FEMS Microbiology Letters 144 (1996) 267-275
Adaptive surface antigen variation in Mycoplasma bovis to the host immune response
Dominique Le Grand &, Michel Solsona b, Renate Rosengarten , Franqois Poumarat bj *
a EC& Nation& V&tPrinaire de Lyon, Pathologic du B&ail, B.P. 83, 69280 Marcy-IEtoile, France
b CNEVA-Lyon, Luboratoire de Pathologic Bovine de Lyon, B.P. 7033, 69342 Lyon Cedex 07, France
Institut fcr Bakteriologie und Tierhygiene, Veteriniirmedizinische Universitiit Wien, Josef-Baumann-Gasse 1, 1210 Vienna, Austria
Received 14 May 1996; revised 26 August 1996; accepted 9 September 1996
The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of i14. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.
Keywords: Mycoplasma bows; Variable surface protein; Antigenic variant; Immune modulation; Immune evasion
Mycoplasma bovis is considered one of the most pathogenic mycoplasma species in cattle. It is well established as the etiologic agent of mastitis [l], ar-
thritis , and pneumonia [3,4], and has also been reported to cause diseases of the genital tract , abscesses  and meningitis . M. bovis diseases occur all over the world leading to extensive eco- nomic losses for both dairy and meat production
* Corresponding author. Tel: +33 78 72 65 43; Fax: +33 78 61 91 45.
[8,9]. Their incidence is growing in Europe with the increase of the beef trade during the past years [9,10]. Due to their resistance to antibiotic therapy and the lack of commercially available sensitive diagnostic tools and effective vaccines, M. bovis-induced dis- eases are difficult to prevent and to control.
A major characteristic of most mycoplasma infec- tions, including those caused by A4. bovis, is that they are usually chronic in nature. Although the ba- sis for this chronicity is not yet well understood, it has become apparent during the last few years that mycoplasmas may possess immune evasion mechan- isms, which enable them to rapidly change the struc-
0378-1097 /96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00377-l
268 ,!I Lr Grund (1 ul. I FEMS Mrrohwlog~ Lrtten 144 /1996) 267-275
CA CA CACACACA
1 2 3 4 5 6
CACACA CACA CACA CA
1 2 1 2 1 2 1 2
Fig. I. In vitro modulation of surface antigen expression in M. hovi~ strain 1067 by serum antibodies from a calf (M019) immunized with the same strain. a: Western blots of total cell protein from a population of organisms resulting from different inocula immunostained with bovine serum M019: both lanes (1) corresponding to culture from 10 CFU/ml inoculum; (2), lOa CFUlmI inoculum; (3) lo3 CFUl ml inoculum; (4), lo5 CFU/ml inoculum; (5) lo6 CFUlml inoculum: (6) IO7 CFU/ml inoculum. Lanes A (assay): culture with MO19 se- rum antibodies; lanes C (control): culture with bovine control serum. The position and size (in kDa) of immunostained proteins are indi- cated (right panel). b, c, d, e: Western blots of total cell protein from a population of organisms resulting from a 10 CFU/ml (lanes 1) and 10 CFUlml (lanes 2) inocula, respectively immunostained with mAbs: lA1 (b), lE5 (c), 5D7 (d) and Ia (e). Lanes A: culture with MO19 serum antibodies: lanes C: culture with bovine control serum. The position and size (in kDa) of immunostained proteins are indi- cated (right panel).
D. Le Grand et al. I FEMS Microbiology Letters 144 (1996) 267-275 269
ture and expression of some of their membrane sur- face proteins exposed to the host immune system [l l-131. Our previous investigations have shown that M. bovis employs two surface antigenic varia- tion systems which could act independently to facil- itate immune evasion by this organism [14-171. The first represents a family of membrane surface lipo- proteins designated Vsps (variable surface proteins) , with as yet three defined members (VspA, VspB, VspC) which undergo a high rate of phase and size variation in vitro. A second, unrelated membrane surface protein designated pMB67  proved to be as highly variable as the Vsps. Both types of membrane proteins are predominant antigens recog- nized during M. bovis infection and disease [14,15,17-l However, neither in M. bovis nor in other mycoplasma species in which variable surface pro- teins have been identified, has the function of these proteins in immune evasion in the host been unequi- vocally demonstrated. Only very recently, Citti and Wise [l&19] have reported that antigenic variants of the swine pathogen M. hyorhinis are very likely to be subject to random selection, i.e., they arise sponta- neously and independently of the host immune re- sponse, but their subsequent survival is determined by immune selection.
In the present study we have explored the possibil- ity that M. bovis may be able to avoid the host immune system by changing its surface antigenic mo- saic as soon as the immune system recognizes it, i.e., whether an effective humoral immune response in the host can repress expression of the target protein or induce switching to expression of an antigenically distinct protein. To address this issue, we have devel- oped an immune pressure assay to examine the effect of sera from calves immunized or experimentally in- fected with M. bovis and of specific monoclonal anti- bodies (mAbs) on the pattern of surface antigen ex- pression in vitro.
2. Materials and methods
2.1. Monoclonal antibodies
Four mAbs directed toward M. bovis variable sur- face antigens were used in this study (as ascites fluids or hybridoma culture supematants). The construc-
tion and characteristics of mAb lE5 have recently been described in detail [ 151. It is an immunoglobulin (Ig) M isotype and recognizes a surface epitope on VspA, VspB and VspC . mAbs 5D7  and 1Al (IgGl) have been developed within a collaborative research project of the CNEVA Lyon and the Istitu- to Zooprofilattico Sperimentale in Brescia (Italy). Both mAbs were prepared against M. bovis type strain PG45 and react with VspA and VspC (mAb lA1) and, respectively, other as yet undefined pro- teins of the Vsp family (mAbs 1Al and 5D7; Rosen- garten, Poumarat, Le Grand and Yogev, unpub- lished results), which have been previously classified as antigen cluster I . mAbs 12 and Ns have been constructed by Vetoquinol Biotechnologie in colla- boration with CNEVA Lyon . Both mAbs were prepared against M. bovis strain 1067 (see below). While mAb Is (IgGl) reacts with a surface epitope on the phase- and size-variant Vsp-unrelated surface protein pMB67 , mAb Ns recognizes a surface protein of 45 kDa that is present in most, but not all M. bovis strains tested .
2.2. Bovine sera
Two calves, designated MO19 and M021, were immunized with M. bovis strain 1067 as follows. Briefly, each calf was injected intramuscularly with 1 X lOlo formalized mycoplasmas emulsified in alumi- num hydroxide as adjuvant, followed by a second injection of the same dose and by the same route 4 weeks later. Three weeks after the second injection, the calves were challenged by inoculation of 2 x lo7 viable mycoplasmas into the carpal joint . In par- allel experiments, two other calves which were not immunized, designated MO1 5 and M017, received the same dose. Corresponding sera, designated M019, M021, MO15 and M017, respectively, were obtained 15 days after inoculation, Serum anti- body titers were determined by the indirect hemag- glutination test (IHT) [21,22]. IHT titers of the end- point dilution were 1:640, 1:640, 1:80 and 1:40, re- spectively, for the four sera.
2.3. Mycoplasmas and culture conditions
M bovis strain 1067 was isolated in 1983 from a case of bovine mastitis  and successfully used to
270 D. Le Grand et al. I FEMS Microbiology Letters 144 (IYY6) 267-275
experimentally induce reproducible arthritis in calves . Clonal variants of M. bovis type strain PG45 expressing a single size variant of VspA (65 and 63 kDa), VspB (46,44.5,43 or 42 kDa) or Vsp C (79 or 75 kDa), or lacking all three Vsps (designated ABC- OK) have been previously described . Each organ- ism was propagated at 37C in a standard mycoplas- ma medium . Stocks were prepared from mid- exponential phase cultures and stored at -80C.
2.4. Immune pressure test
Fresh, broth-grown organisms from primary pas- sages of stocks (late exponential phase) containing approximately log CFU/ml were serially diluted lo- fold to lo-. 160-ml aliquots of each dilution were distributed in duplicate in 96-well microtiter plates. To each test well, 40 ml of IHT-positive bovine sera or 40 ml of ascites or hybridoma culture superna- tants containing mAbs were add