MICROBIOLOGY
LETTERS
ELSEVIER
FEMS Microbiology Letters 143 (1996) 159-165
Variable expression and geographic distribution of
Mycoplasma agalactiae surface epitopes demonstrated
with monoclonal antibodies
Dominique
Bergonier ‘, Franc0 De Simone b, Pierre Russo ‘, Michel Solsona d,
Maurice Lambert ‘, FranGois Poumarat d,*
a EC&
Nationale
V&Prinaire, Dipartement
b Istituto Zooprojilattico
’ CNEVA
Sophia Antipolis, Laboratoire
’ CNEVA
Lyon, Laboratoire
Sperimentale
Elevage et Prod&s,
della Lombardia
31076
Toulouse Cedex, France
e dell’Emilia, via Bianchi 7, Brescia, Italy
de Pathologic des Petits Ruminants et des Abeilles, BP 111, 06902 Sophia Antipolis Cedex, France
de Pathologic bovine, 31 avenue T. Garnier. BP 7033, 69342 Lyon Cedex 07, France
Received30 April 1996; accepted 29 July 1996
Abstract
Species-specific monoclonal antibodies (MAbs) were developed against Mycoplasma agalactiae reference strain PG2 and
French isolate P89 to study the in vitro expression of surface epitopes and to probe the antigenic profiles of 245 field isolates
originating from 10 different countries. Colony immunostaining with MAbs on clonal lineage showed that 4 out of 9 speciesspecific epitopes exhibited a high rate of variation, demonstrating that M. agalactiae possesses a capacity for phenotypic
diversification of its surface antigenicity. The emphasis was on dot immunobinding screening of the field isolates with MAbs
recognizing permanently expressed epitopes. Eight different profiles could be defined. Great differences in epitope conservation
were demonstrated with some area-specific strains completely lacking certain specific determinants. These results indicate that
the antigenic variability of M. agalactiae relies not only upon surface switching mechanisms but also upon true epitope
differences, partially related to the geographic origin of the isolates.
Keywords:
Contagious
agalactia;
Mycoplasma
agalactiae;
Antigenic
1. Introduction
Mycoplasma agalactiae is the ‘classical’ causative
agent of contagious agalactia (CA), a severe mycoplasmosis affecting sheep and goats throughout the
world [l]. Although chronic forms occur frequently
* Corresponding author. Tel.: +33 78 72 65 43;
Fax: +33 78 61 91 45.
0378-1097 / 96/$12.00 Copyright
PIISO378-1097(96)00301-l
0 1996 Published
variability;
Dot immunobinding
assay; Monoclonal
antibody
in enzootic
areas, M. agalactiae primoinfection
causes rapid acute mastitis and agalactia, often associated with arthritis and keratitis, and therefore extensive economic losses particularly
in large dairy
flocks and transhumance
breeding systems. One of
the major problems in CA control is that antibiotherapy usually proves ineffective, so that limitation of disease spread in a flock or summer pasture
depends on early identification
and removai of infected animals [2]. This would be greatly assisted if
by Elsevier Science B.V. All rights reserved
160
D. Bergonirr
et ul. I FEMS
MmdCdo,qv
a specific, sensitive and rapid diagnosis technique
could be developed, capable of detecting all M. agaluctiae strains. It should be possible to improve detection considerably by using monoclonal antibodies
(MAbs) [3], but the ‘universal’ applicability of these
latter must be assessed by studying the geographic
variability of the recognized epitopes, since some
antigenic differences have already been reported
within French M. agaluctiae isolates [4].
Mycoplasma surface antigens are not only of interest for diagnosis (generally comprising more species-specific determinants than internal antigens), but
they have also been shown in several species to possess remarkable ability to undergo phenotypic variations [5,6]. Such mechanisms of surface diversification have recently been described in M. bovis, which
is genetically, antigenically and pathologically closely
related to M. uguluctiue [7-91. Although considerable
interest has been focused over the past few years on
these basic antigenic properties of mycoplasmas,
there is no evidence of such variability in A4. agulactiae. MAbs have been shown to be key tools for
monitoring diversity on the mycoplasma cell surface.
The purpose of this study was to use a panel of M.
ugalactiae species-specific MAbs to characterize by
immunobinding
assays the in vitro expression and
distribution of surface epitopes within clonal lineage
of the same strain and among field isolates, to determine the possible existence of restricted recognition
of certain strains. In order to characterize an hypothetical antigenic diversity, we selected 245 M.
agalactiae isolates associated with various clinical
signs (typical, atypical and asymptomatic
CA) and
isolated from 1951 to 1995 in different countries. The
emphasis was on the assessment of geographic heterogeneity, to determine the extent to which strains
isolated from different countries, different independent breeding areas and different flocks from enzootic areas varied antigenically, and to see whether any
variations in either epitope correlated with differences in virulence.
2. Materials
and methods
2.1. Mycoplasma
Organisms
culture and strums
were grown in modified
PPLO broth
Letters
143 (1996)
159-165
(Difco). Dot immunobinding
assays (DIBA) were
performed directly with primocultures.
For the colony immunostaining
assay (CISA), cultures in late
exponential
phase were filtered through a 0.2 urn
membrane (Millipore) and serially diluted to lo-“.
Then, 0.1 ml of each dilution was spread evenly
over the surface of agar plates, which were incubated
for 3 days at 37°C.
International
reference strains of 28 sheep, goat
and cattle Mollicutes species were assayed for the
of M. agalactiae
identification
species-specific
MAbs: M. capricolum ssp. cupricolum, M. mycoides
ssp. mycoides LC, M. putrefaciens, M. arginini, M.
mycoides ssp. cupri, Mycoplasma species PG50, M.
bovigenitalium, M. caltfornicum, M. verecundum, Mycoplusma species 2D, M. cupricolum ssp. cupripneumoniue, M. conjonctivae,
M. ovipneumoniae,
M. ad-
leri, M. yeatsii, M. auris, M. cottewii, M. canadense,
M. alkalescens,
M. gallisepticum,
M. bovirhinis, M.
mycoides ssp. mycoides SC, M. canis, Acholeplusma
(A.)
laidlawii, A. axanthum,
Ureaplasma
diversum,
M. agulactiae (PGZ) and M. bovis (PG45 Donetta).
41 M. bovis field isolates from the CNEVA Lyon
collection were also tested. The geographic distribution of the MAb-recognized
antigens was studied
with a total of 245 M. ugaluctiae field isolates from
10 countries and 16 geographically and commercially
independent sheep or goat breeding areas. Greek isolates were kindly provided by Dr. A. Giannaty-Stefanou (Institute of Infectious and Parasitic Diseases,
Thessaloniki),
Spanish isolates by Dr. J. Marco
(SIMA, Derio), Swiss isolates by Prof. J. Nicolet
(Institute for Veterinary Bacteriology, Bern), Italian
isolates by Dr. F. de Simone (IZS della Lombardia e
dell’Emilia,
Brescia), and Portuguese,
Ethiopian,
Turkish and Ivory Coast isolates by Dr. F. Thiaucourt (CIRAD-EMVT,
Montpellier, France). Other
strains came from CNEVA collections. The area origins of the isolates are given in Table 3. The Rumanian strain was a vaccinal one (Agl). The Aveyron
strains were avirulent, and isolated at the time of
routine checking of milk bacteriological
quality in
an area where CA has never been observed. 2% of
the 245 strains were isolated from arthritic joints,
1.2% from pneumonic lung, 0.8% from fetuses and
96% from mastitic milk. Isolation dates ranged from
1951 to 1995. All strains were identified as M. agaluctiae by membrane filtration dot immunobinding
D. Bergonier et al. IFEMS Microbiology Letters 143 (1996) 159-165
nal Antibody
Mannheim).
Isotyping
161
Kit
Isostrip
(Boehringer
2.3. Immunoassays
- 45
- 31
- 21.5
123456789
Fig. 1. Immunoblots of whole cell proteins of M agalactiae field
isolate P89 and reference strain PG2 obtained with species-specific MAbs. MAbs SAl and SA2 were produced from P89 strain.
MAbs 2B12, 3H2, 3F2, lC4, lD4, 4G12 and 3B3 were produced
from PG2 strain. Two pairs of MAbs reacted with the same
bands (lC4 and lD4, 21 kDa; 3H2 and 2B12, 38 kDa). Lanes
1,2: immunoblots
of strain P89 with MAbs SA2 (1) and SAl
(2); lanes 3-9: immunoblots of strain PG2 with MAbs SA2 (3),
SAl (4), 3B3 (5), 4G12 (6), lD4 (7), 3F2 (8) 3H2 (9).
with rabbit hyperimmune
PG2 strain [lo].
serum ‘prepared
against
2.2. Monoclonal antibodies
Two groups of h4. agalactiae MAbs were produced and assayed: the first was prepared from the
PG2 strain and the second from a French (PyreneesAtlantiques) strain (P89) isolated in 1989 from a ewe
with clinical signs of CA. MAbs production
and
supernatant
ELISA screening methods have already
been described [11,12]. ELISA screenings were performed with homologous antigens. Immunoblotting
for determination
of MAb-recognized
antigen molecular mass position was performed with PG2 and
P89 strains, as previously described [13,14]. MAb
isotypes were determined using the Mouse Monoclo-
DIBA was performed according to [lo] for MAbs
and isolate screening and surface-exposed
epitope
identification,
the first step consisting of trapping
the mycoplasmas by vacuum filtration on a nitrocellulose membrane. CISA was used as previously described [5] to characterize the in vitro expression of
MAb-recognized
epitopes in the course of generations from one PG2 clone. The colonies were coated
onto nitrocellulose membranes by agar plate printing. After these antigen attachment steps, an immunoperoxidase assay was performed according to [ 131
for DIBA and CISA. A positive single colony was
localized after CISA, for each MAb, by positioning
the immunostained
membrane above the agar plate.
These clones were then picked and inoculated individually in 1 ml of broth; when growing actively,
cultures were filtered through a 0.2 pm membrane
and were spread over the surface of agar plates.
The subclones were tested by CISA after 3 days
and a sample of 100 colonies examined to determine
the percentage of partially stained colonies. Intensity
Table 1
Dot immunobinding
assessment of epitope MAb recognition
within 245 M. agalactiae field isolates (expressed in percentages)
and molecular masses of epitope-bearing
proteins (PG2 strain)
MAb
Molecular
Permanently
2B12
3H2
lC4
lD4
SAl
Variably
4G12
3B3
3F2
SA2
mass (kDa)
expressed epitopes
38
38
21
21
21-25-38
expressed epitopes
19-39
19-27-32-39
39
38
Percentage
recognized
of strains
84.1
84.1
93.9
93.9
85
88.2
98.4
11.8
93.1
Mycoplasmas were trapped by vacuum filtration on a nitrocellulose membrane. An immunoperoxidase
assay was then performed
with species-specific MAbs recognizing variably or permanently
expressed M. agalactiae epitopes, as defined by colony immunostaining assay. MAb, monoclonal antibody.
D. Brrgonier et al. I FEMS
162
Microbiology
Letters 143 11996) 159-165
variations in DIBA positive results have not been
taken into account in this paper.
3. Results and discussion
Characterization
and screening of MAbs were
firstly carried out by subjecting the supernatants
to
ELISA screening. 43 PG2-induced
and 4 P89-induced clones reacted with the homologous antigens.
DIBA screening of these ELISA positive clones permitted selection of 29 and 3 positive ones, respectively. Nine of these MAbs were M. agalactiae species-specific (7 and 2, respectively). These 9 MAbs
did not show cross-reaction
with PG45 Donetta,
but one (lD4) gave weak to very weak reactions to
11 of the 41 M. bovis field isolates tested. Other
studies reported cross-reactions of MAbs to A4. hovis
with PG2 strain [3,15].
The epitope-bearing
proteins showed two immunoblot pattern types with PG2 or P89 (Fig. 1).
MAbs 3B3, 4G12 and SAl recognized epitopes
were present on several proteins, the major one having a molecular mass position of 38-39 kDa (Table
1). The 6 other epitopes seemed to be borne by single
proteins. Finally, 8 out of the 9 MAbs reacted with
38-39 kDa bands. A previous study had already
identified a strongly antigenic 37 kDa band recognized by a MAb reacting with M. agalactiae and
M. bovis [7].
Subtyping revealed that all MAbs possessed light
chains of the kappa-isotype and heavy chains of the
Table 2
Definition of permanently
expressed epitope profiles
within a collection of 245 M. ugdactiar field isolates
Profiles
2812
ID4
SAI
A
B
c
D
E
F
G
H
+
+
_
_
+
+
+
+
+
+
+
_
_
_
_
obtained
+
+
_
+
These profiles were defined using combinations
of dot immunobinding assay results with the 3 MAbs showing complete and uniform colony immunostainings.
A
B
c
Fig. 2. Colony immunostaining
assay (CISA) of M. agductiue reference strain PG2 with monoclonal antibodies. CISA was used
to characterize the in vitro expression of MAb-recognized
epitopes in PC2 clonal lineage. For each MAb, a positive single colony was localized after CISA by positioning the immunostained
membrane above the agar plate. These clones were picked and
cultured individually (I ml of broth): cultures were then filtered
(0.2 urn membrane), spread over the surface of agar plates and
tested by USA. (A) Single staining with MAb SA2. (B,C) Double staining with MAb SAI (recognizing permanently expressed
epitope) and MAb 4Gl2 (recognizing variably expressed epitope:
black colour). (A,C) Selective staining with sectoring of colonies,
(B,C) Partial or complete staining of colonies.
IgGl isotype, except 3F2 (lambda and IgM isotypes)
and SAI (kappa and IgG2b isotypes).
CISA assessment
of epitope
hypervariability
showed partial
staining
and sectoring
in 3B3,
4G12, 3F2 and SA2 stained cultures (Fig. 2)
whereas other MAbs gave uniform staining of the
M. agalactiae colonies in successive assays. The percentages of partially stained colonies ranged from 75
to 95%. The variation frequency in M. agalactiae as
D. Bergonier et al. I FEMS Microbiology Letters 143 (1996) 159-165
in h4. bovis seems to depend on the MAb [13]. The
existence of selective MAb staining is actually one of
the simplest but most reliable demonstrations
of mycoplasma surface epitope switching. Equivalent systems have been described in M. hyorhinis as variable
surface proteins (Vsp) [16,17], and also in M. pulmonis [18], U. urealyticum [19], A4. gullisepticum [20] and
ikf. bovis, in which two such systems have been well
defined [13,21,22]. Such antigenic systems for surface
diversification have never been reported for M. agaluctiue. It has been suggested, particularly
in M. bovis, that reversible switching of membrane
protein
antigens expression on the mycoplasma
surface is
an adaptative
strategy, permitting
effective escape
from a competent immune system, and possible interaction with different host tissue types and infection stages. In addition to the close genetic and
antigenic relationship
between M. bovis and M.
uguluctiae, considerable similarities in tissue tropisms
(udder, joints, lung, genital tract) and the importance
of chronic evolutions despite the demonstrable presence of circulating
antibodies
should be stressed.
This may partially be explained by similar or related
types of adaptative strategies, although surface anti-
Table 3
Comparison
international
of geographic origin and antigenic
reference strain PG2)
Country
Area
GrfXOZ
Italy
Rumania
Spain
Portugal
France
France
France
France
France
Switzerland
Turkey
Ivory Coast
Ethiopia
PG 2b
Total
Macedonia
Lombardia,
Navarra,
profile distribution
A
Em.”
Cast.’
Pyretrees-AU.”
Aveyron
Savoies
Poitou-Char.”
various
Ticino
_
_
_
_
163
genicity altering mechanisms are probably of lesser
extent or frequency in M. agalactiae (Bergonier and
Poumarat, unpublished
results). Modulation
of M.
agalactiae surface antigen expression by host immune response is currently under investigation.
DIBA screening of a total of 245 M. ugaluctiae
field isolates firstly showed the same geographical
distribution
for two pairs of MAbs-recognized
epitopes (lC4 and lD4; 3H2 and 2B12). The degree of
representativeness
of each epitope is given in Table
1. The recognition of a very well conserved epitope
(3B3: 98.4% of isolates), which could be a potential
candidate for development of detection tests or specific immunogens,
was of particular interest but future works must take into account its variability in
successive generations. All other MAbs except one
(3F2) were shown to react with most of the isolates
(84.1 to 93.9%). Variations between MAbs in the
number of strains recognized have already been reported in M. gallisepticum [20,23,24] and in M. bovis
D31.
In order to propose a serological grouping of M.
agalactiae strains, we identified several DIBA antigenie profiles, using either the 9 species-specific
of 246 M. agalactiae strains (245 field isolates and
within a collection
B
35
4
1
23
1
114
16
C
5
1
1
3
D
E
2
3
17
2
5
F
G
H
5
2
1
I
1
1
1
1
195
1
9
26
4
7
1
3
Total
35
4
1
38
1
114
16
17
2
11
3
1
1
1
1
246
Antigenic profiles were defined using combinations of dot immunobinding
assay results with the 3 MAbs detecting permanently expressed
epitopes.
“Lombardia,
Em., Lombardia, Emilia; Navarra, Cast., Navarra, Castilla-Leon, Castilla-La Mancha; Pyrenees-Atl., Pyrbnees-Atlantiques;
Poitou-Char.,
Poitou-Charentes.
bPG2, M. agalactiae reference strain.
164
D. Bergonier et al. IFEMS
Microbiology Letters 143 (1996) 159-165
MAbs or the three that showed constant expression
in CISA (Table 2). In the first case, we obtained 21
whole MAbs profiles (WP), four of these being major ones (representing 84.5% of isolates). However,
because four epitopes were shown to undergo phenotypic variations, grouping strains on the basis of
WP might be unreliable. Consequently,
for characterization of geographic heterogeneity,
we focused
on constant expression MAbs profiles (CEP) variations. With this second kind of classification, 8 profiles were identified, although one of them comprised
79.3% of M. ugalactiue isolates (Table 3). Nevertheless, the quantitative importance of each profile must
be interpreted with caution, because not every country or area was equally represented in our collection.
At a general (international)
level, we found that
profiles were present in several countries (at least 2)
and all the more in different areas, except for two
profiles comprising a single French isolate. At a national level, our results revealed some intra-country
heterogeneity.
French isolates, being the most numerous and showing the best spatial distribution,
gave a good illustration of this geographic diversity.
Two groups of isolates, based on CEP or WP, could
be distinguished
in this country:
the south-west
group (Pyrenees-Atlantiques
and Aveyron) belonging to the A profile, and the remaining regions belonging to the B, C, D, F, G and H profiles. None of
the 7 profiles comprised strains from both geographic groups. These systematic geographic differences are probably related to traditional
breeding
characteristics
and to commercial transaction
customs in France (and many Mediterranean countries).
South-west group strains were isolated from dairy
ewe flocks of local breeds, which had not had any
physical or commercial contacts with other areas or
breeds, whereas strains from the second group were
isolated from goat flocks in a French enzootic area
or in other departments, where breeders are generally
used to constituting
or enlarging their flocks with
goats from different, sometimes very distant regions.
Thus, when CEP are considered, true antigenic
differences seem to exist within A4. agaluctiue species,
some of them closely related to geographic origin.
Both regional and epitope switching variabilities
may also be valid at the same time: the 3F2-recognized epitope has been demonstrated
by CISA to
undergo variations
in successive generations,
but
was only exhibited (Table 1) by Greek and Italian
isolates (11.8%). This kind of country or area-specific
reactivity is likely to be relevant in the geographic
classification of M. agalactiae isolates, since this epitope was never exhibited by isolates from other
countries cultured in the same broth and subcloning
conditions.
At an enzootic area level, we observed an extensive or even total homogeneity among isolates. In
these regions, self-sufficiency breeding systems are
very frequent, and outbreaks often originate from
contagion during transhumance.
Finally, including CEP and 3F2 distribution,
the
geographic diversity of the M. agalactiae antigenic
determinants
consisted, with our MAbs and isolate
collections, of identification of the following profiles :
a major Iberian and south-west French profile, a
Savoies profile (sporadically
present in Spain), a
Greek and Italian profile and several ‘occasional’
profiles. Such antigenic heterogeneity of geographic
origin has not been reported for M. his
[13] or
other species. Determination
of the immunogenicity
and location of the recognized epitopes on representative strains from each area by immunoblotting
is
now in progress.
Virulent and avirulent or vaccinal strains did not
differ in their MAb-recognized
antigenic properties.
The animal host-related differences are not worth
investigating
as strongly correlated with the geographic differences.
In conclusion,
this work provides information
about the antigenic
variability
of mycoplasmas,
based on the characteristics of small ruminant breeding systems, which traditionally
develop with few
exogenous
commercial
transactions,
especially in
sheep due to the existence of local breeds. Phenotypit switching of epitopes probably also occurs in
vivo for M. agaluctiae, and may be one of the mechanisms permitting
the existence of post-clinical
healthy carriers of great epidemiological importance
in CA, as in the majority of enzootic areas stamping
out is not achieved. Random subcloning of M. agaluctiae for in vitro studies may lead to cultures lacking important epitopes and to poorly reproducible
results. We therefore suggest that antigen and immunogen preparations
are made with known and/or
standardized passage numbers and culturing conditions.
D. Bergonier
et al. IFEMS
Microbiology
Letters 143 (1996)
Acknowledgments
The authors gratefully thank the French
ment Veterinary Laboratories for supplying
lates.
Departthe iso-
References
PI Da Massa, A.J., Wakenell, P.S. and Brooks, D.L. (1992) Mycoplasmas of goats and sheep. J. Vet. Diagn. Invest. 4, lOI113.
PI Lambert, M. (1987) Contagious agalactia of sheep and goats.
Rev. Sci. Tech. Off. Int. Epiz. 6, 6999711.
131 Heller, M., Berthold, E., Ptutzner, H., Leirer, R. and Sachse,
K. (1993) Antigen capture ELISA using a monoclonal antobody for the detection of Mycoplasma bovis in milk. Vet.
Microbial. 37, 1277133.
141 Poumarat, F., Solsona, M. and Martel, J.L. (1992) Genotype,
protein and antigen variability among strains of Mycoplasma
agalactia! In: IOM Letters, Vol. 2, p. 326, 9th International
Congress of the International Organisation for Mycoplasmology, Ames, IA, USA, August 2-7.
151 Rosengarten, R. and Wise, K.S. (1990) Phenotypic switching
in mycoplasmas:
phase variation of diverse surface lipoproteins. Science 247, 315-318.
VI Wise, K.S., Yogev, D. and Rosengarten, R. (1992) Antigenic
variation, In: Mycoplasmas,
Molecular Biology and Pathogenesis (Maniloff, J., MacElhaney,
R.N., Finch, L.R. and
Baseman, J.B., Eds.), pp. 4733489, American Society for Microbiology, Washington, DC.
[71 Berthold, E., Heller, M., Pfutzner, H., Leirer, R. and Sachse,
K. (1992) Preparation
and characterization
of monoclonal
antibodies against Mycoplasma bovis. J. Vet. Med. B 39,
353-361.
D. and Martel, J.L. (1992) Ap181 Poumarat, F., Longchambon,
plication of dot immunobinding
on membrane filtration (MF
dot) to the study of relationships within ‘M. mycoides cluster’
and within ‘glucose and arginine-negative
cluster’ of ruminants mycoplasmas. Vet. Microbial. 32, 375-390.
191 Mattsson, J.G., Guss, B. and Johansson, K.-E. (1994) The
phylogeny of Mycoplasma bovis as determined by sequence
analysis of the 16s rRNA gene. FEMS Microbial. Lett. 115,
3255328.
D. (1991) IdenUOI Poumarat, F., Perrin, B. and Longchambon,
tification of ruminant mycoplasmas by dot immunobinding
on
membrane filtration (MF dot). Vet. Microbial. 29, 329-338.
Ull Brocchi, E., Gamba, D., Poumarat, F., Martel, J.L. and De
Simone, F. (1993) Improvements in the diagnosis of conta-
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
159-165
165
gious bovine pleuropneumonia
through the use of monoclonal
antibodies. Rev. Sci. Tech. Off. Int. Epiz. 12, 559-570.
Lambert, M. (1985) Application dune technique ELISA infonnatide
au strodiagnostic
de 1’Agalactie Contagieuse des
petits ruminants. Rev. Med. Vet. 136, 303-306
Poumarat, F., Solsona, M. and Boldini, M. (1994) Genomic,
protein and antigenic variability of Mycoplusma bovis. Vet.
Microbial. 40, 305-321.
Bencina, D. and Bradbury, J.M. (1991) Indirect immunoperoxidase assay for detection of antibody in chicken Mycoplasma infection. Avian Pathol. 20, 113-124.
Ball, H.J., Mackie, D.P., Finlay, D., Gunn, J., Mac Farland,
E.A., Reilly, G.A.C. and Pollock, D. (1994) An antigen-capture ELISA for the detection of Mycopfasma bovis in milk.
Irish Vet. J. 47, 4552.
Rosengarten,
R. and Wise, KS. (1991) The Vlp system of
Mycopfasma hyorhinis: combinatorial
expression of distinct
size variant lipoproteins
generating high-frequency
surface
antigenic variation. J. Bacterial. 173, 47824793.
Yogev, D., Rosengarten, R. and Wise, K.S. (1991) Variation
and genetic control of surface antigen expression in Mycoplasmas: the Vlp system of Mycoplasma hyorhinis. Zentralbl. Bakteriol. 278, 275-286.
Watson, H.L., Mac Daniel, L.S., Blalock, D.K., Fallon, M.T.
and Cassel, G.H. (1988) Heterogeneity among strains and a
high rate of variation within strains of a major surface antigen
of Mycoplasma pulmonis. Infect. Immun. 56, 1358-1363.
Watson, H.L., Blalock, D.K. and Cassel, G.H. (1990) Variable antigens of Ureaplasma urealyticum containing both serovar-specific and serovar-cross-reactive
epitopes. Infect. Immun. 58, 3679-3688.
Bencina, D., Kleven, S.H., Elfaki, M.G., Snoj, A., Dove, P.,
Dorrer, D. and Russ, I. (1994) Variable expression of epitopes
on the surface of Mycoplasma gallisepticum demonstrated with
monoclonal antibodies. Avian Pathol. 23, 19-36.
Behrens, A., Heller, M., Kirchhoff, H., Yogev, D. and Rosengarten, R. (1994) A family of phase and size-variant membrane surface lipoprotein antigens (Vsp) of Mycoplasma bovis.
Infect. Immun. 62, 507555084.
Behrens, A., Poumarat, F., Le Grand, D., Heller, M. and
Rosengarten, R. (1996) A novel inununodominant
membrane
protein (pMB67) involved in Mycoplasma bovis surface antigenie variation. FEMS Immunol. Med. Microbial., in press.
Panangala, VS., Morsy, A.M., Gresham, M.M. and ToivioKinnucan, M. (1992) Antigenic variation of Mycoplasma gallisepticum, as detected by use of monoclonal antibodies. Am.
J. Vet. Res. 53, 1139-1144.
Garcia, M., Elfaki, M.G. and Kleven, S.H. (1994) Analysis of
the variability in expression of Mycoplasma gallisepticum surface antigens. Vet. Microbial. 42, 1477158.