenotype. All colonies found on a primary isolation plate inoculated by tracheal swabbing of an infected chicken were positive with mAb lE5 (panel c) and were negative with mAb 12D8 (data not shown). This suggests that a selection process occurs during early stages of infection, generating a uniform population in which PvpA is expressed while p67a is switched OFF.

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Fig. 2. In vivo selection of M. gallisepticum antigenic variants during experimental infection with strain R. A representative portion of a colony immunoblot of strain R was immunostained with mAb lE5 to PvpA (panel a) and restained with mAb 12D8 (panel b) Comparison is made with a corresponding colony blot of a reisolated population, obtained by inoculating a trachel swab taken 8 days PI directly on to agar medium, immunostained with mAb lE5 (panel c). Clonal variants of strain R inoculum showing the antigenic phenotype PvpA-p67a+ am indicated in panels a and b by arrows. An arrowhead is used in panels a and b to indicate a colony with the phenotype PvpA-p67a+ containing a PvpA-p67asector and another colony with the phenotype PvpA-p67a-. All other colonies in this portion of the blot show the PvpA+p67af phenotype. Colonies with the PvpA+p67aphenotype are not seen. All colonies on the primary isolation plate shown in panel c have the phenotype PvpA+p67a-. Negative reaction of the colony blot in panel c with mAb 12D8 is not shown.

More evidence indicating in vivo antigenic variation in M. gallisepticum was obtained in additional experiments. A specific clonal variant (R/E) derived from the R strain by colony immunoblotting and exhibiting the phenotype PvpA+p67a(Yogev et al., 1994) was used as inoculum in experimental respiratory infection (Fig. 3). Two sequential reisolates were recovered from the same infected chicken and subjected to western immunoblot analysis using mAbs lE5 and 12D8 (panel a) or with the reference chicken antiserum (panel b) . Immunostaining with the relevant mAbs confirmed the predicted phenotype of variant R/ E (Fig. 3, panel a, lane 1) but also demonstrated variation with respect to p67a expression in the two successive reisolates recovered at different times (5 weeks or 8 weeks PI) from the same chicken (panels a and b, lanes 2 and 3). Notably, PvpA is detected by mAb lE5 in the inoculum strain and in the two reisolates (panel a), and also weakly by the reference antiserum (panel b) . However, other immunogenic proteins (p47, ~50, ~72, ~79) detected by the reference serum showed variation in expression among the inoculum R/E and the two reisolates (panel b) . In additional experiments we examined temporal variation in host antibody response by testing the reaction between antiserum collected at different times PI and the M. gallisepticum reisolates from the same bird infected with variant R/E (as in Fig 3). SDS-PAGEseparated total proteins of inoculum variant R/E and the two sequential reisolates were immunostained with serum samples collected from the same chicken at four different times

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p67albe PvpA+

Fig. 3. In vivo antigenic variability of M. gallisepticum clonal variant R/E derived from strain R. Panels a and b. Western immunoblot analysis of total proteins of clonal variant R/E lane 1) and two reisolates recovered sequentially at 5 weeks (lane 2) and 8 weeks (lane 3) PI from a chicken aerosol-inoculated with variant R/E. Blots were inmumostained consecutively with mAbs lE5 and 12D8 (panel a) or with reference anti-M. gallisepticum chicken antiserum (panel b) The two sequential isolates are bracketed at the top. The invariant expression of p67b (panel a, lanes 1 to 3 ) was confirmed by restaining the blot in panel a with mAb A3 (data not shown). PvpA and p67a as well as other immunogenic proteins (p47, ~50, p72 and ~79) undergoing in vivo phase variation in their expression are indicated. Another variable protein of 46 kDa which is poorly immunogenic is indicated by an asterisk (panel b, lane 1) .

PI. During this period striking changes in the antigen expression profile and in antibody recognition were observed (Fig. 4). A 72 kDa protein of M. gallisepticum R/E was the first recognized by the host immune system, as shown by the reaction of the 8 day PI serum with inoculum R/E (Fig. 4, panel a, lane 1) . The level of this antibody is stable during infection as seen in lane 1 of panels a to d. However only a weak band of this protein is seen in the reisolates from the chicken (Fig. 4, panels a-d, lanes 2 and 3). Other than ~72, the most immunogenic protein is p67b which is readily recognized by host antibodies at day 15 PI and is also present in the two reisolates (panel b, lanes l-3). The circulating antibodies to p67b start to disappear at 30 days PI even though the antigen is invariably expressed in the various isolates (panel c, lanes l-3). This may be seen by the reaction of the reisolates when tested with serum taken at 64 days PI (panel d, lanes 2 and 3). In contrast, no antibodies to p67a are present in the early stages of infection (Fig 4, panels a to c) , as can be seen by comparison of these data with those obtained for the same reisolates with anti-p67a and reference M. gallisepticum antiserum (Fig. 3, panels a and b). Another immunogenic antigen undergoing in vivo variation is a 50 kDa antigen strongly expressed only in the isolate recovered at 8 weeks PI but recognized by antisera collected at all 4 times (Fig. 4, panels a-d, lane 3). Variation in expression of other proteins (p46, p47, and p79), which was previously described in Fig. lb and Fig. 3b, also occurs in the sequential reisolates shown in Fig. 4.

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d 12-“-T

Cl’2

72+

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50,

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15

30

PvpA

64

Fig. 4. Temporal variation in serum antibody response of the chicken host to experimental infection with hf. gaZlisepticum variant R/E. Western immunoblot analysis of SDS-PAGE-separated total cell proteins of variant R/E inoculum (lane 1) and two sequential reisolates recovered 5 weeks (lane 2) or 8 weeks (lane 3) PI. Blots were immunostained with 4 serum samples collected from the same aerosol-inoculated chicken at the time in days PI, as indicated at the bottom (panels a-d). Sequential isolates are indicated by brackets at the top. Positions and molecular masses (in kDa) of selected proteins, including p67a and p67b, showing either a strong (arrows) or weak (arrowheads) i~~ologi~ reaction at the different times after inoculation are indicated on the left. PvpA (55 kDa) is indicated in panels c and d by arrowheads on the right.

In contrast and relative to all other variable proteins described above, PvpA is only poorly immunogenic. This is shown by the weak immunostaining in Fig. 4, panels c and d, although the presence of this protein in M. galLiseptic~m R/E and in the reisolates is clearly demonstrated by specific i~unost~ning with mAb lE5 (Fig. 3, panel a). I~unost~ning with mAb lE5 distinguishes PvpA from a closely migrating protein of about 57 kDa expressed by all three strains and weakly detected by the serum collected at 15 days PI (Fig. 4, panel b, lanes l-3), and more strongly at 30 and 64 days PI (panel c and d, lane l-3).

4. Discussion Evidence accumulated during the past few years indicates that mycoplasmas possess a remarkable ability to alter antigenic com~nents of their surface (Wise et al., 1992). This strategy apparently serves as an effective means of avoiding the host immune response, allowing the pathogenic organisms to constantly maintain surviving subpopulations during the course of infection, Investigation of in vivo antigenic variation of the pathogen occurring in its natural host is, therefore, of great interest. Several species-specific immunogenic proteins of M. gal~~se~tic~m(~82, p67, ~64, ~56, ~54, ~35, p26 and ~24) have been previously described ( Avakian et al., 1991; Avakian and Ley, 1993; Czifra et al., 1993; Forsyth et al., 1992; Markham et al., 1992; Yogev et al., 1994). Some of these immunogenic antigens were also reported to undergo in vitro ( BenEina et al., 1994; Garcia et al., 1994; Markham et al., 1992) or in vivo (BenEina et al., 1993)

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phenotypic switching. However, detailed investigations of variation in antigen expression occurring in vivo have not been fully described. The use of mAbs specific to M. gallisepticum surface antigens in conjunction with a reference anti-M. gallisepticum chicken antiserum was shown in this study to be a valuable tool for identification of immunogenic antigens of M. gallisepticum. Used together with sera obtained from infected chickens during different stages of infection, it was possible to monitor variation during the course of infection. It is important to emphasize that apparent changes in antigen expression may also occur due to the state of the culture used for immunoblot analysis. In order to avoid this possibility, all protein samples analyzed here represent equivalent amounts of organisms from mid-logarithmic phase cultures grown under standard conditions. The data presented here suggest that changes in antigen expression on the mycoplasma cell surface also occur in vivo in the chicken host. Several noteworthy findings revealed in this study support this concept. Firstly, the interesting observation that p67a, a phase variable and major immunogenic protein of pathogenic strain R (Yogev et al., 1994), was present in the inoculum for experimental infection but was not detected in reisolates of M. gallisepticum at 3 weeks or 8 weeks PI (Fig. 1) . A similar situation was found in chickens infected with M. gallisepticum clonal variant R/E which possesses a p67a negative phenotype and in which the immunodominant protein is p72 (Fig. 3). In the case of infection with R or R/E there was the emergence during infection of a population of M. gallisepticum organisms which differ markedly from the infecting organisms with respect to presentation of the immunodominant surface antigen. Several models may be proposed by which this variation can be generated. One possible explanation focuses on the level of gene expression. Recognition of the immunodominant antigen by the chicken immune system may repress the corresponding gene activity, thus affecting the expression of the protein. Simultaneously, repression of this gene may turn ON genes encoding other immunogenic antigens. For example, while expression of p67a was not found in an isolate made 3 weeks PI, other proteins (p46, p47, ~72, ~75, and ~79) were expressed and could be easily detected by chicken serum (Fig. 1) . An alternative model suggests that the reisolates exhibiting a p67a-negative phenotype may represent spontaneous p67a-escape variants which are resistant to the mycoplasmacidal activity of cognate antibodies directed against p67a and other related surface proteins. In the case of either of these models for surface diversity, the possibility exists that p67a variation, as well as variation of p46, ~47, ~50, ~75, and ~79, occurs in response to some host environmental factors other than immune modulation of antigen expression. Specific host environmental conditions developing during the course of infection could result in phenotypic changes. Interestingly, variant R/E and certain other variants studied in our laboratory were relatively stable during in vitro passage in contrast to the variation observed in vivo (Yogev, D. and Menaker, D., unpublished results). Additional evidence supporting the occurrence of in vivo antigenic variation emerged from immunostaining of colonies on a primary isolation plate inoculated by a tracheal swab taken 8 days PI. The finding that the heterogeneous population of the inoculum strain R was completely replaced by a homogeneous population exhibiting a uniform phenotype of PvpA+p67a(Fig. 2) argues for an in vivo selection process occuring during the early stages of infection. Recent studies of the cloned and sequenced pvpA gene revealed that the predicted PvpA amino acid sequence shows adhesin-like properties, suggesting a possible

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function in adherence (Yogev et al., submitted for publication). Interestingly, in contrast to the immunodominant p67a or p72 proteins, the immune recognition of PvpA is weak, which may explain the lack of modulation of this antigen by the immune system. This raises the possibility that a variety of processes, including those mentioned above, are involved in generating surface diversity and may occur simultaneously in vivo. One implication of the high degree of surface diversity generated in vivo is that use of proteins undergoing high frequency variation as the basis for diagnostic reagents may be ill-advised. In addition, the practice of random selection of single colony for identification or laboratory studies may inadvertently result in the loss of the epitope of interest. Previous studies ( Avakian and Ley, 1990) demonstrated the heterogeneity of the antibody response among individual infected chickens. This was found also in the present study, but we have also demonstrated the heterogeneity of the antigen presentation by the M. gallisepticum reisolates from different birds (Fig. 1) or from the same bird at different times after infection (Fig. 4). The variation in expression during infection of several major immunogenic proteins (p46, p47, ~50, p67a, ~72, ~75 and ~79) underscores the complexity of in vivo surface antigenic variation in this organism. Taken together our results show that surface antigenic variation of major immunogenic proteins of M. gallisepticum occurs in vivo. The results support the concept of the mycoplasma cell surface as a complex and changing mosaic of epitope-bearing antigens and further suggest that the repertoire of surface antigens is subject to modulation by host environmental factors, most probably the host immune response.

Acknowledgements The work was supported by Grant No. 847-0253-92 from the Agricultural Research Council of Israel (S.L.), a fellowship (Ro 73915-1) from the Deutsche Forschungsgemeinschaft (R.R.) and a grant from the Hebrew University Authority for Research and Development (D.Y .) The authors wish to thank Dr. S.H. Kleven and Dr. Gy. Czifra for kindly sharing monoclonal antibodies used in this study.

References Anonymous, 1989. National poultry improvement plan and auxiliary provisions, Publication VS-91-40, U.S.D.A., Hyattsville, MD. Avakian, A.P., Kleven, S.H. and Ley, D.H., 1991. Comparison of Mycoplasma gallisepticum strains and identification of immunogenic integral membrane proteins with Triton X-l 14 by immunoblotting. Vet. Microbial., 29: 319-328. Avakian, A.P. and Ley, D.H., 1993. Inhibition of Mycopkrsma gallisepticum growth and attachment to chick tracheal rings by antibodies to a 64 kDa membrane protein of M. gallisepticum. Avian Dis., 37: 706-714. Behrens, A., Heller, M., Kirchhoff, H., Yogev, D. and Rosengarten, R., 1994. A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bouis. Infect. Immun.,62: 5075-5084. Bencina, D., Xleven, S.H., Elfaki, M.G., Snoj, A., DOVE,P., Dorrer, D. and Russ, I., 1994. Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies. Avian Pathol., 23: 19-36.

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