Institute for Veterinary Bacteriology,
University of Berne, CH-3012 Berne,
Switzerland1; CIRAD-EMVT, Campus
International de Baillarguet, F-34032 Montpellier,
France2; and Facultad de
Veterinaria, Universidad de Las Palmas, E-35071 Las Palmas,
Spain3
Received 29 June 1998/Returned for modification 6 November
1998/Accepted 4 December 1998
 |
INTRODUCTION |
The Mycoplasma mycoides
subsp. mycoides large-colony type (LC) and M. mycoides subsp. capri strains belong to the
M. mycoides cluster, a group of six closely related
mycoplasmas (13). M. mycoides subsp.
mycoides LC causes mastitis, keratoconjunctivitis, polyarthritis, pneumonia, and septicemia in goats (13, 14, 34,
36). It has also been isolated, rarely, from cattle (20, 27) and sheep (25). M. mycoides subsp.
capri is reported to cause a pattern of diseases similar to
those induced by M. mycoides subsp. mycoides
LC specifically in goats, including mastitis, arthritis, and pulmonary
diseases (13, 21, 23, 28, 40, 41). From an epidemiological
point of view, differential identification of the members of the
M. mycoides cluster is of major importance. An accurate and
rapid means of identification of the subspecies or subtypes of
mycoplasmas of the M. mycoides cluster is prerequisite for
the differentiation, since the different members of this cluster show
very strong differences in virulence and epidemiological impact.
However, many methods fail in specificity because they are hampered by
strong serological cross-reactions between the different members of the
M. mycoides cluster (7, 12, 15, 17, 32, 39).
It has been reported that M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri are antigenically very similar as assessed by
numerical analysis of one-dimensional sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein patterns
(11, 22). Identical results have been obtained by
two-dimensional PAGE protein patterns, which confirm that M. mycoides subsp. mycoides LC strains are more closely
related to M. mycoides subsp. capri strains than to M. mycoides subsp. mycoides small-colony type
(SC) strains (26, 35).
A DNA probe based on a randomly chosen genomic fragment was developed
for the differentiation of the different members of the M. mycoides cluster into four groups. This DNA probe grouped M. mycoides subsp. mycoides LC strains together with
M. mycoides subsp. capri and distinguished them
from the other members of the M. mycoides cluster. However,
it did not allow differentiation between these two mycoplasmas
(38). DNA-DNA hybridization studies revealed variable values
for DNA homology between M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri (75 to 94%), between M. mycoides subsp.
mycoides LC and M. mycoides subsp.
mycoides SC (88 to 93%), and between M. mycoides
subsp. capri and M. mycoides subsp.
mycoides SC (75 and 93%) depending on experimental
conditions (2, 9). Phylogenetic studies based on sequence
analysis of 16S rRNA genes (rrs) revealed 99.9% similarity
between M. mycoides subsp. mycoides LC and
M. mycoides subsp. capri. These results suggested that these two mycoplasmas could be grouped into a single subspecies, one distinct from M. mycoides subsp.
mycoides SC (29).
With the objective of getting a better insight into the antigenic and
genetic differences among the different members of the M. mycoides cluster, the major surface-located lipoprotein antigens of M. mycoides subsp. mycoides SC, P72, and
of Mycoplasma sp. bovine group 7, P67, had been
characterized (8, 16). Immunoblot analysis of type and field
strains of the different species from the M. mycoides cluster by using monospecific polyclonal antibodies against each of these proteins revealed that these two proteins were
antigenically species specific.
Southern blot hybridization with a gene probe for P72 revealed
that the M. mycoides subsp. capri strain PG3
and the M. mycoides subsp. mycoides LC
strain Y-goat as well contained an analogous gene. The aim of the
present study was to clone, sequence, and analyze the genes
encoding the major lipoproteins from M. mycoides subsp.
capri and M. mycoides subsp.
mycoides LC.
| |
MATERIALS AND METHODS |
Strains and growth conditions.
Mycoplasma strains used in
this study and their origins are listed in Table
1. The Mycoplasma species were
cultured in standard mycoplasma medium at 37°C (5). Cells
were pelleted by centrifugation at 20,000 × g for 20 min, washed in TES buffer (10 mM Tris, 1 mM EDTA, 0.8% NaCl [pH
8.0]) and resuspended in TES buffer to reach a cell concentration of
109 cells ml
1. Escherichia coli
XL1-Blue MRF' {
(mcrA)183
(mcrCB-hsdS-mrr)173 endA1 supE44 thi-1
recA1 gyrA96 relA1 lac [F' proAB
laclqZM15
Tn10(Tetr)]} and XLOLR
{(mcrA)183
(mcrCB-hsdS-mrr)173 endA1 thi-1 recA1 gyrA96 relA1 lac [F' proAB
laclqZM15
Tn10(Tetr)]
r,
Su} (Stratagene, La Jolla, Calif.) were grown on
Luria-Bertani broth at 37°C (3). E. coli YN2980
(leuUGA lacZ659UGA
trpA9605UAG his-29UAG
ile thyA metB argH rpoB rpsL prfB3[pISM3001])
was used as a suppressor strain for UGATrp and was grown
as described (37). Expression vector pBK-CMV phagemid
(Stratagene) was propagated in XLOLR strain.
DNA manipulation, construction and screening of gene library, and
PCR.
Genomic DNA was extracted by the guanidinium
thiocyanate method as indicated (31). DNA from the
M. mycoides subsp. mycoides LC strain Y-goat
and the M. mycoides subsp. capri strain PG3
was partially digested with SauIIIA and used to construct
ZAP Express phage banks (Stratagene) as described (8).
The gene libraries were screened with a digoxigenin-11-dUTP-labeled DNA
probe, and positive clones were transformed to phagemids as indicated
(16). All standard techniques of molecular biology have been
described previously (3). PCR amplifications were carried
out as indicated (16), by using the oligonucleotide
primers listed in Table 2.
Nucleotide sequencing and sequence analysis.
Plasmids
pJFFMMLC2 (from M. mycoides subsp. mycoides
LC) and pJFFMMCA4 (from M. mycoides subsp.
capri) containing 2.0-kb and 4.5-kb inserts, respectively,
were sequenced on both strands. Sequencing was performed with an
Applied Biosystems DNA Sequenator AB373 by using the Taq Dye
Deoxy Terminator Cycle kit (Applied Biosystems/Perkin Elmer, Norwalk,
Conn.), using oligonucleotide primers matching T3 and T7 promoter
sequences contained on the cloning vector (Table 2). Full-length
sequencing of the insert was obtained by producing an ordered nested
set of deletion mutations in the same cloning vector with a Nested
Deletion kit according to the directions of the supplier (Pharmacia
Biotech, Uppsala, Sweden) and subsequent DNA sequence analysis of the
plasmids with the T3 and T7 primers.
The DNA sequences and their deduced amino acid sequences were analyzed
with the program PROSITE (4) of the DNA analysis software
PC/Gene and with the program PSORT (24) and were aligned with the software GCG (Genetics Computer Group, Madison, Wis.). Sequence comparisons with GenBank/EMBL and NBRF databases were performed with the BLAST programs (1).
Preparation of recombinant LppA[MmymyLC] antigen and production
of polyclonal mouse anti-LppA[MmymyLC] serum.
The
lppA gene of M. mycoides subsp.
mycoides LC cannot be expressed in its full length in
E. coli K-12, due to the mycoplasma-specific UGATrp codons (42), which are read in E. coli as stop codons. We have therefore used an expression system
which has been developed for efficient suppression of
UGATrp for the expression of mycoplasmal proteins in
E. coli (37). Strain YN2980 (37) was
transformed with plasmid pJFFMMLC2 and grown to the
mid-exponential growth phase. Expression of the cloned
lppA[MmymyLC] gene from the vector's plac promoter was induced by addition of 1 mM
isopropyl-
-D-thiogalactopyranoside and growth for 4 h further. Cells were then harvested, washed with TES buffer, and
resuspended in a 0.1 volume of TES buffer. The resuspended cells were
sonicated on ice with a Branson Sonifier 250 (Branson Ultrasonics,
Danbury, Conn.) equipped with the Microtip at an output of 3 for 1 min.
The protein concentration of the suspension obtained was measured by
the method of Bradford (6) and reached 400 µg
ml1.
In order to obtain polyclonal anti-LppA[MmymyLC] serum, 150 µl of
the suspension described above containing 60 µg of protein per
administration was mixed with an equal volume of Freund's complete
adjuvant (Difco Laboratories, Detroit, Mich.) as described (18) and used to immunize mice. After 3 weeks the mice were booster immunized with the same amount of protein, but Freund's incomplete adjuvant was used. Blood for serum was taken 2 weeks later.
The serum was adsorbed with E. coli YN2980 carrying the empty vector pBK-CMV. The antigen for adsorption was prepared as
indicated (8). Immunoblot analysis was done according to standard protocols (3, 8). The mouse serum was diluted
1:2,000, and reaction products were visualized with affinity-purified
goat phosphatase-labeled anti-mouse immunoglobulin G (heavy and light chains) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.)
diluted 1:2,000.
Nucleotide sequence accession numbers.
The GenBank/EMBL
sequence accession numbers for the lppA[MmymyLC] gene from
M. mycoides subsp. mycoides LC and the
lppA[Mmyca] gene from M. mycoides subsp.
capri are AF072714 and AF072715, respectively.
| |
RESULTS |
Cloning of the lppA genes of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri.
Southern blot analysis of
HindIII-digested genomic DNA from the type and
reference strains of all members of the M. mycoides cluster was performed with a digoxigenin-labeled
gene probe for the P72 (lipoprotein) gene of M. mycoides subsp. mycoides SC (8). These
hybridization results showed the presence of genes analogous to the P72
gene in all members of the M. mycoides cluster.
Gene libraries of M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri containing about 106 phage clones
ml1 were constructed with phage ZAP Express vector
and screened. From both libraries, plasmids which were shown to
contain the entire genes of the lipoproteins named
lppA[MmymyLC] and lppA[Mmyca], as
assessed from sequencing data of the extremities of the inserts, were
retained. Plasmid pJFFMMLC2 contained a 2.0-kb insert from M. mycoides subsp. mycoides LC, and plasmid
pJFFMMCA4 contained a 4.5-kb insert from M. mycoides
subsp. capri (Fig. 1). The
integrity of the inserts of these plasmids was verified by PCR
amplification of the corresponding fragments from genomic DNA of
M. mycoides subsp. mycoides LC and from
M. mycoides subsp. capri by using primers matching the sequenced extremities of the inserts (Table 2).
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FIG. 1.
Locations of the lppA gene (black boxes) and
surrounding genes on plasmids pJFFMMLC2 containing a 2.0-kb insert
cloned from the DNA of the M. mycoides subsp.
mycoides LC strain Y-goat (a) and pJFFMMCA4 containing a
4.5-kb insert cloned from the DNA of the M. mycoides
subsp. capri strain PG3 (b). The ORFs are represented by
white boxes, and arrowheads indicate the direction of transcription and
translation. The locations of the oligonucleotide primers are shown by
arrows. Filled triangles indicate the location of
plac promoter of the cloning vector.
|
|
DNA sequence analysis of the lipoprotein genes and of
flanking genes.
The inserts of plasmids pJFFMMLC2 and
pJFFMMCA4 were sequenced in both directions. The nucleotide
sequence of the insert pJFFMMLC2 contained an open reading frame (ORF)
encoding the lipoprotein LppA[MmymyLC] precursor of 526 amino acid
(aa) residues with a predicted molecular mass of 60.288 kDa (Fig.
2). It is preceded by a consensus
sequence for a ribosome binding site (RBS) located five nucleotides
(nt) upstream of the initiation codon, AUG. The analogous ORF in
plasmid pJFFMMCA4 encoded the lipoprotein LppA[Mmyca] precursor,
which shows 523 aa residues and a predicted molecular mass for
LppA[Mmyca] of 59.822 kDa (Fig. 2). This ORF is preceded 6 bp
upstream by a canonical RBS sequence. The two lppA
genes contain mycoplasma-specific UGATrp codons
corresponding to aa 147 and 415 and to aa 144 and 412, respectively,
and are terminated by UAA stop codons. Analysis of the amino acid
sequences of LppA indicated a consensus sequence for a potential
recognition site of a prokaryotic signal peptidase II (19)
at aa 21 to 25 and a lipid attachment site at the cysteine residue
starting at aa 25 (Fig. 2). A potential transmembrane region is located
at aa residues 7 through 23 of the leader sequence. The amino acid
sequences of the mature LppA[MmymyLC] and LppA[Mmyca]
are virtually the same. They have 94% identical and 95%
positive (identical + similar) amino acids. Differences are
mainly located at the N terminals of the proteins, where a few
deletions and substitutions are located (Fig. 2). The mature LppA
proteins of both M. mycoides subsp. mycoides
LC and M. mycoides subsp. capri showed a
similarity (42 to 44% identity and 50 to 52% positivity for aa
residues) to P72 of M. mycoides subsp.
mycoides SC.
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FIG. 2.
Amino acid sequence comparison of LppA precursors from
the M. mycoides subsp. mycoides LC strain
Y-goat and from the M. mycoides subsp. capri
strain PG3. Underlined amino acids correspond to the consensus sequence
for the signal peptidase II recognition site. The arrow indicates the
potential cleavage site for this peptidase. Vertical bars show
identical amino acids, and dots between sequences show similar amino
acids.
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|
The sequence analysis of both plasmids pJFFMMLC2 and pJFFMMCA4 revealed
a part of an ORF, ORF1, upstream of lppA. Its function is
currently unknown. However, ORF1 in both M. mycoides
subsp. mycoides LC and M. mycoides subsp.
capri showed a high similarity to the ORF upstream of the
gene encoding P72 in M. mycoides subsp. mycoides SC (8). The ORF1 of M. mycoides subsp. mycoides LC showed 52% identical amino
acids and 69% identical nucleotides at the DNA level with the ORF1 of
M. mycoides subsp. capri. In both plasmid
clones, the lppA gene is followed by a hairpin structure representing a potential transcriptional termination signal (Fig. 1).
In pJFFMMCA4, the hairpin structure was followed by two ORFs that
sequenced in the opposite direction; one, ORFX, encodes a polypeptide
with unknown function, and the second has 63% identical nucleotides
with a DNA methylase in Spiroplasma species (accession no.
X17195) (33) (Fig. 1). Upstream of the potential DNA methylase gene we found a hairpin structure that acts as a
transcription stop signal and is part of an ORF showing 93% identical
nucleotides to the mtlD (which encodes mannitol-1-phosphate
dehydrogenase) gene analogue found in M. mycoides
subsp. mycoides SC (8) (Fig. 1). In pJFFMMLC2, an
ORFX was found downstream of the transcription stop signal following
lppA. It showed 89% identical nucleotides to the analogous
location in M. mycoides subsp. capri (Fig.
1).
Expression and serological specificity of LppA of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri.
The expression and the
antigenic specificity of LppA in M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri were analyzed on immunoblots by using adsorbed
polyclonal mouse antibodies directed against recombinant
LppA[MmymyLC]. Whole cells of the M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri type strains and field strains, as well as the type
and reference strains of the other members of the M. mycoides cluster (Table 1), were solubilized in SDS sample buffer
and used as antigens on the immunoblots. The mouse
anti-LppA[MmymyLC] serum strongly reacted with a 62-kDa band or a
60- and 62-kDa doublet for all strains of M. mycoides subsp. mycoides LC and M. mycoides subsp.
capri tested except strain WK354/80. The apparent molecular
mass of LppA on SDS polyacrylamide gels (62 kDa) is slightly
higher than the expected molecular mass as calculated from
its DNA sequence-deduced amino acid composition (60 kDa).
Strain WK354/80, for which the taxonomic classification is
not clear, showed weak reactions with a doublet band of 58 and 60 kDa.
The other mycoplasmas of the M. mycoides cluster did not react with anti-LppA[MmymyLC] antibodies, thus showing
the antigenic specificity of LppA[MmymyLC] to M. mycoides subsp. mycoides LC and M. mycoides subsp. capri (Fig.
3).
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FIG. 3.
Expression of lipoprotein LppA[MmymyLC] and
LppA[Mmyca] in mycoplasmas of the M. mycoides
cluster (a) and in the M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri field strains (b and c, respectively). Immunoblots
containing total antigens of the different mycoplasmas (Table 1) were
probed with anti-LppA[MmymyLC] serum from a mouse immunized with
recombinant LppA[MmymyLC]. The positions of the lipoproteins
LppA[MmymyLC] and LppA[Mmyca] and of molecular mass
standards (broad range) (Bio-Rad Laboratories) are indicated. Certain
strains showed a doublet band reacting with anti-LppA[MmymyLC]
antibodies (b). We interpret these doublets to be due to
posttranslational modification of these lipoproteins. In certain
strains, no doublets are visible, which might be due to the fact that
the cells were taken in a late stage of cell growth. Secondary bands
with molecular masses not within the 60 to 62 kDa range are interpreted
as background reactions due to nonspecific serological reaction of the
mouse sera. The faint doublet at 58 and 60 kDa seen with strain
WK354/80 is supposed to be due to a lipoprotein closely related to, but
different from, LppA[MmymyLC] and LppA[Mmyca].
|
|
Specificity of lppA to M. mycoides
subsp. mycoides LC and M. mycoides subsp.
capri.
The presence of lppA[MmymyLC] and
lppA[Mmyca] was studied in M. mycoides
subsp. mycoides LC and M. mycoides subsp.
capri strains and in the other members of the M. mycoides cluster by PCR amplification with the primer pair
MMMLC2-L and MMMLC1-R (Table 2) by using as the templates chromosomal
DNA from all mycoplasma strains used in this study (Table 1). All
strains of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri showed
the expected DNA fragment of 1.05 kb with the exception of strain
WK354/80, which amplified a smaller fragment (0.85 kb). The PCR product
obtained from WK354/80 was analyzed further by DNA sequencing. DNA
sequencing results showed that the edges of the PCR fragment but not
the central part of the segment were identical to
lppA[MmymyLC] and lppA[Mmyca]. In
total, this 0.85-kb fragment showed 63 to 64% similarity to lppA[MmymyLC] and lppA[Mmyca], 95%
similarity to the P67 gene of the Mycoplasma sp.
bovine group 7, and 93% similarity to the P72 gene of
M. mycoides subsp. mycoides SC. None of the
other mycoplasmas of the M. mycoides cluster analyzed
showed any amplification product (Fig. 4
and Table 1). These results confirmed that the primer pair MMMLC2-L and
MMMLC1-R amplified a specific 1.05-kb fragment from M. mycoides subsp. mycoides LC and M. mycoides subsp. capri. For further analysis, the
PCR amplification products of the lppA[MmymyLC]
and lppA[Mmyca] genes were digested with the restriction
enzyme AluI and examined (Fig.
5). Profiles obtained from the different
strains showed some variations, indicating a certain heterogeneity of
the lppA gene within the different strains. The profiles
show that the same differences are found within strains of the same
subspecies as well as among strains of different subspecies and hence
do not differentiate M. mycoides subsp.
mycoides LC strains from M. mycoides subsp.
capri strains.
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FIG. 4.
PCR fragments obtained from amplification with the
primer pair MMMLC2-L and MMMLC1-R and genomic template DNA from type
and reference strains of the M. mycoides cluster and
from the M. mycoides subsp. mycoides LC and
M. mycoides subsp. capri field strains
(Table 1). As size markers, HindIII-digested DNA
fragments were used. Their sizes are given in kilobases on the left
side.
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FIG. 5.
Restriction fragment analysis of PCR-amplified
lppA genes from the M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri strains (Table 1). PCR products of DNA from type
strains and field strains were digested with AluI and
analyzed by electrophoresis on 8% polyacrylamide gels. The standards
used were HinfI-digested pBR322 fragments. Their sizes are
indicated in base pairs.
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| |
DISCUSSION |
Sequence data show that the lipoproteins LppA[MmymyLC] and
LppA[Mmyca] form a family together with the major lipoproteins of
M. mycoides subsp. mycoides SC, P72, and of
Mycoplasma sp. bovine group 7, P67, and are hence suggested
to have a function which is analogous to the two latter lipoproteins.
Accordingly, we propose that P72 and P67 be renamed lipoproteins
LppA[MmymySC] and LppA[Mbgr7], respectively.
Analysis of LppA from both M. mycoides subsp.
mycoides LC and M. mycoides subsp.
capri showed that the potential transmembrane region located
in the leader sequence of LppA[Mmyca] is identical to that found
in LppA[MmymySC] (P72) of M. mycoides subsp.
mycoides SC and in LppA[Mbgr7] (P67) of
Mycoplasma sp. bovine group 7 (8, 16) and differs
only in a single amino acid (residue 11) from the leader sequence of
LppA[MmymyLC]. The corresponding gene fragment is also highly
conserved and could therefore serve as a valuable probe for the cloning
of other mycoplasmal lipoprotein genes.
DNA sequence analysis of the lppA genes of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri revealed a very high degree of
similarity which is also reflected antigenically, as shown on
immunoblots. Minor variations, which can be observed in the
lppA genes of various field strains, are not specific to the
two subspecies. The differences seem to have only minor phenotypic impact since they are not reflected antigenically as
revealed by immunoblots. Genetic variation among different field
isolates from M. mycoides subsp. mycoides LC
and M. mycoides subsp. capri has also been
detected in other gene loci (40a). Hence, LppA seems to be a
common and specific antigen of the two closely related mycoplasmas
M. mycoides subsp. mycoides LC and
M. mycoides subsp. capri. The chromosomal
location of the lppA genes seems to be conserved in the
vicinity of the mtlD gene (which encodes
mannitol-1-phosphate dehydrogenase).
The specific PCR using the primer pair MMMLC2-L and MMMLC1-R
amplified the lppA genes from M. mycoides subsp. mycoides LC and M. mycoides subsp. capri field strains of various
geographic origins but not those from other members of the
M. mycoides cluster. We therefore propose that this PCR
be used for the identification of the phylogenetic and antigenic entity
M. mycoides subsp. mycoides LC/M.
mycoides subsp. capri.
Interestingly, for strain WK354/80, PCR amplification of the
lppA gene resulted in a shorter-than-expected fragment which was shown to be most similar to the lppA[Mbgr7] (P67) gene
of Mycoplasma sp. bovine group 7. WK354/80 strain was
first described in the literature as Mycoplasma sp.
bovine group 7 (10) and was later retyped by us as
M. mycoides subsp. capri. Sequence analysis
of the rrs gene of strain WK354/80, on the other hand, showed its strong similarity to that of M. mycoides
subsp. mycoides LC. These results reflect the ambiguous
taxonomic status of WK354/80, which must be located intermediate
between those of Mycoplasma sp. bovine group 7 and
M. mycoides subsp. mycoides LC/M.
mycoides subsp. capri. It illustrates further the good
discriminatory potential of the lppA genes, which encode the
major lipoproteins, in the differentiation of the members of the
M. mycoides cluster.
We are grateful to Yvonne Schlatter for technical assistance with
DNA sequence analysis and PCR and to Margrit Krawinkler for expert help
with identification and cultivation of mycoplasmas. We thank Chris
Minion, Ames, Iowa, for the kind gift of strain YN2980, which proved to
be most helpful for the expression of cloned mycoplasmal genes, and
Shmuel Razin, Jerusalem, Israel, Kevin Dybvig, Birmingham, Alabama, and
Karl-Erik Johansson, Uppsala, Sweden, for their helpful suggestions in
naming lipoproteins.
This study is part of European COST action 826 on ruminants'
mycoplasmoses and was supported by grant C96.0073 of the Swiss Ministry
of Education and Science and by the Swiss Federal Veterinary Office.
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Abstract
Introduction
