ELSEVIER
IMMUNOLOGY AND
MEDICAL
MICROBIOLOGY
FEMS Immunology and Medical Microbiology 16 (1996) 173-181
Characterization of monoclonal antibodies that recognize
common epitopes located on 0 antigen of lipopolysaccharide of
serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae
J.I. Rodriguez Barbosa a, C.B. Gutihez Martin a, R.I. Tasch
K.R. Mittal b, E.F. Rodriguez Ferri a.*
a, O.R. Gonzilez
a,
a Department of Animal Pathology: Animal Health (Microbiology and Immunology), Faculty of Veterinary Medicine, Uniuersify of L&n,
24071 L.&n, Spain
b Department qf Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montrkzl, Saint-Hyacinthe,
Que. J2S 7C6, Canada
Received 29 May 1996; revised 25 July 1996; accepted 21 August 1996
Abstract
Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae
(reference strain Shape 4074) were produced and characterized.
All hybridomas secreting mAbs were reactive with
whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5lX that failed to recognize serotype 9.
They did not react with other taxonomically
related Gram-negative
organisms tested. The predominant isotype was
immunoglobulin (Ig) Ivl, although IgG,,, IgG,,, and IgG, were also obtained. The epitopes identified by these mAbs were
resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however,
they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection
assay showed that mAbs
could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H.5 and
llC3) recognized a ladder-like banding profile consistent with the 0 antigen of lipopolysaccharide
(LPS) from smooth
strains. Group II (mAbs 3BlO and 9Hl) recognized a long smear of high molecular weight which ranged from 60 to 200
kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been
classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis,
immunodiffusion
and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of
those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacdllus pleuropneumoniae which were located on the 0 antigen of LPS.
Keywords: Actinobacillus pleuropneumoniae;
Monoclonal antibodies; Bacterial lipopolysaccharide; Serotype 1; Serotype 9; Serotype 11;
Serotyping
1. Introduction
* Corresponding author. Tel.: +34 (87) 291 203; fax: +34
(87) 291 194.
Actinobacillus
pleuropneumonia,
pleuropneumoniae
causes porcine
which is a highly contagious res-
092%8244/96/$15.00
Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII SO928-8244(96)00081-8
174
J.1: Rodriguez Barbosa et al. / FEMS Immunology and Medical Microbiology 16 (1996) 173-181
piratoty disease. Infected pigs develop an acute infection with high morbidity and mortality rates. Surviving animals become carriers and act as reservoirs
of the disease. The disease is prevalent in countries
with an intensive pig industry where it causes substantial economic losses [ 1,2]. Twelve serotypes of
A. pleuropneumoniae
have been described. Serotypes
1 and 5 are the most prevalent in Canada and United
States [3-51. Serotypes 9 and 11 have not been
isolated in Canada or in the United States whereas
these serotypes are often identified in France and the
Netherlands
[6-91. Serotypes 1 and 9 have been
described in Spain, although with a low prevalence
[lo,1 11.
A. pleuropneumoniae
type specific epitopes are
located on capsular polysaccharide and somatic antigen of lipopolysaccharide
(LPS). The 0 antigen is
responsible for cross-reactions among serotypes 1, 9
and 11 [8,9,12,13] and between serotypes 4 and 7
[ 14,151. Perry et al. [ 161 have reported that serological cross-reactions
among different serotypes coincide with the structural similarity of O-antigens.
Serotyping of A. pleuropneumoniae
involves a variety of serological techniques (such as immunodiffusion, coagglutination,
indirect haemagglutination
or
counterimmunoelectrophoresis)
and the use of rabbit
polyclonal antisera and crude antigens [4]. Cross-reactions can result from impure antigenic preparations
and rabbit polyclonal sera containing both serotypespecific antibodies and antibodies against common
antigens of A. pleuropneumoniae
or related species.
From an epidemiological
point of view, a rapid
and precise identification of the serotype involved in
an outbreak is indispensable
for the control of the
disease, because whole inactivated bacterial vaccines
protect only against the homologous serotype [2].
Monoclonal
antibodies
have been developed
against a variety of organisms particularly
Gram
negative as specific reagents for identification
and
serotyping [17]. They are useful tools which complement conventional techniques using polyclonal antisera.
The purpose of this study was the production and
characterization of mAbs against surface epitopes of
the 0 antigen of LPS of serotype 1 of A. pleuropneumoniae for use as tools for serotyping and for
further elucidation of surface epitopes involved in
cross-reactions with other serotypes.
2. Materials
and methods
2.1. Animals
BALB/c
mice used in the study were obtained
from a breeding colony maintained in our vivarium
and according to the standard guidelines of animal
care at the Faculty of Veterinary Medicine (Leon
University).
2.2. Bacterial strains and growth conditions
The A. pZeuropneumoniae reference strains representing serotypes 1 through 12 and the 96 field
isolates used in this study are listed in Table 1. The
serotypes of the field isolates were as follows: 70
isolates of serotype 1, 11 of each serotypes 9 and 11
and two isolates of each serotypes 5 and 12. The
isolates of serotypes 1, 5 and 12 were from diagnostic laboratories in Canada, the United States and
Spain. The serotype 9 and 11 isolates were from
diagnostic laboratories in France and the Netherlands. The field isolates were recovered from pulmonary tissues of pigs which had died of acute
pleuropneumonia.
Primary identification of A. pleuropneumoniae isolates was based on bacteriological
characteristics as described by Kilian et al. [18]. The
cultures were stored at - 80°C and grown on PPLO
agar before serological testing. The serotype of each
isolate was previously established using a battery of
serological tests including coagglutination,
indirect
hemagglutination,
immunodiffusion
and counter-immunoelectrophoresis
using hyperimmune
polyclonal
rabbit sera against serotypes 1 to 12 [4,9]. Other
bacterial strains used are also listed in Table 1 and
were grown on Trypticase Soy Agar plates at 37°C
for 18 h, except for Haemophilus parasuis and
Haemophilus
taxon ‘minor group’, which were
grown as described for A. pleuropneumoniae
strains.
Whole bacterial cells (WBC) were washed and suspended in phosphate-buffered
saline (PBS; pH 7.2)
and the suspensions adjusted to an optical density
(OD) of 1 at 650 nm (approximately
IO9 CPU/ml)
using a spectrophotometer
model ‘Spectronic 601’
(Bausch and Lomb). The path length of the cell was
1 cm.
2.3. A4Ab production
Four 8 week old-male BALB/c mice were immunized intraperitoneally
with 0.3 ml of a 1: 1.2 mixture
J.I. Rodriguez Barbosa et al./ FEMS bnmunology and Medical Microbiology 16 (1996) 173-181
of 10’ formalin-fixed
A. pleuropneumoniae serotype
1 cells (strain Shope 4074) and Freund’s incomplete
adjuvant (Difco). Tlhe mice were boosted intraperitoneally 3 times at 2 week-intervals
with 2 X 10’
formalinised
bacterial cells. Blood was taken from
each mouse and the antibody response was measured
by enzyme-linked
immunosorbent
assay (ELISA).
The mouse with the highest serum antibody titer was
selected as the spleen donor and was intravenously
given a final booster injection of 4 X lo8 viable cells
suspended in PBS. Three days later, hybridization
was performed according to Kijhler and Milstein [19]
with slight modifications
[ZO]. Non-Ig-secreting
murine myeloma P3 X63 Ag8.653 cells [21] were
Table 1
Reference
175
fused with spleen cells in a ratio of 1:2.5 in the
presence of 50% (w/v> polyethylene
glycol 1500
(Baker, Phillipsburg) as the fusing agent. The fused
cells were cultured in a 96 well culture plate for 24 h
in the presence of tissue culture medium containing
13% (v/v) fetal calf serum (FCS) @era-Lab, Sussex), 2 mM glutamine (Sigma), 1 mM pyruvate
and 100 IU/ml
(Sigma), 30 p g / m 1 gentamycin
penicillin (Antibioticos, S.A., Spain). The tissue culture medium was replaced the next day with selective medium containing (10m5 M azaserine (Sigma)
and 10e4 M hypoxanthine (Sigma)) and 13% FCS.
Murine thymus was used as feeder cells to facilitate
hybridoma growth and survival. Twelve days after
and field strains used in this study
Strain
Description
Source a (reference)
Actinobacillus pleuropneumoniae
Shope 4074
Shope 4226
Shope 1421
M62
K17
L20
Fern tf~
wF83
405
CVJ13261
D13039
56153
8329
70 strains
11 strains
11 strains
2 strains
2 strains
Bordetella bronchiseptica ATCC19395
Escherichia coli MC1061
Klebsiella pneumoniae P.TCC13883
Pasteurella haemolytica ATCC33396
Pasteurella multocida subsp. multocida CCM6077
Pasteurella multocida subsp. multocida CCM6080
Haemophilusparasuis ATCC19417
Haemophilus taxon ‘minor group’ G 112
Actinobacillus suis CCM5586
Listeria iuanouii ATCCl9119
serotype 1, reference strain
serotype 2, reference strain
serotype 3, reference strain
serotype 4, reference strain
serotype 5a, reference strain
serotype 5b, reference strain
serotype 6, reference strain
serotype 7, reference strain
serotype 8, reference strain
serotype 9, reference strain
serotype 10, reference strain
serotype 11, reference strain
serotype 12, reference strain
serotype 1, field isolates
serotype 9, field isolates
serotype 11, field isolates
serotype 5, field isolates
serotype 12, field isolates
reference strain
reference strain
reference strain
reference strain
capsular type A, reference strain
capsular type D, reference strain
reference strain
field isolate
reference strain
reference strain
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
R. Nielsen
KRM, UMC
KRM, UMC
KRM, UMC
KRM, UMC
KRM, UMC
collection
M. Simonet
collection
collection
collection
collection
collection
MIS, FVM, ULE
collection
collection
a R. Nielsen: National V’eterinary Laboratory, Copenhagen, Denmark; KRM and UMC: K.R. Mittal, University of Montreal, Canada; M.
and Immunology Section,
Simonet: Faculty of Medicine ‘Necker-Enfants
Malades’, Paris, France; MIS, FVM and ULE: Microbiology
Faculty of Veterinary Medicine, University of Le6n, Spain.
176
J.I. Rodriguez Barbosa et al. / FEMS Immunology and Medical Microbiology 16 (1996) 173-181
the fusion, hybridoma culture supematants were examined for the presence of antibodies by ELISA,
using a suspension of A. pleuropneumoniae
strain
Shope 4074 as antigen. Positive hybridomas were
selected and subcloned
by the limiting dilution
method. Hybridomas producing mAbs were allowed
to grow until they died and the supematant
was
harvested by centrifugation at 1,000 X g for 10 min
and stored at - 80°C until used.
2.4. isotyping of mAb
The isotypes of the mAbs were determined by
ELISA (Sigma) with a mouse monoclonal subisotyping kit containing rabbit anti-mouse immunoglobulin
IgG,, IgG,,, IgG,,, IgG,, IgM and IgA following
the manufacturer’s instructions.
2.5. Enzyme-linked
immunosorbent
assay (ELISAl
Indirect ELISA was performed essentially as described
by Rodriguez
Barbosa
et al. [22].
Polyestyrene microtiter plates (Nunc, Denmark) were
coated with 5 X lo6 cells of serotype 1 (reference
strain Shope 4074) per well (that is, 100 ~1 of
washed, unfixed whole bacterial ceils (A,,, = 1) per
well were diluted 1: 10 in distilled water and were
allowed to dry overnight at 37°C). The wells were
blocked with 300 ,~l of 2% (w/v) of bovine serum
albumin (BSA) (Serva, Germany) in PBS at 37°C for
30 min. Hybridoma supernatants were added (100 ~1
per well) to the ELISA plates and incubated for 2 h
at room temperature. This was followed by three
washes with washing solution (PBS + 0.05% Tween
20 (PBST)) and the addition of 100 ~1 of either
rabbit anti-mouse
IgG (heavy plus light chains)horseradish
peroxidase
or goat anti-mouse
IgM
(Fc)-horseradish
peroxidase conjugate (Nordic Immunology,
The Netherlands),
diluted 1:2,500 and
15,000 respectively, in PBS (pH 7.2) containing 1%
BSA. Next, plates were incubated at room temperature for 30 min, and unreacted enzyme conjugates
were removed from the wells by five washes with
PBST solution. Finally, 100 ~1 of substrate solution
containing 5-aminosalicylic
acid in 0.005% H,O,
(Merck) was added to each well and the plates were
incubated at room temperature for 30 min in the
dark. The reaction was stopped by the addition of 50
~1 of 1N NaOH, and the optical density was read at
450 nm on a Titertek Multiscan plate reader (Flow
Laboratories, England). mAbs were scored as positive when results exceeded the mean value of the
negative control culture supematant
by at least
twofold. Two unrelated bacteria were used as a
negative controls (Escherichia
coli MC1061 and
Listeria ivanouii ATCC 10119).
Culture supematants were also tested in a heterologous ELISA against reference strains of other
serotypes of A. pleuropneumoniae
and against other
closely related Gram-negative microorganisms
listed
in Table 1.
2.6. Rapid dot ELISA (RDE)
The culture supematants
of two of the mAbs
developed (4B7 and 9H5) were tested by RDE against
the 96 field isolates [4,9]. Volumes of 10 ~1 of
optimally diluted WBC suspensions from each field
isolate were spotted onto nitrocellulose
membranes
and allowed to dry at room temperature. The membrane was incubated with 5% (w/v) skim milk in
PBST and then incubated with mAb supematants for
one hour at room temperature. Dots were washed
and incubated with sheep anti-mouse
horseradish
peroxidase conjugate (Biorad) for 1 h at room temperature and washed. Membranes
were incubated
with 4-chloro-1-naphthol
substrate solution (Sigma).
Color was allowed to develop and excess substrate
was removed by washing in distilled water [23].
2.7. Slide agglutination
test (SAT)
The SAT was carried out as described by Mittal et
al. [24] using WBC suspensions of reference strains
as antigens. SAT was performed by mixing 100 ~1
of undiluted hybridoma supematants with an equal
volume of a bacterial suspension adjusted to an OD
of 1 at 650 nm. Positive agglutinations
were observed for 2 to 3 min at room temperature.
2.8. Enzymatic and chemical treatments of whole cell
antigen
The WBC suspensions
were treated with proteinase K (Sigma) following the method of Thomas
J.I. RodriguezBarbosaet al. / FEMS Immunologyand MedicalMicrobiology16 (1996) 173-181
177
and Selwood [25]. Periodate oxidation was performed by treating tihe WBC antigens with 40 mM of
sodium periodate (Sigma) followed by a wash in
PBS according to the procedure described by Woodward et al. [26]. Antigens prepared from homologous
and from heterologous strains with and without proteinase K and sodiulm periodate treatments were used
as controls.
blotted off. The blot was covered with Saran-Wrap
and exposed to Hyperfilm-ECL (RPN2104, Amersham) for 1 to 5 min.
2.9. Sodium
One fusion was performed, yielding over 1000
hybridomas. Seven hybridomas were selected, cloned
twice, expanded and allowed to grow until they died
to obtain culture supematant enriched in mAb. The
immunoglobulin (Ig) type and subtype were determined. Four of the mAbs (57.1%) were of the IgM
isotype, although IgG,,, IgG,, and IgG, were also
detected (Table 2). These mAbs were further characterized by slide agglutination test (SAT). Only those
mAbs belonging to the IgM isotype were able to
agglutinate the WBC suspension of reference strains
of serotypes 1, 9 and 11, except for mAb 5D6 which
only reacted with reference strains of serotypes 1 and
11 (Table 2).
trophoresis
dodecyl
s&fate-polyacrylamide
(SDS-PAGE),
hanced chemiluminescence
elec-
western blotting and en(ECL) immunodetection
WBC suspensions of serotype 1 (reference strain,
Shope 4074) and serotype 4 (reference strain M62:
negative control) were washed twice in sterile PBS
and adjusted to an OD of 0.6 at 525 nm. The
bacterial suspensions were centrifuged at 100 X g
for 3 min. The pellets were dissolved in 50 ~1 of
lysis buffer (2% SDS, 4% 2-mercaptoethanol, 10%
glycerol, 1 M Tris (pH 6.8) and 0.01% bromphenol
blue) and boiled for 10 min. Finally, the lysate was
treated with 25 pug proteinase K dissolved in 10 ~1
of lysis buffer for 60 min at 60°C [27]. The solubilized antigens were: separated by SDS-PAGE using
the method of Laemmli [28] on a 4% polyacrylamide
stacking gel and a 10% separating gel.
Separated bands from the gel were electrotransferred on to a nitrocellulose (NC) membrane with
transfer buffer (T-buffer) consisting of 25 mM Trizma
base and 192 mM Iof glycine (pH 8.3) [29]. The gel
and NC membrane were sandwiched between layers
of Whatman filter paper no. 3 (Whatman International, England), soaked in T-buffer and placed in
the semidry transfer apparatus (LKB 2117 Multiphor
II, Pharmacia, LKB, Sweden). Proteins were transferred by applicatiosn of 100 V for 1 h (two minigels
at a time).
ECL immunodetection of LPS bound to NC sheets
was carried out using Amersham Life Science
reagents and protocols (Amersham, England). A
28SL miniblotter device with a manifold (Immunetits, MA) was used to incubate culture supematants.
Peroxidase activity was detected using an ECL kit
(Amersham) according to the instructions of the
manufacturer. Briefly, equal volumes of the ECL A
and B reagents were mixed and allowed to gently
wash over the blolt for 1 min. Excess reagent was
3. Results
3.1. Production
and characteristics
3.2. ELBA and RDE for determining
and classifying field isolates
of mAbs
mAb specificity
All the seven hybridomas (except mAb 5D6)
produced antibodies which gave positive reactions in
Table 2
Some characteristics
of the mAbs used in this study
Monoclonal
antibody
Isotype
3ElO
4B7
9H5
llC3
3BlO
9Hl
5D6
IgG2b
IgG2a
IgG3
IgM
IgM
IgM
IgM
Positive reactions with
serotype reference strains
SAT a
ELISA ’
1,9andllb
1,9andll
1,9andll
land11
1,9and 11
1,9 and 11
1,9and 11
1,9and 11
1,9andll
1,9andll
1 and 11
a Slide agglutination test.
b Appearance of macroscopic clumps after 2 to 3 min of mixing
undiluted culture supematants
with a suspension of whole-cell
antigen.
’ mAbs were scored positive when the optical density were twofold the mean value of the negative control culture supematant.
178
J.I. Rodriguez Barbosa et al./ FEMS Immunology and Medical Microbiology 16 (1996) 173-181
ELISA and RDE with reference strains of serotypes
1, 9 and 11 but not with the remaining serotypes.
mAbs 3B 10 and 9H 1 gave much lower OD values at
450 nm for serotype 9 than for serotype 1 (data not
shown). mAb 5D6 failed to react with serotype 9.
None of the seven antibodies reacted with other
Gram-negative
bacteria tested including
Huemophilus parasuis, Pasteurella haemolytica, P. multocida capsular types A and D, Haemophilus taxon
‘minor group’, Actinobacillus suis, Klebsiella pneumoniae and Bordetella bronchiseptica.
The reactivities of mAbs 4B7 and 9H5 were also
tested against 96 field isolates. Both recognized all
the isolates of serotypes 9 and 11. They failed to
react with 34% of the isolates belonging to serotype
1. Neither reacted with any serotype 5 or 12 isolates
(Table 3).
3.3. Proteinase K and sodium periodate sensitivity of
epitopes recognized by mAbs
Sodium periodate treatment completely abolished
the reactivity of antigens with all the seven mAbs.
Proteinase K treatment did not affect their reactivities (data not shown).
3.4. ECL-immunodetection
The epitope specificity of the mAbs was investigated by ECL-immunodetection
using serotype 1
antigens blotted on NC sheets from a 10% separating
gel loaded with proteinase K-treated WBC lysate
[27]. Bacterial cell lysate of serotype 4 (strain M62)
Table 3
Number of field isolates of Actinobacillus pleuropneumoniae
reacting with mAbs 4B7 and 9H5 by rapid dot ELISA
Serotype a
(field strains)
1
9
11
5
12
Number of positive
reactions with mAbs (%)
4B7
9H5
46 (65.7)
ll(100)
ll(100)
46 (65.7)
ll(100)
ll(100)
0 (0)
0 (0)
0 (0)
0 (0)
Number of
field isolates
tested
70
11
11
2
2
Total: 96
a Serotyping was performed by immunodiffusion,
counterimmunoelectrophoresis
and coagglutination using poiyclonaf rabbit sera.
nb
cd
ef
EPI
km
ij
3h
^, /*
,.
2 -.,
!,
_<)
69
-
,-
9.)
3:;“:
46-
>/
2,
30
1
.P!
’
‘!
21
14
3BlO 487
9H5
UC3
3810
9Hl
Fig. 1. Enhanced
chemiluminiscence
(ECL)-immunodetection
analysis
of mAbs against
Actinobacillus pleuropneumoniae
serotype 1 (reference strain Shope 4074). Lanes a, c, e, g, i, k, m,
and o: reactivity of mAbs against A. pleuropneumoniae serotype
1 (serotype 1 Shope 4074); lanes b, d, f, h, j, 1, n, and p: negative
control: reactivity of MAbs against A. pleuropneumoniae serotype
4 (reference strain M62).
was used as a negative control. Two patterns of
reactivities were found. The mAbs of group I (3E10,
4B7, 9H5, and llC3) gave a ladder-like banding
pattern, characteristic of smooth type LPS (Fig. I).
mAbs of group II (3B 10 and 9Hl) recognized a long
smear of high molecular weight with mobility corresponding to 60 to 200 kDa (Fig. 1). mAb 5D6 failed
to react with any bands on immunoblots.
4. Discussion
We obtained a panel of seven hybridomas secreting mAbs against serotype 1 of A. pleuropneumoniae (reference strain Shope 4074). All the mAbs
identified
surface exposed epitopes on reference
strains of serotypes 1, 9 and I 1. None of the mAbs
reacted in ELISA or RDE with serotypes 2, 3, 4, 5,
6, 7, 8, 10 and 12 of A. pleuropneumoniae or any
other taxonomically related Gram-negative species.
Capsular polysaccharide
and somatic LPS of A.
pleuropneumoniae are considered serotype-specific
antigens [16]. All the seven mAbs described in this
study were directed against epitopes sensitive to
sodium periodate oxidation. This is evidence that
they are located at non-reducing
terminal carbohydrate antigenic sites and that they are carbo-
J.I. Rodriguez Barbosa ef al. / FEMS Immunology and Medical Microbiology 16 (1996) 173-181
hydrates [26]. Immunoblot
analysis using a whole
cell lysate indicated that the epitopes were resistant
to proteinase K and heat treatment for 4 min in the
presence of SDS and reducing conditions. This is
also consistent with the carbohydrate nature of these
antigenic determinants.
To assess the potential of these mAbs for serotyping, they were tested against 96 field isolates previously classified by conventional methods. All of the
mAbs recognized the same reference strains (except
n-&b 5D6 that did not react with the serotype 9
reference strain). This implies that the epitopes identified were well-conserved in the O-antigen of reference strains LPS. H:owever, two of the mAbs tested
(4B7 and 9H5) were not able to recognize 34% of
the serotype 1 isolates suggesting antigenic heterogeneity. There may be two or more subtypes within
serotype 1 of A. pleuropneumoniae:
one subtype
including
all the field isolates sharing O-antigen
epitopes with the reference strain Shope 4074 recognized by mAbs 4B’7 and 9H5 and the other subtype
including isolates with different O-antigen epitopes.
Mittal et al. [30] have already demonstrated
the
existence of antigenic heterogeneity among field isolates of serotype 1 of A. pleuropneumoniae.
They
reported that strains of serotype 1 could be subdivided into 2 groups (designated as A and B), depending on the presence of type specific antigens resistant
to heating at 121°C detected by coagglutination
test.
Similarly, Jolie et ad. [5J reported two different antigenie subtypes (designated as 1A and 1B) within
serotype 1 of A. pr’europneumoniae by coagglutination and immunoblot
tests using a set of 75 field
isolates isolated in the United States. They concluded
that LPS O-antigens from serotype 1A seemed to be
more similar to serotype 9 than to either serotype 1B
or serotype 11.
The structures of the O-antigen of serotypes 1 to
12 of A. pleuropneumoniae
have been described
[16]. Biochemical analysis suggests that the structure
of serotype 1 O-antigen is identical to that of serotype
11. The O-antigen of serotype 9 differs from that of
serotypes 1 and 11, since the common trisaccharide
backbone repeating unit was partially substituted by
terminal 2-acetoamido-2-deoxi-@-glucose
residues
linked to rhamnopyranosyl
units in the main chain.
Conventional
serological methods based on rabbit
polyclonal sera (immunodiffusion,
indirect hemag-
179
glutination
and coagglutination)
[8,12,13,24] have
shown that the putative epitopes responsible for the
cross-reactivities
among serotypes 1, 9 and 11 of A.
pleuropneumoniae
might be located on the somatic
antigen of LPS. Our findings are consistent with
these structural and serological data. Our mAbs recognized epitopes located on the O-antigen of LPS in
immunoblots. Furthermore, serotypes 1, 9 and 11 of
Actinobacillus
pleuropneumoniae
all had common
epitopes.
Our panel of mAbs displayed two patterns of
immunoreaction
in immunoblots. Thus, mAbs classified into group I (3E10, 4B7, 9H5 and llC3) detected a ladderlike profile characteristic of smooth
type LPS whereas those mAbs included in group II
(3BlO and 9Hl) reacted with a high-molecularweight material giving a single smear from 60 to 200
kDa (Fig. 1). mAb 5D6 failed to react in this technique. These two groups may correspond to at least
two distinct epitopes or different degrees of mAbs
affinity. All the mAbs of the IgG isotype gave a
ladder whereas those of the IgM isotype did not
(except for mAb llC3 which gave a weak ladder).
Thus, our observations are consistent with previous
reports [9,12,16,31], providing a serological evidence
of the implication of LPS O-antigen carrying immunodominant
epitopes involved in cross-reactivities
among strains of serotypes 1, 9 and 11.
Byrd and Kadis [32] using SDS-PAGE and silver
staining reported that LPS from A. pleuropneumoniae Shope 4074 was of semirough type. ECL-immunodetection with proteinase K-treated whole-cells
and the mAbs of group I (Fig. 1) strongly suggest
that LPS O-antigens of serotypes 1, 9 and 11 displayed a ladderlike banding pattern similar to that of
smooth type LPS in accordance with the profile
reported by Beynon et al. [13] and Jolie et al. [5].
According to Perry et al. [16], specific O-antigen
of LPS is always associated with a specific capsular
polysaccharide
in A. pleuropneumoniae
strains. To
obtain a better and reliable serotyping scheme, we
propose that the serotypes of A. pleuropneumoniae
could be more rigorously defined by specification of
both their polysaccharide and O-antigen of LPS. This
would be in accordance with tbe nomenclature introduced for members of the family Enterobacteriaceae
included in Section 5 of Bergey’s Manual of Systematic Bacteriology [33]. Thus, serotypes 1, 9 and 11
J.I. Rodnlguez Barbosa et al./ FEMS bnmunology ana’ Medical Microbiology 16 (1996) 173-181
180
could be designated as Kl:Ol, K9:Ol and Kll:Ol,
respectively. The current scheme should be revised
and the isolates should be classified according to
their specific serotype antigens.
Finally, the mAbs described in this study may
provide reliable specific tools for rapid serological
diagnosis of animals infected with serotypes 1, 9 and
11 by inhibition or competition ELISA. Moreover,
these mAbs could be useful as a complementary
specific reagents to polyclonal sera for serotyping of
field isolates belonging
to A. pleuropneumoniae
serotypes 1, 9 and 11.
In conclusion,
we have developed a panel of
seven mAbs that identified common polysaccharide
epitopes located on O-antigen of LPS which are
shared among serotypes 1, 9 and 11 of Actinobacillus (Haemophilus) pleuropneumoniae.
Acknowledgements
This research was supported by grant AGF93-0684
from the ‘Programa National
de Investigation
y
Desarrollo Ganadero’, CICYT, Spain. J.I.R.B. and
R.I.T. were recipients of a predoctoral long-term
fellowship from the ‘Ministerio
de Education
y
Ciencia’ and O.R.G.LL. was recipient of a predoctoral fellowship from ‘Junta de Castilla y Leon
(Spain).
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