Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major...

transcript

Antigen heterogeneity among Mycoplasma

mycoides subsp. mycoides SC isolates:

discrimination of major surface proteins

RosaÂrio Gonc,alvesa,*, JoseÂ Regallaa, Jacques Nicoletb,Joachim Freyb, Robin Nicholasc, John Bashiruddind,

Paola de Santisd, Aires Penha Gonc,alvesa

aLaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Estrada de Benfica 701, 1500 Lisboa, PortugalbInstitut for Veterinary Bacteriology, University of Berne, Laenggasstrasse 122,

CH-3012 Berne, SwitzerlandcDepartment of Bacteriology, Central Veterinary Laboratory, Woodham Lane, Addlestone,

Surrey KT15 3NB, UKdIstituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise `G. Caporale', via Campo Boario,

64100 Teramo, Italy

Received 3 February 1998; accepted 1 June 1998

Abstract

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma

mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel

electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia

antisera and hyperimmune rabbit sera against several representative strains revealed some

differences in protein profiles and variability in antigens among strains from different geographic

regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48,

44, 39 and 38 kDa. There were differences among European strains, where a larger group coming

from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from

Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic

profiles, with quantitative differences and in a few strains some antigenic bands were absent. The

group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/

70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp.

mycoides SC membrane proteins were characterised by Triton X-114 partitioning and p110, p98,

p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these

five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential

Veterinary Microbiology 63 (1998) 13±28

* Corresponding author. Tel.: +351-1-7162075; fax: +351-1-7163964.

0378-1135/98/$ ± see front matter Crown Copyright # 1998 Published by Elsevier Science B.V.

PII: S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 2 1 4 - 4

of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by

the trypsin treatment. Crown Copyright # 1998 Published by Elsevier Science B.V.

Keywords: Mycoplasma mycoides; Contagious bovine pleuropneumonia; Antigens; Immunoblotting

1. Introduction

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp.

mycoides SC (M. mycoides SC), is a serious disease in some parts of the world. In the

African continent it causes important economic losses, while in Europe outbreaks have

been reported in Portugal, Italy and Spain in the past decade. Approaches to study the

mechanisms involved in the pathogenesis of M. mycoides SC have been directed towards

identifying and characterizing the major antigens in order to distinguish strains of high

and low virulence. Antigenic variation of M. mycoides SC of bovine origin has been

demonstrated (Costas et al., 1987; Poumarat and Solsona, 1995) with isolates from

different animal species and geographic locations (Gonc,alves et al., 1994b, 1996). These

preliminary studies showed differences among strains isolated from cattle, small

ruminants and water buffalo originating from European countries, in particular Italy. The

results obtained led us to extend this work to more strains and a wider range of polyclonal

The aims of this study were: (i) to differentiate strains of M. mycoides SC from Europe,

Africa and Australia, including the type strain PG1 by immunoblotting with a panel of

polyclonal antibodies to establish the major immunogenic components; (ii) to identify

major surface membrane proteins with Triton X-114 partitioning; and (iii) to study the

membrane topology of immunodominant antigens.

2. Materials and methods

2.1. Strains and growth conditions

Forty-six strains of M. mycoides SC from European countries (Portugal [n�24], Spain

[n�6], France [n�4] and Italy [n�12]), 10 strains from African countries (Senegal

[n�4], Burkina Faso [n�1], Chad [n�1], Ethiopia [n�1], Rwanda [n�1], Sudan [n�1]

and Tanzania [n�1]), three strains from Australia, and the type strain PG1 were used in

this study (Table 1). M. mycoides SC cells, from log-phase cultures growing in modified

Gourlay medium at 378C, were harvested by centrifugation at 14 600�g for 1 h at 48C,

followed by three washes in phosphate-buffered saline solution (PBS, 0.1 M Na2HPO4,

0.1 M NaH2PO4, 0.15 M NaCl, pH 7.2). Washed cells were resuspended in the same

buffer and stored at ÿ208C.

2.2. Hyperimmune sera and bovine sera

Seven sera from naturally infected cattle with CBPP consisted of: three from Portugal

(#806, #845, #925), three from Italy (#1, #2, #3) and one from Uganda (animal Kikara);

14 R. Gonc,alves et al. / Veterinary Microbiology 63 (1998) 13±28

Table 1Strains of Mycoplasma mycoides subsp. mycoides SC used

Strain Collectiona Origin Isolated Host

B103 LNIV Portugal 1986 Cattle/lung

B345 LNIV Portugal 1993 Cattle/pleural fluid

B356 LNIV Portugal 1993 Cattle/lymph node

B675/1 LNIV Portugal 1993 Cattle/lung

B773/124 LNIV Portugal 1991 Cattle/semen

B773/125 LNIV Portugal 1991 Cattle/semen

B820/123 LNIV Portugal 1991 Cattle/prepucial washing

B820/124 LNIV Portugal 1991 Cattle/prepucial washing

C305 LNIV Portugal 1993 Goat/lung

C425 LNIV Portugal 1993 Goat/lung

O326 LNIV Portugal 1993 Sheep/milk

PB90 LNIV Portugal 1990 Cattle/lung

2059 LPB Spain 1984 Cattle/lung

O697 LNSPA Spain 1989 Cattle

R898 LNSPA Spain Cattle

U110 LNSPA Spain Cattle

PO2 CIRAD France 1980 Cattle/lung

2022 LPB France 1984 Cattle/lung

L2 IVBBE Italy 1992 Cattle/lung

Bf138 IZSTE Italy 1992 Buffalo

57/13 IZSBRE Italy 1992 Cattle

130/20 IZSTE Italy 1992 Cattle/pleural fluid

80 IZSTE Italy 1992 Cattle

466 IZSTE Italy 1992 Cattle (fetus)

921/1 IZSTE Italy 1992 Cattle

6467 IZSBRE Italy 1992 Cattle/lung

AfadeÂ CIRAD Chad 1968 Cattle

Fatick CIRAD Senegal 1968 Cattle

Filfili CIRAD Senegal preÂ- 1988 Cattle

R. Gonc,alves et al. / Veterinary Microbiology 63 (1998) 13±28 15

and sera from cattle submitted to contact exposure with endobronchially infected animals

consisted of: one from Portugal (#11) contact exposed to field strain (Correia et al., 1990)

and one from a Swiss cow (#502) contact exposed to Italian strain L2 (Miserez et al.,

1996). These were collected at days 101 and 112 post-contact and had complement

fixation test titres of 1/1280 and 1/160, respectively. Hyperimmune rabbit serum to a

Portuguese strain (B345) absorbed with whole cells of M. mycoides SC Italian strain 466

(monospecific serum to 98 kDa antigen) (Gonc,alves et al., 1996) and rabbit antisera

against 17 M. mycoides SC isolates representing different antigenic profiles and countries

of origin were produced. The monospecific serum was prepared by adsorbtion as follows:

equal parts of hyperimmune rabbit serum were mixed with the sediment obtained after

the centrifugation of the Italian strain 466 for 4 h at room temperature (RT) and overnight

at 48C. The amount of original cell mass used for adsorption was equivalent to 1513 mg of

protein. Serum collected by centrifugation at 13 000�g for 60 min was adsorbed twice in

order to avoid non-specific immunostaining. Hyperimmune antisera to whole

mycoplasma organisms were raised in rabbits by injections of 200 mg protein per dose

per animal in Freund's incomplete adjuvant. The rabbits were injected intradermally and

intramuscularly twice with a 3-week interval, and boosted 3 weeks later with intravenous

injections five or six times every 48 h. Rabbits were bled 4 days after administration of

the last injection. All the preinoculated rabbit sera were negative both by CFT and

immunoblotting procedures using B103 antigen. Sera and antisera were diluted 1:10 or

1:100 in PBS containing 0.1% skim milk and 0.1% ovalbumin (dilution buffer) for

Western blots, the antisera being first absorbed with culture medium, in equal parts, to

avoid non-specific reactions.

Table 1(Continued)

Strain Collectiona Origin Isolated Host

Gemu Goffa CIRAD Ethiopia 1974 Cattle

KH3J CIRAD Sudan 1940 Cattle/vaccine strain

T1Sr CIRAD Tanzania 1952 Cattle/vaccine strain

2144 Dakar CIRAD Senegal preÂ- 1968 Cattle

2162 CIRAD Senegal preÂ- 1968 Cattle

87137-9 Ouagadougou CIRAD Burkina Faso 1987 Cattle

94111 CIRAD Rwanda 1994 Cattle

DVZ IVBBE Australia 1965 Cattle

Gladysdale NCTC Australia Cattle

V5 IVBBE Australia 1965- 8 Cattle/vaccine strain

PG1 (NCTC 10114) NCTC Unknown 1931 Cattle

a LNIV, LaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Lisboa, Portugal; LNSPA, Laboratorio Nacional deSanidad y ProduccioÂn Animal, Santa FeÂ, Granada, Spain; LPB, Laboratoire de Pathologie Bovine, Lyon, France;CIRAD, CIRAD-EMVT, Maisons-Alfort, Franc,a; IZSTE, Istituto Zooprofilattico Sperimentale dell'Abruzzo edel Molise, Teramo, Italy; IZSBRE, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia,Brescia, Italy; IVBBE, Institute for Veterinary Bacteriology, Berne, Switzerland; NCTC, National Collection ofType Cultures, PHLS, London, UK.

2.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting

The protein content of thawed mycoplasma cells was estimated by the method of

Lowry et al. (1951). Samples were mixed with lysis buffer (500 mM Tris/HCl pH 6.8,

4.6% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol and 0.004%

bromophenol blue) and boiled for 5 min. Equal amounts of lysates (20 mg wellÿ1) were

separated by SDS-PAGE in a 1.5 mm thick, 4% stacking/5±15% gradient-resolving gel

and the electrophoresis was run at a constant current of 40 mA according to Laemmli

(1970) and visualised with a Coomassie blue stain.

Separated proteins were transferred to 0.45 mm nitrocellulose membranes (NC) at 70 V

constant voltage for 1.5 h (Towbin et al., 1979). The membranes were blocked in PBS

containing 1 M glycine, 1% egg albumin and 5% skim milk for 2 h at RT then allowed to

react with diluted sera at 378C under agitation for 2 h. After washing at RT for three

15 min washes in 0.1% (v/v) Tween 20 in PBS and again for 10 min in PBS alone, the NC

sheets were incubated for 1 h at RT with appropriate dilutions of peroxidase-conjugated

sheep anti-bovine-IgG (H�L chains) or anti-rabbit-IgG (H�L chains) in dilution buffer.

After washing as above, the substrate comprising 30 mg 4-chloro-1-naphthol dissolved in

10 ml methanol jointed to 50 ml PBS and 30 ml H2O2 was added and left for 5±15 min in

the dark.

2.4. Fractionation of membrane components

The PG1 strain was subjected to Triton X-114 (TX-114) phase partitioning as

described by Bordier (1981), and applied to mycoplasmas (Riethman et al., 1987; Wise

and Kim, 1987). The strain was thawed and adjusted to 2 mg protein mlÿ1 in PBS.

Prewashed, condensed TX-114 was added to give a final concentration of 1% (w/v) and

incubated for 30 min at 48C. After centrifugation at 48C for 5 min at 13 000�g to remove

the insoluble material, the supernatant was incubated for 5 min at 378C to induce rapid

condensation of TX-114, and then centrifuged at 228C for 5 min at 10 000�g. The

aqueous (upper) phase was transferred to a second tube and brought to 1% TX-114,

whereas the TX-114 (lower) phase was brought to the original volume with PBS. Both

suspensions were then dissolved by incubation at 48C for 15 min and phase partitioning

was repeated three times. Both phases were used for subsequent electrophoretic and

immunoblotting analysis with a Portuguese CBPP serum (animal #806).

2.5. Treatment of mycoplasma proteins with trypsin

Trypsin at two final concentrations was used (Riethman et al., 1987) to digest whole

cells of the PG1 strain. Protocol 1: Washed cells of a 48 h culture incubation were

suspended in TS buffer (10 mM Tris±HCl pH 7.5, 150 mM NaCl), to a final concentration

of 0.4 mg mlÿ1 of trypsin (Sigma) and incubated at 378C for 30 min. After centrifugation

at 48C for 5 min at 13 000�g, pelletted cells were washed twice with TS. Protocol 2:

Washed cells of a 48 h culture incubation were suspended in PBS with trypsin to a final

concentration of 1.0 mg mlÿ1 plus the protease-inhibitor phenylmethylsulfonyl fluoride

to a final concentration of 1 mM and incubated at 378C for 30 min. Mycoplasmas were

sedimented by centrifugation at 48C for 5 min at 13 000�g and the cell pellet washed

twice with PBS. Sample lysis buffer to the original volume was immediately added to

pellets treated as described. Aliquots of mycoplasma cells in buffers without enzyme

were treated similarly. Samples were heated under reducing conditions and submitted to

SDS-PAGE and western blotting. Blots were immunostained with a Portuguese CBPP

serum (animal #806).

3. Results

SDS-PAGE separation of proteins of M. mycoides SC strains stained with Coomassie

blue produced 50±60 bands corresponding to molecular weights of 212±14.4 kDa. In one

Portuguese strain and 10 Italian strains the 98 kDa band was not present. A similarity in

protein profiles in the range of 94±67 kDa and 53±34 kDa was observed between Spanish

strain 2059 and African strain 94111. The amounts of particular proteins differed in

African vaccine strains. In Australian strains the absence of 95 kDa in Gladysdale and

30 kDa protein in vaccine strain V5 was observed. PG1 strain lacked the 54 and 30 kDa

proteins, and had a similar profile to the V5 strain.

In immunoblotting the major conserved antigenic bands ranged from 110±38 kDa

designated p110, p95, p80, p69, p62, p60, p48, p44, p39 and p38 according to their

molecular sizes. The bands identified by bovine sera were always lower in number than

those by rabbit antisera. When Portuguese strains were analysed with infected animal sera

and hyperimmune sera, one strain (B675), differed from all the others. This strain was

further cloned and one clone (B675/3) had a similar profile to most Italian strains, that is,

it lacked the 98 kDa protein. Sera from naturally infected Italian bovines and the serum

from a bovine infected experimentally with strain L2 (Swiss bovine #502) did not

recognise the 98 kDa antigen (Gonc,alves et al., 1996). Two Italian strains studied (6467

and 6472) presented the 98 kDa (Fig. 1). Strains 2059 and 94111 produced a distinctive

profile with serum from animal #806 that distinguished them from all others: a strong

reaction at the level of 81.5, 77.5 and 65 kDa. The p95 antigen was not stained in the

isolate from prepucial washing, B820/123 (Gonc,alves, 1994) and Gladysdale strain, and

in the French strain 2091 this polypeptide was always situated at a lower level comparing

to the other strains (see arrow in Fig. 1). In strain PG1 the p45 and p38 were weakly

recognised by sera from naturally infected bovines. Serum from an African bovine

detected most of the antigens detected by sera from European bovines but with some

differences: 71.5/70 kDa polypeptides were recognised in African, Australian strains and

PG1 but not in European strains (Fig. 2). The same profile was obtained with all the

hyperimmune rabbit sera produced with African, Australian and PG1 strains (Figs. 3±5).

It is important to clarify that, in the present study, the molecular weight of the

immunogenic protein P72 (Cheng et al., 1996) was slightly lower, 69/67 kDa (data not

shown). The monospecific serum to 98 kDa antigen, as described above, reacted only

with the 98 kDa polypeptide in Portuguese, Spanish and French strains and not with any

proteins of the Italian strains tested (Gonc,alves et al., 1996). Strains isolated from bull

semen, specially B774/124 strain (Gonc,alves, 1994), showed a larger number of

immunoreactive bands. Also, the hyperimmune rabbit serum produced against the other

al#806).

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right.

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strain from semen (B773/125) detected a larger number of antigenic bands in all the

M. mycoides SC strains studied. In the Italian strain 6467 the 48 kDa protein was not

antigenic when reacted with all available antisera although a similar protein was present

in electrophoretic profile. The 37 kDa polypeptide of the Senegalese strain Filfili was not

revealed with any sera. Anti-Filfili serum did not recognise the 37 kDa polypeptide of any

strain. Differences observed in immunoblots of all strains stained with hyperimmune

rabbit sera produced against African vaccine strains were: 76, 65 and 37 kDa antigens

were not identified by anti-KH3J, with the exception of a 37 kDa antigen in PG1 strain.

PG1 hyperimmune rabbit serum did not recognise the polypeptide of 54 kDa in all

strains. A weak reaction in the 45 and 39/38 kDa antigens was observed in all strains

immunoblotted with this antiserum and there was an absence of the staining of 30 kDa

polypeptide.

To investigate the possibility that the variable antigens detected in several strains of

M. mycoides SC might be membrane proteins, PG1 strain was subjected to TX-114 phase

fractionation and further staining with a CBPP serum to determine whether these proteins

were immunogenic in the natural host. Immunoblots of SDS-PAGE profiles of TX-114

treated proteins of PG1, showed the presence of 11 proteins in the detergent phase, of

which p110, p98, p95, p80, p62, p48 and p44 were strongly recognised as immunogenic

by a CBPP serum, as well as three soluble proteins in aqueous phase (p55, p38, p37). On

the basis of their highly selective hydrophobic properties, antigens p110, p98, p95 and

p62 were provisionally classified as integral membrane proteins. The appearance of p69/

p67 in both TX-114 phases is likely to be due to an incomplete partitioning as P72 has

been shown to be a lipoprotein (Cheng et al., 1996).

Stained protein profiles and immunoblots of strain PG1 treated with trypsin showed

that some of the above immunogenic bands (p110, p98, p95, p62) were removed with a

greater amount of trypsin (Fig. 6(A),(B), lane 3) whereas mycoplasma cells in buffers

without trypsin yielded the native proteins (data not shown). In spite of the failure of the

total hydrophobicity of bands at 69 and 67 kDa their surface location was confirmed by

their removal with trypsin treatment (Fig. 6(A),(B), lane 3). The surface membrane

position of antigen p48 could not be demonstrated conclusively. A polypeptide of about

48 kDa was stained after trypsin treatment, but was probably the residue of proteolytic

degradation of another protein. However, in Coomassie blue-stained gels it appeared to be

membrane-associated.

4. Discussion

In the work reported here differences in protein profiles between M. mycoides SC

strains usually reflected variations in the concentrations of individual proteins, but, the

absence of specific proteins was noted in some cases. In a previous study (Gonc,alves et

al., 1994a), the type strain PG1 of unknown origin was characterised by the absence of

54 kDa protein. In this study, protein profiles of PG1 and the Australian strain V5 were

similar, both lacking the 30 kDa band.

European strains form a genetic clonal lineage (Cheng et al., 1995), and antigenic

differences within them are seen in the Portuguese (with one exception), Spanish and

French strains which together form one group with nearly identical immunoprofiles

which are distinct from the Italian strains. The detection of two distinct clones in the same

culture of a Portuguese strain, one of them antigenically similar to the Italian group,

demonstrates a mixed population in the same isolate. Previous studies have demonstrated

the difference in most Italian strains which lack the 98 kDa protein when compared with

other European strains (Gonc,alves et al., 1996). The results presented here suggest that

the 10 Italian strains may not synthesise the polypeptide corresponding to the 98 kDa

antigen or its antigenic structure is unrelated and they are a variant clone from the same

geographic origin. A different antigenic profile around 100 kDa was also observed in

92% of Italian strains (Poumarat and Solsona, 1995). To correlate these findings and to

have a better epidemiologic knowledge of M. mycoides SC in Europe it is imperative to

analyse a higher number of strains, especially Spanish and French strains.

Though the European strains have genetic patterns different from African strains

(Cheng et al., 1995), observations have verified an antigenic similarity between a Spanish

strain (2059) and an African strain from Rwanda after immunostaining with sera from

naturally infected Portuguese cattle. This African strain also forms a genetic pattern

different from the second cluster (Cheng et al., 1995). In agreement with these genetic

studies, immunologically, PG1-type strain is closely related to the African and Australian

strains, with a strong band at level 71.5/70 kDa, which is not found in European strains.

These results give strong support to the hypothesis formulated by several researchers

(Cheng et al., 1995; Poumarat and Solsona, 1995) that re-emergent CBPP outbreaks in

the last 15 years in Europe were not from imported African cattle. Together these findings

also point out that both the genetic and the protein banding similarities between

Australian and African strains diverge from the generally accepted proposition that CBPP

in Australia was imported from Europe (Provost et al., 1987; ter Laak, 1992).

Alternatively, and more likely, the Australian and African strains have arisen from strains

which no longer exist in Europe today.

Additionally, our observations with PG1 strain, show that the 98 kDa is confined to the

hydrophobic TX-114 phase and disappears with trypsin treatment of whole cells, thereby

confirming its surface membrane localization. The majority of the proteins recognised by

the CBPP serum in the detergent phase were also immunodominant in previous studies

with whole cells (Regalla et al., 1994). These data showed that IgG immunoprofiles of

sera from naturally infected cattle in all stages of the CBPP contained from 10 to 35

reactive proteins. Of these the simultaneous presence of five proteins ± p110, p98, p95,

p62, and p48 ± were common to all the sera examined and, therefore, were considered the

minimum CBPP-specific profile of immunodominant antigens. This common immuno-

logical pattern is very encouraging as much as it indicates the diagnostic potential of the

immunoblotting test: even in CFT-negative sera from infected animals this profile was

common (Regalla et al., 1996). A strong IgA reaction to the membrane lipoprotein P72 of

M. mycoides SC, in bronchial lavage samples of cattle experimentally infected, confirmed

the induction of a specific local immune response (Abdo et al., 1997). Furthermore, these

studies showed that other reacting antigens with IgA, such as 110, 95 and 48 kDa, are

lipoproteins.

Proteins partitioning into TX-114 phase have been demonstrated previously to be

important immunogenic surface components recognised by host antibodies during

infection (Riethman et al., 1987; Rosengarten and Wise, 1991). The function of antigenic

variation in several mycoplasmas like M. hyorhinis (Rosengarten and Wise, 1991),

M. gallisepticum (Yogev et al., 1994), M. bovis (Sachse et al., 1992; Rosengarten et al.,

1994), has been attributed to either immune evasion or, for structural proteins,

microorganism±host interactions essential for pathogenesis. Antigenic variation, an

important mechanism of infection, is due to membrane surface proteins (Vsps) mainly

lipoproteins (Wise et al., 1993). Further studies to demonstrate if M. mycoides SC

lipoproteins have a similar role in virulence are in progress.

Moreover, from these preliminary studies, it appears that in PG1 a peripheral

membrane protein with an apparent molecular weight of 37 kDa which partitions in the

aqueous phase contributes to the variation of the antigenic profile in Filfili strain of M.

mycoides SC. Similar proteins have been demonstrated in other species of mycoplasmas

(Rosengarten et al., 1995).

Acknowledgements

We are grateful to Rosinda Martinho, Joaquim Galharda and Paula Cortes for their

expert technical assistance. This study is part of the European COST action 826 on

ruminants' mycoplasmoses. It has been carried out with the financial support of the

Commission of the European Communities, Agriculture and Fisheries (FAIR) specific

RTD programme, CT95-0711 `Development of new and improved diagnostic tests for

Contagious Bovine Pleuropneumonia (CBPP) in Europe'. It does not necessarily reflect

its views and in no way anticipates the Commission's future policy in this area.

References

Abdo, El-M., Nicolet, J., Miserez, R., Gon, , c,alves, R.Regalla, J., Griot, C., Bensaide, A., Krampe, M., Frey, J.,

1997. Humoral and bronchial immune responses in cattle experimentally infected with Mycoplasma

mycoides subsp. mycoides SC. Vet. Microbiol. 59, 109±122.

Bordier, C., 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256,

1604±1607.

Cheng, X., Nicolet, J., Poumarat, F., Regalla, J., Thiaucourt, F., Frey, J., 1995. Insertion element IS1296 in

Mycoplasma mycoides subspecies mycoides small colony identifies an European clonal line distinct from

African and Australian strains. Microbiol. 141, 3221±3228.

Cheng, X., Nicolet, J., Miserez, R., Kuhnert, P., Krampe, M., Pilloud, T., Abdo, El-M., Griot, C., Frey, J., 1996.

Characterisation of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp.

mycoides small colony type. Microbiol. 142, 3515±3524.

Costas, M., Leach, R.H., Mitchelmore, D.L., 1987. Numerical analysis of PAGE protein patterns and the

taxonomic relationships within the `Mycoplasma mycoides cluster'. J. Gen. Microbiol. 133, 3319±3329.

Correia, I., Regalla, J., Ferreira, H., Correa de SaÂ, I., Albuquerque, T., Vacas de Carvalho, J., Penha Gonc,alves,

A., 1990. Comparative study of immunological tests: Complement fixation test, passive haemagglutination

and radial reverse immunodiffusion as applied to the serological diagnosis of contagious bovine

pleuropneumonia, In: Regalla, J. (Ed.) Contagious bovine pleuropneumonia, Commission of European

Communities, EUR 12065 EN, Luxembourg, pp. 125±143.

Gonc,alves, M.R., 1994. Isolation and identification of Mycoplasma mycoides subsp. mycoides SC from bull

semen and sheath washings in Portugal. Vet. Rec., 135, 308±309.

Gonc,alves, R., Regalla, J., Penha Gonc,alves, A., 1994a. Immunoblotting and electrophoretic analysis of

Mycoplasma mycoides subsp. mycoides SC strains isolated from bovine and small ruminants, Int. Organ.

Mycoplasmol. Lett. 3, 64±65.

Gonc,alves, R., Regalla, J., Penha Gonc,alves, A., 1994b. Intraspecies antigenic variation in Mycoplasma

mycoides subsp. mycoides SC strains from different sources detected through immunoblots with naturally

infected bovine sera, XX ReuniaÄo da Sociedade Portuguesa de Imunologia, Lisboa, 64 pp.

Gonc,alves, R., Regalla, J., Nicolet, J., Bashiruddin, J., DeSantis, P., Penha Gonc,alves, A., 1996. A 98 kDa

antigenic band as a possible epidemiological marker differentiating European isolates of Mycoplasma

mycoides subsp. mycoides SC, In: Frey, J., Sarris, K., (Eds.), COST 826 ± Mycoplasmas of Ruminants:

Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, European Commission, EUR 16934

EN, Luxembourg, pp. 49±51.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

Nature 227, 680±685.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol

reagent. J. Biol. Chem. 193, 265±275.

Miserez, R., Pilloud, T., Krampe, M., Griot, C., Bruckner, L., Blum, J., Tontis, A., Frey, J., Nicolet, J., 1996.

Experimental infections in cattle with a European strain and an African strain of Mycoplasma mycoides

subsp. mycoides SC, In: Frey, J., Sarris, K. (Eds.) COST 826 - Mycoplasmas of Ruminants: Pathogenicity,

Diagnostics, Epidemiology and Molecular Genetics, European Commission, EUR 16934 EN, Luxembourg,

pp. 147±149.

Poumarat, F., Solsona, M., 1995. Molecular epidemiology of Mycoplasma mycoides subsp. mycoides biotype

small colony, the agent of contagious bovine pleuropneumonia. Vet. Microbiol. 47, 305±315.

Provost, A., Perreau, P., Breard, A., Le Goff, C., Martel, J.L., Et Cottew, G.S., 1987. PeÂripneumonie contagieuse

bovine. Rev. Sci. Tech. Off. Int. Epiz 6(3), 565±624.

Regalla, J., Gonc,alves, R., Penha Gonc,alves, A., 1994. Immunodominant antigens of Mycoplasma mycoides

subsp. mycoides SC agent of contagious bovine pleuropneumonia, XX ReuniaÄo da Sociedade Portuguesa de

Imunologia, Lisboa, 65pp.

Regalla, J., Gonc,alves, R., Niza Ribeiro, J., Duarte, L., Penha Gonc,alves, A., 1996. Use of Immunoblotting test

for carrier state definition in contagious bovine pleuropneumonia, In: Frey, J., Sarris, K., (Eds.), COST 826

± Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics,

European Commission, EUR 16934 EN, Luxembourg, pp. 72±74.

Riethman, H.C., Boyer, M.J., Wise, K.S., 1987. Triton X-114 phase fractionation of an integral membrane

surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis. Infect. Immun. 55, 1094±

Rosengarten, R., Wise, K.S., 1991. The Vlp system of Mycoplasma hyorhinis: Combinatorial expression of

distinct size variant lipoproteins generating high-frequency surface antigenic variation. J. Bacteriol. 173,

4782±4793.

Rosengarten, R., Behrens, A., Stetefeld, A., Heller, M., Ahrens, M., Sache, K., Yogev, D., Kirchhoff, H., 1994.

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of

diverse membrane surface proteins. Infect. Immun. 62, 5066±5074.

Rosengarten, R., Levisohn, S., Yogev, D., 1995. A 41 kDa variable surface protein of Mycoplasma gallisepticum

has a counterpart in Mycoplasma imitans and Mycoplasma iowae. FEMS Microb. Lett. 132, 115±123.

Sachse, K. Grajetzki, C., Pfutzner, H., Hass, R., 1992. Comparison of Mycoplasma bovis strains based on SDS-

PAGE and immunoblot protein patterns. J. Vet. Med. B 39, 246±252.

ter Laak, E.A., 1992. Contagious bovine pleuropneumonia. A review. Vet. Quart. 14, 104±110.

Towbin, S.T., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrilamide gels to

nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350±4354.

Wise, K.S., Kim, M.F., 1987. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively

modified by covalently bound lipid. J. Bacteriol. 169, 5546±5555.

Wise, K.S., Kim, M.F., Theiss, P.M., Lo, S.-C., 1993. A family of strain-variant surface lipoproteins of

Mycoplasma fermentans. Infect. Immun. 61, 3327±3333.

Yogev, D., Menaker, D., Strutzberg, K., Levisohn, S., Kirchhoff, H., Hinz, K.-H., Rosengarten, R., 1994. A

surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and

Mycoplasma bovis. Infect. Immun., 62, 4962±4968.

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major...

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