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Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins
Veterinary Microbiology 63 (1998) 13±28
Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins RosaÂrio Gonc,alvesa,*, Jose Regallaa, Jacques Nicoletb, Joachim Freyb, Robin Nicholasc, John Bashiruddind, Paola de Santisd, Aires Penha Gonc,alvesa a
LaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Estrada de Benfica 701, 1500 Lisboa, Portugal b Institut for Veterinary Bacteriology, University of Berne, Laenggasstrasse 122, CH-3012 Berne, Switzerland c Department of Bacteriology, Central Veterinary Laboratory, Woodham Lane, Addlestone, Surrey KT15 3NB, UK d Istituto 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 * 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
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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 sera. 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 [n24], Spain [n6], France [n4] and Italy [n12]), 10 strains from African countries (Senegal [n4], Burkina Faso [n1], Chad [n1], Ethiopia [n1], Rwanda [n1], Sudan [n1] and Tanzania [n1]), 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 600g 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);
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Table 1 Strains of Mycoplasma mycoides subsp. mycoides SC used Strain
Collectiona
Origin
Isolated
Host
B103 B345 B356 B360 B421 B523 B526 B530 B674 B675/1 B675/2 B675/3 B773/124 B773/125 B820/123 B820/124 B991 B994 C305 C425 O326 O512 O526 PB90 2059 6363 6364 O697 R898 U110 PO2 2022 2091 2117 L2 Bf138 57/13 130/20 80 95 170 466 921/1 6467 6472 6479 Afade Fatick Filfili
LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LPB LPB LPB LNSPA LNSPA LNSPA CIRAD LPB LPB LPB IVBBE IZSTE IZSBRE IZSTE IZSTE IZSTE IZSTE IZSTE IZSTE IZSBRE IZSBRE IZSBRE CIRAD CIRAD CIRAD
Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Spain Spain Spain Spain Spain Spain France France France France Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Chad Senegal Senegal
1986 1993 1993 1993 1993 1993 1993 1993 1993 1993 1993 1993 1991 1991 1991 1991 1993 1993 1993 1993 1993 1993 1993 1990 1984 1991 1991 1989
Cattle/lung Cattle/pleural fluid Cattle/lymph node Cattle/lymph node Cattle/lung Cattle/lymph node Cattle/lymph node Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/semen Cattle/semen Cattle/prepucial washing Cattle/prepucial washing Cattle/lung Cattle/lung Goat/lung Goat/lung Sheep/milk Sheep/milk Sheep/milk Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle Cattle Cattle Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/lung Buffalo Cattle Cattle/pleural fluid Cattle Cattle Cattle Cattle (fetus) Cattle Cattle/lung Cattle/lung Cattle/lung Cattle Cattle Cattle
1980 1984 1984 1984 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1968 1968 preÂ- 1988
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Table 1 (Continued) Strain
Collectiona
Origin
Isolated
Host
Gemu Goffa KH3J T1Sr 2144 Dakar 2162 87137-9 Ouagadougou 94111 DVZ Gladysdale V5 PG1 (NCTC 10114)
CIRAD CIRAD CIRAD CIRAD CIRAD CIRAD CIRAD IVBBE NCTC IVBBE NCTC
Ethiopia Sudan Tanzania Senegal Senegal Burkina Faso Rwanda Australia Australia Australia Unknown
1974 1940 1952 preÂ- 1968 preÂ- 1968 1987 1994 1965
Cattle Cattle/vaccine strain Cattle/vaccine strain Cattle Cattle Cattle Cattle Cattle Cattle Cattle/vaccine strain Cattle
1965- 8 1931
a LNIV, LaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Lisboa, Portugal; LNSPA, Laboratorio Nacional de Sanidad 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 e del Molise, Teramo, Italy; IZSBRE, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, Italy; IVBBE, Institute for Veterinary Bacteriology, Berne, Switzerland; NCTC, National Collection of Type Cultures, PHLS, London, UK.
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 000g 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.
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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 (HL chains) or anti-rabbit-IgG (HL 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 000g 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 000g. 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 000g, 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
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sedimented by centrifugation at 48C for 5 min at 13 000g 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
Fig. 1. Immunoblotting profiles of several M. mycoides SC strains with a Portuguese CBPP serum (animal #806). The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 2. Immunoblotting profiles of several M. mycoides SC strains with an African CBPP serum (Kikara). The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 3. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against KH3J strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 4. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against Gladysdale strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 5. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against PG1strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 6. Trypsin treatment of M. mycoides SC (PG1 strain): (A) SDS-PAGE Coomassie blue staining; (B) Immunostaining of SDS-PAGE samples, prepared as panel A, with Portuguese CBPP serum (animal #806). Samples of whole lysed organisms (lane 1), proteins subjected to 0.4 mg mlÿ1 (lane 2) and 1.0 mg mlÿ1 (lane 3) trypsin concentrations. Protein and antigen identifications shown at right in both panels.
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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
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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 immunological 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
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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.
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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± 1100. 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 SDSPAGE 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 surface proteins RosaÂrio Gonc,alvesa,*, Jose Regallaa, Jacques Nicoletb, Joachim Freyb, Robin Nicholasc, John Bashiruddind, Paola de Santisd, Aires Penha Gonc,alvesa a
LaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Estrada de Benfica 701, 1500 Lisboa, Portugal b Institut for Veterinary Bacteriology, University of Berne, Laenggasstrasse 122, CH-3012 Berne, Switzerland c Department of Bacteriology, Central Veterinary Laboratory, Woodham Lane, Addlestone, Surrey KT15 3NB, UK d Istituto 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 * 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
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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 sera. 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 [n24], Spain [n6], France [n4] and Italy [n12]), 10 strains from African countries (Senegal [n4], Burkina Faso [n1], Chad [n1], Ethiopia [n1], Rwanda [n1], Sudan [n1] and Tanzania [n1]), 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 600g 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);
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Table 1 Strains of Mycoplasma mycoides subsp. mycoides SC used Strain
Collectiona
Origin
Isolated
Host
B103 B345 B356 B360 B421 B523 B526 B530 B674 B675/1 B675/2 B675/3 B773/124 B773/125 B820/123 B820/124 B991 B994 C305 C425 O326 O512 O526 PB90 2059 6363 6364 O697 R898 U110 PO2 2022 2091 2117 L2 Bf138 57/13 130/20 80 95 170 466 921/1 6467 6472 6479 Afade Fatick Filfili
LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LNIV LPB LPB LPB LNSPA LNSPA LNSPA CIRAD LPB LPB LPB IVBBE IZSTE IZSBRE IZSTE IZSTE IZSTE IZSTE IZSTE IZSTE IZSBRE IZSBRE IZSBRE CIRAD CIRAD CIRAD
Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Portugal Spain Spain Spain Spain Spain Spain France France France France Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Chad Senegal Senegal
1986 1993 1993 1993 1993 1993 1993 1993 1993 1993 1993 1993 1991 1991 1991 1991 1993 1993 1993 1993 1993 1993 1993 1990 1984 1991 1991 1989
Cattle/lung Cattle/pleural fluid Cattle/lymph node Cattle/lymph node Cattle/lung Cattle/lymph node Cattle/lymph node Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/semen Cattle/semen Cattle/prepucial washing Cattle/prepucial washing Cattle/lung Cattle/lung Goat/lung Goat/lung Sheep/milk Sheep/milk Sheep/milk Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle Cattle Cattle Cattle/lung Cattle/lung Cattle/lung Cattle/lung Cattle/lung Buffalo Cattle Cattle/pleural fluid Cattle Cattle Cattle Cattle (fetus) Cattle Cattle/lung Cattle/lung Cattle/lung Cattle Cattle Cattle
1980 1984 1984 1984 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1968 1968 preÂ- 1988
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Table 1 (Continued) Strain
Collectiona
Origin
Isolated
Host
Gemu Goffa KH3J T1Sr 2144 Dakar 2162 87137-9 Ouagadougou 94111 DVZ Gladysdale V5 PG1 (NCTC 10114)
CIRAD CIRAD CIRAD CIRAD CIRAD CIRAD CIRAD IVBBE NCTC IVBBE NCTC
Ethiopia Sudan Tanzania Senegal Senegal Burkina Faso Rwanda Australia Australia Australia Unknown
1974 1940 1952 preÂ- 1968 preÂ- 1968 1987 1994 1965
Cattle Cattle/vaccine strain Cattle/vaccine strain Cattle Cattle Cattle Cattle Cattle Cattle Cattle/vaccine strain Cattle
1965- 8 1931
a LNIV, LaboratoÂrio Nacional de Investigac,aÄo VeterinaÂria, Lisboa, Portugal; LNSPA, Laboratorio Nacional de Sanidad 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 e del Molise, Teramo, Italy; IZSBRE, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, Italy; IVBBE, Institute for Veterinary Bacteriology, Berne, Switzerland; NCTC, National Collection of Type Cultures, PHLS, London, UK.
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 000g 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.
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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 (HL chains) or anti-rabbit-IgG (HL 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 000g 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 000g. 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 000g, 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
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sedimented by centrifugation at 48C for 5 min at 13 000g 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
Fig. 1. Immunoblotting profiles of several M. mycoides SC strains with a Portuguese CBPP serum (animal #806). The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 2. Immunoblotting profiles of several M. mycoides SC strains with an African CBPP serum (Kikara). The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 3. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against KH3J strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 4. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against Gladysdale strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 5. Immunoblotting profiles of several M. mycoides SC strains with the hyperimmune serum against PG1strain. The identification of strains is given on top of each row and is described in Table 1. Pt ± Portugal; Sp ± Spain; Fr ± France; It ± Italia; Af ± Africa; Au ± Australia. Molecular weight markers at left and antigens at right.
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Fig. 6. Trypsin treatment of M. mycoides SC (PG1 strain): (A) SDS-PAGE Coomassie blue staining; (B) Immunostaining of SDS-PAGE samples, prepared as panel A, with Portuguese CBPP serum (animal #806). Samples of whole lysed organisms (lane 1), proteins subjected to 0.4 mg mlÿ1 (lane 2) and 1.0 mg mlÿ1 (lane 3) trypsin concentrations. Protein and antigen identifications shown at right in both panels.
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25
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
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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 immunological 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
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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.
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R. Gonc,alves et al. / Veterinary Microbiology 63 (1998) 13±28
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± 1100. 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 SDSPAGE 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.