ELSEVIER

MICROBIOLOGY LETTERS

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-I’Etoile, 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

Abstract

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 bow’s; Variable surface protein; Antigenic variant; Immune modulation; Immune evasion

1. Introduction

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 [2], and pneumonia [3,4], and has also been

reported to cause diseases of the genital tract [5], abscesses [6] and meningitis [7]. 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

a

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)

[16], 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 [17] 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 [15]. mAbs 5D7 [14] 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 [15]. mAbs 12 and Ns have been

constructed by Vetoquinol Biotechnologie in colla-

boration with CNEVA Lyon [14]. 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 [17], mAb Ns recognizes a surface protein of 45 kDa that is present in most, but not all M. bovis strains tested [14].

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 [20]. 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 [24] and successfully used to

270 D. Le Grand et al. I FEMS Microbiology Letters 144 (IYY6) 267-275

experimentally induce reproducible arthritis in calves

[20]. 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 [16]. Each organ-

ism was propagated at 37°C in a standard mycoplas- ma medium [23]. Stocks were prepared from mid-

exponential phase cultures and stored at -80°C.

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 added. Similarly, 40 ml of IHT-negative bovine serum or of mAb-nega-

tive ascites was added to each control well. Bovine

test and control sera were heat-inactivated at 56°C for 30 min prior to use to destroy any complement-

mediated mycoplasmacidal effect. Growth was di-

rectly estimated by measuring medium optical den- sity at 460 nm. After incubation at 37°C for 7 days, each well content was prepared for Western immu-

noblot analysis as described below.

2.5. Western blot analysis

Organisms from each well (200 ml of culture) were

harvested by centrifugation at 13 000 X g for 30 min

at 4°C. The procedures for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and

subsequent Western blotting of mycoplasma proteins

have been previously described [14]. For SDS- PAGE, cell pellets were heated at 100°C for 5 min under reducing conditions and samples correspond- ing to 20 ml of culture (containing total proteins from approximately lo7 CFU) loaded onto 10% polyacrylamide gels. For Western blot analysis, blots were blocked for 50 min with Tris-buffered saline (TBS; Tris-HCl, 0.01 M; NaCI, 0.15 M; pH 7.2) containing 10% (v/v) horse serum, washed once with TBS containing 0.05% (v/v) of Tween 20 and once with TBS only, and then incubated for 2 h at

33°C with the primary antibodies diluted 1:50 (bo-

vine sera), 1 :lOO (mAb lE5), 1:400 (mAb 5D7), I:500 (mAb Nz), 1: 1000 (mAb 12) or 1: 1500 (mAb 1Al) in TBS supplemented with 5% (v/v) horse ser-

um (TBS-HS). After washing as before, blots were incubated for 1 h at 33°C in peroxidase-labelled sec-

ondary antibody against mouse IgM, mouse IgG or

bovine immunoglobulins, each diluted 1:600 in TBS- HS. After repeated washing, the blots were devel- oped using 4-chloro- 1 -naphthol as chromogenic sub-

strate.

3. Results

3.1. lZJkct of bovine serum antibodies jiom immunized

or experimentally infected calves on the pattern

of surface antigen expression in M. bovis strain

1067

When organisms of M. bovis strain 1067 were cul-

tivated in the presence of M. bovis-specific bovine

immune sera lacking complement activity, there

was independently from the mycoplasma inoculum size and the serum tested never any growth inhibiting

effect observed. To determine whether the serum antibodies could induce alterations in expression of

surface antigens, the mycoplasma cells were har- vested and the patterns of antigen expression exam-

ined in Western immunoblot with MO19 serum anti-

bodies. The antigen pattern of such a culture of strain 1067 grown in the presence of MO19 serum antibodies (A) compared to a control culture (C) is

shown in Fig. la. When the initial CFU/ml value

(inoculum) of the mycoplasma culture was 107-lo”, no significant alteration in expression of immuno-

dominant (antibody binding) antigens (with a major

antigen migrating at 50 kDa) were detected (Fig. la, lanes 3-6). However, when the inoculum size was only 10 CFU/ml, the exposure of strain 1067 to M. bovis-specific serum antibodies resulted in ex- pression of a 45 kDa major immunogenic antigen (Fig. la, lane lA), while the 50 kDa major reactive band of the original strain (data not shown) and of the control culture (Fig. la, lane 1C) disappeared. The same differences in the antigen profile of strain 1067 were seen when identical blots of total protein were immunostained with Vsp-specific mAbs 1Al

D. Le Grand et al. IFEMS Microbiology Letters 144 (1996) 267-275 271

a

-60

-17

CACACACA

-50

-45

Fig. 2. In vitro modulation of pMB67 and Vsp surface antigen

expression in M. bovis strain 1067 by mAbs Is (a) and 5D7 (b).

Western blots of total cell protein from a population of organ-

isms resulting from different inocula were immunostained with

mAbs Is (a) and 5D7 (b). Lanes A: culture grown in the pres-

ence of mAb; lanes C: control culture grown in the absence of

mAb. a: both lanes (1) corresponding to culture from 10s CFUl

ml inoculum; (2), lo4 CFU/ml; (3), lo3 CFU/ml. A second step

of immune pressure (4) was performed from cuhure shown on

lane A2 as indicated by arrows. b: both lanes (1) corresponding

to culture from lo6 CFUlml inoculum; (2), lo5 CFU/ml; (3), lo4

CFU/ml. A second step of immune pressure (4) was performed

from culture shown on lane A2 as indicated by arrows. After im-

munostaining with mAb Is (a) or 5D7 (b), the blots were re-

stained with mAb 1Al (lower panels) to ensure equivalent

amounts of total protein per channel. The position and size (in

kDa) of immunostained proteins are indicated (right panels). c

(Fig. lb) or lE5 (Fig. lc), indicating that the variant

antigens recognized by the bovine immune serum (Fig. la) are members of the Vsp surface lipoprotein

family, which under the influence of specific antibod-

ies either decrease in length or are replaced by an antigenically distinct variant Vsp protein. In con-

trast, when mAbs 5D7 (Fig. Id) and Is (Fig. le)

were used as probes in replicate blots, one other

effect of serum antibodies on the antigen profile of M. bovis was detected, namely the repression of ex-

pression of certain surface antigens, which involve Vsp proteins (as identified by mAb 5D7, Fig. Id,

lanes 1A and 2A), as well as the Vsp-unrelated sur- face protein pMB67 that occurs in strain 1067 in a

36 kDa size form (Fig. le, lanes 1A and 2A). While the various alterations of surface antigen expression

in M. bovis strain 1067 induced by serum antibodies

MO19 were easily identified and highly reproducible, those obtained with bovine sera MO15 and MO17 were less striking and restricted to extinction of ex-

pression (data not shown) of pMB67 (MO15 and

M017) and the mAb 5D7-defined Vsps (M015). In one case (bovine serum M021), no altered pattern of

expression was detected (data not shown).

3.2. Eflect of mAbs 12 and 507 on expression of pM367 and Vsp proteins in M. bovis strain 1067

In contrast to the bovine serum antibodies tested, mAbs Is and 5D7 showed a significant growth inhib- ition of M bovis strain 1067 when the initial CFU value of the mycoplasma culture was less than 500

272 D. Le Grand et al. IFEMS Microbio1og.v Letters 144 119961 267-275

Fig. 3. In vitro modulation of Vsp surface antigen expression by

mAb lE5 in clonal variants M. hovis type strain PG45 expressing

distinct size variants of the 63 kDa VsuA (a) and the 75 kDa

a

VspC (b). Western blots of total cell protein from a population

of organisms resulting from different inocula were immuno-

stained with mAb lE5: both lanes (1) corresponding to culture

from lo2 CFUlml inoculum; (2) 10 CFUlml. Lanes A: culture

grown in the presence of mAb lE5; lanes C: control culture

grown in the absence of mAb lE5. A second step of immune

pressure was performed from culture shown on lanes IA and 2A

as indicated by arrows: (3) from 1A and (4) from 2A. Each lane

represents equivalent amounts of total protein as demonstrated

by the relative immunostaining intensity of an invariant 45 kDa

protein with mAb N2 (lower panel). The position and size (in

kDa) of immunostained Vsps are indicated (right panels). VspA.

VspB and VspC are indicated by letters A, B and C.

and 5000 cells respectively. Both antibodies clearly

repressed expression of the target proteins, namely

mAb 12 the 36 kDa size version of the nonlipopro-

tein pMB67 (Fig. 2a, lanes lA, 2A and 3A). and mAb 5D7 certain as yet undefined Vsps (Fig. 2b,

ACAC AC AC

lanes 2A and 3A). The effect of removal of the in- 1 2 3 4 ducing mAb was determined by passaging the myco-

plasma cells in fresh mAb-free broth medium and

examining their antigen expression patterns in Wes- tern immunoblots with the corresponding mAb (Fig. 2a,b, lanes 4C). This analysis revealed that in the absence of specific immune pressure, the target anti-

gen can either reappear (pMB67; Fig. 2a, lane 4C)

or remain in the repressed state (mAb 5D7-defined Vsps; Fig. 2b, lane 4C). To ensure that equal

amounts of protein, i.e., equal numbers of organ- isms, were loaded per sample, the blots were re-

stained with mAb 1Al (Fig. 2a,b, lower panels).

3.3. Effect of‘ Vsp-speciJic mAh lE5 on Vsp surface

antigen expression in clod variants of A4. bovis

type strain PG45 expressing single size variants

of VspA, VspB or VspC

When clonal variants of M. bovis type strain PG45 expressing a single VspA, VspB or VspC size variant were cultivated in the presence of Vsp-specific mAb lE5, no significant growth inhibition was shown. There were two different effects observed with an initial value of less than lo3 CFU/ml, depending on the expressed Vsp product. While expression of VspA (Fig. 3a, lanes 2A, 3A and 4A) and VspC (Fig. 3b, lanes 3A and 4A) size variants on the M. bovis

CACA AC AC

1 2 3 4

-45

D. Le Grand et al. IFEMS Microbiology Letters 144 (1996) 267-275 273

a

C A

-45

C A

Fig. 4. In vitro modulation of Vsp surface antigen expression by

mAb 1Al in an M. bovis strain PG45 clonal variant lacking ex-

pression of VspA, VspB and VspC. Western blot analysis of a

culture (10s CFU/ml inoculum) grown in the absence (lanes C)

or in the presence (lanes A) of mAb 1Al. Blots were immuno-

stained with mAb 1Al (a) or mAb lE5 (b). Each lane represents

equivalent amounts of total protein as demonstrated by the rela-

tive immunostaining intensity of an invariant 45 kDa protein

with mAb Ns (lower panel). The position and size (in kDa) of

immunostained Vsps are indicated (right panels). VspA and

VspB are indicated by letters A and B.

cell surface was repressed by the mAb, expression of

VspB (data not shown) remained unaffected even after two steps of immune pressure. VspC nearly

disappeared only after a second step of immune pres-

sure (Fig. 3b, lanes 3A and 4A) indicating that ex- pression of VspC was down-regulated but not com- pletely repressed. When the selective immune pressure was removed by passaging the cells in broth medium without the mAb, VspA and VspC were

again expressed in the same abundance and in full

length as in the original clone (Fig. 3a,b, lanes 3C and 4C). Interestingly, removal of mAb lE5 resulted not only in reappearance of the original VspA or VspC size form, but also in simultaneous expression

of one or two additional, previously unexpressed

Vsps (e.g., a 42 kDa VspB in Fig. 3a,b, lanes 3C and 4C, and in some experiments a 75 kDa VspC,

data not shown). Immunostaining of corresponding blots with mAb Ns to a Vsp-unrelated 45 kDa pro-

tein revealed that the apparent level of this protein in the cultures analyzed was unaltered (Fig. 3a,b, lower panels), thereby confirming that gels were similarly

loaded with equivalent amounts of organisms.

3.4. Effect of Vp-specific mAb IA1 on Vsp surface

antigen expression in a clonal variant qf A4. bovis

type strain PG45 lacking VspA, VspB and VspC

To further examine the influence of cognate anti- bodies on the pattern of Vsp surface antigen expres- sion, a clonal variant of M bovis type strain PG45

that lacked VspA, VspB and VspC (as demonstrated by the lack of immunostaining in Western immuno-

blots of control cultures with mAb lE5; Fig. 4b, lane C), but expressed other as yet undefined Vsps of the

previously described antigen cluster I [15], was ex- posed to mAb 1Al that recognizes these additional

Vsps (Fig. 4a, lane C). This mAb treatment did not lead to significant growth inhibition. For an initial

inoculum value of less than lo3 CFU/ml, it resulted

in the disappearance of the expressed Vsps and the

appearance of two previously lacking Vsp proteins, which according to their reaction profile with mAbs 1Al (Fig. 4a, lane A) and lE5 (Fig. 4b, lane A)

could be identified as VspA and VspB. In contrast to mAb lA1, mAb lE5 did not induce any altered

patterns of Vsp expression (data not shown). As de- scribed above, the Vsp-unrelated mAb Ns-defined 45 kDa protein was used as control marker for similar

protein quantities loaded (Fig. 4, lower panels).

4. Discussion

Although increasing numbers of studies with my- coplasmas, including M pulmonis [25], M. hominis [26], M. arthritidis [27] and M. gallisepticum [28],

274 D Le Grmrl ct ul. I FEMS Microhwlog~~ Letters 144 (1996) 267 ~275

have demonstrated the occurrence of mycoplasma surface antigenic variation in vivo, it is as yet un-

known if this in vivo variation is a mechanism to

avoid destruction by the host immune system and

how it is regulated. Previous reports [l 1,12,18,19] have suggested that immune evasion by mycoplas- mas in the host may likely be accomplished by the

occurrence of antigenic variant clones which arise spontaneously and independently of the host im-

mune response. Very recently Levisohn et al. [28]

pointed out the possibility that the repertoire of my- coplasma surface antigens in vivo may be subject to immune modulation. Preliminary experimental data

by other investigators reporting the in vitro selection of antibody-induced antigenic variants of M. horninis

[29] and M. hovis [30] support this idea. The present

study with M. bovis confirms and extends these in- itial observations. It was clearly demonstrated (i)

that growth of M. bovis in the presence of M. ho-

v&specific serum antibodies from immunized or ex-

perimentally infected calves or monoclonal antibod- ies which specifically react with predominant M.

bows surface antigens, namely the Vsp lipoproteins or the Vsp-unrelated protein pMB67, may result in

repression of expression or truncation of the target protein or in switching to expression of an antigeni-

tally distinct variant protein, while (ii) removal of

the inducing antibody may cause reversion to the

original phenotype.

In the first place these observations suggest that under the specific immune pressure preexisting ‘es- cape’ variants of the culture inoculum which are re-

sistant to antibody-mediated inhibition may be se- lected. This kind of process may be illustrated by

mAbs Is and 5D7 which respectively showed a growth inhibitory effect for less than 5 X lo2 to 5 X IO” cells inocula. These results suggested that

the incidence of such ‘escape’ variants within the initial mycoplasma population was approximately

10-2-10~3 for 12 and 10-s-lo-’ for 5D7. The fre- quency of pMB67 variation was reported to be 10~“~ lo-* per cell per generation [17], this is relatively lower than our results suggesting another process that random selection might occur for 12. But further investigations must nevertheless be carried out. On the other hand, modifications induced by the mAbs

lE5 and 1Al cannot be explained by selection of low incidence ‘escape variants’. No growth inhibition oc-

curred and significant modifications appeared only for inocula of I-100 cells. The presence of such ‘es-

cape variants’ within an initial mycoplasma popula-

tion of only 1llOO cells is statistically unlikely since

the frequency of Vsps was reported to range between I OF” and 1O-5 per cell per generation [16]. We there- fore concluded that the observed changes in Vsp sur-

face antigen expression were indeed induced by the respective antibody as sensory signal. In some cases,

however, bovine serum antibodies from immunized

calves (e.g., M021) failed to induce any obvious al- terations in M. bovis surface antigen expression, a result which might be explained by the heterogeneity

of the antibody response among individual calves. Thus, the mechanisms used by M. bovis for survi-

val within the host may involve numerous pathways.

One such pathway is certainly the high-frequency mutational variation [16] which has been found in

several other mycoplasma species [ 11,12,19] and oc- curs as a random process creating a large repertoire

of antigenic variants which are subject to immune selection. A second mechanism relevant to immune

evasion has emerged from the present study. The results suggest that binding of antibodies to specific antigens on the surface of the organism may directly

affect gene expression, a process which appears to be

reversible under certain circumstances (repression-

-expression) and thus is highly reminiscent of clas-

sical bacterial metabolite induction-repression sys- tems. Although our in vitro immune pressure assay

does not exactly reflect the situation in vivo during infection, due to the absence of several factors (e.g., complement), the data presented here, along with

those recently reported by Torp et al. [29] and Giild- ner et al. [30], may lead to new insights into the pathobiology of M. bovis and other pathogenic my-

coplasma species, which will be important for the rational design of both attenuated live and subunit

vaccines to prevent and control mycoplasma infec-

tions.

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