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Journal of Clinical Microbiology, June 1998, p. 1704-1710, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection and Identification of
Actinobacillus pleuropneumoniae Serotype 5 by
Multiplex PCR
Terry M.
Lo,
Christine K.
Ward, and
Thomas J.
Inzana*
Center for Molecular Medicine and Infectious
Diseases, Virginia-Maryland Regional College of Veterinary Medicine,
Virginia Polytechnic Institute and State University, Blacksburg,
Virginia 24061-0342
Received 3 October 1997/Returned for modification 28 January
1998/Accepted 20 March 1998
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ABSTRACT |
Serotyping of Actinobacillus pleuropneumoniae is based
on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed
for detection of A. pleuropneumoniae and
identification of serotype 5 isolates. DNA sequences specific to the
conserved export and serotype-specific biosynthesis regions of the
capsular polysaccharide of A. pleuropneumoniae
serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA
fragments, respectively. The 0.7-kb fragment was amplified from all
strains of A. pleuropneumoniae tested with the
exception of serotype 4. The 0.7-kb fragment was not amplified from any
heterologous species that are also common pathogens or commensals
of swine. In contrast, the 1.1-kb fragment was amplified from all
serotype 5 strains only. The assay was capable of amplifying DNA from
less than 102 CFU. The A. pleuropneumoniae
serotype 5 capsular DNA products were readily amplified from
lung tissues obtained from infected swine, although the 1.1-kb product
was not amplified from some tissues stored frozen for 6 years. The
multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains
from other serotypes. The use of primers specific to the biosynthesis
regions of other A. pleuropneumoniae serotypes would
expand the diagnostic and epidemiologic capabilities of this assay.
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INTRODUCTION |
Actinobacillus
pleuropneumoniae is the etiologic agent of swine
pleuropneumonia, which is highly contagious and may result in high herd
mortality (24). Current attempts to control the disease have
been unsuccessful and have resulted in large economic losses to the
swine industry (1, 12, 16). Because the disease can be
transmitted quickly throughout the herd, early detection of this
bacterium is important for control and treatment of the disease
(3, 30). A. pleuropneumoniae has been
classified into two biotypes and 14 serotypes that vary in virulence
(15, 17, 20, 21) and predominance in different geographic
regions (3, 23). The serotype specificity of A. pleuropneumoniae is determined by the capsular polysaccharide
present on its surface (10, 15, 18). Serotyping is important
for understanding how the disease is spread, for treatment and
prevention, and for epidemiologic monitoring of the serotypes present
in herds and/or regions. In the United States, serotype 5 of
A. pleuropneumoniae is one of the most commonly
isolated (23). Several serologic assays have been developed
for serotyping of A. pleuropneumoniae, but the
specificities of these assays vary considerably. Cross-reactivities between serotypes 1 and 9, serotypes 3, 6, and 8, serotypes 1 and 5, and serotypes 4 and 7 have been reported (9, 13, 14). These
cross-reactions are most likely due to shared species-specific antigens
such as lipopolysaccharide or membrane proteins (18).
PCR has become a powerful and increasingly popular tool in microbial
identification (19). The capability of PCR to detect genetic
sequences from minute quantities of DNA is advantageous compared to
serologic forms of detection for several reasons: cross-reactions
between antigen and antibody are avoided, strains that have been
previously characterized as untypeable due to autoagglutination may be typeable by PCR, amplification of DNA by PCR makes it extremely sensitive, and PCR can be performed directly on samples without a wait
for culture of the bacteria.
Genes involved in A. pleuropneumoniae serotype 5 capsular polysaccharide export (cpx) and polysaccharide
biosynthesis (cps) were recently cloned and sequenced by
Ward and Inzana (28, 29). The cpx genes are
highly conserved (4), whereas the cps genes are
serotype specific (28). Based on the conserved nature of the
cpx genes and the serotype specificity of the cps
genes, we sought to determine whether DNA primers from these regions
could be used in a multiplex PCR assay to reveal the presence of
A. pleuropneumoniae in samples and, if present,
whether the strain could be identified as serotype 5.
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MATERIALS AND METHODS |
Bacterial strains and culture.
All bacterial strains used in
this study are described in Table 1.
K17-C and J45-C are nonencapsulated mutants of A. pleuropneumoniae serotype 5 produced by chemical mutagenesis.
J45-100 is a nonencapsulated mutant of serotype 5 strain J45 that lacks
a portion of its cps region due to homologous recombination
(28). All bacterial strains were grown at 37°C on brain
heart infusion (BHI) agar plates (Difco Laboratories, Detroit, Mich.)
containing 5 µg of NAD per ml (BHI-NAD) or in BHI-NAD broth.
Tissue and nasal swab samples.
Lung tissue samples were
taken from pigs challenged intratracheally with 5 × 107 CFU of A. pleuropneumoniae serotype 5 (11) and had been stored at 
20°C since 1991.
DNA isolation.
Genomic A. pleuropneumoniae
DNA was purified as previously described (29). Briefly,
A. pleuropneumoniae log-phase cells were suspended in
10 mM Tris-1 mM EDTA, pH 8.0, and incubated at 37°C for 1 h in
0.66% sodium dodecyl sulfate and 100 µg of RNase per ml. The
suspension was then incubated an additional hour at 56°C with 100 µg of proteinase K per ml, and the DNA was purified by repeated
phenol-chloroform (Amresco Inc., Solon, Ohio) extraction. Genomic DNA
was precipitated from the aqueous phase by adding 0.3 volumes of 3 M
sodium acetate and 2.5 volumes of 95% ethanol. The DNA precipitate was
then dried and resuspended in sterile water.
Multiplex PCR sample preparation.
DNA from whole bacterial
cells for PCR amplification was extracted by suspending a loopful of
colonies in 100 µl of water and lysing the suspended cells by heating
them at 100°C for 10 min. The lysed cells were centrifuged, and the
supernatant containing the bacterial DNA was removed and frozen at
20°C until needed. For extraction of bacterial DNA from lung
samples, the tissue was sectioned into thin slices approximately 2 mm
long. The sectioned tissue was then mashed and vortexed in 1 ml of
water and boiled at 100°C for 10 min. The extract was centrifuged,
and the supernatant containing the DNA was removed and frozen at
20°C until used.
DNA primers.
Oligonucleotide primers were selected by using
DNAStar (Madison, Wis.) Primer Select software. Primers A and B were
designed from the cps region of A. pleuropneumoniae serotype 5; primers C and D were designed from
the cpx region of the same A. pleuropneumoniae strain (28, 29). The primers were
selected based on the following properties: primer length, product
length, product location, hairpin formations, dimer formations, and
annealing temperature. The sequences for the oligonucleotide primers
are given in Table 2.
PCR.
PCRs were performed in a total volume of 50 µl and
were based on the procedure described by Saiki et al. (22).
Master mixes for PCR were made fresh in batches of 750 µl. Each final
reaction mix contained 10 mM Tris; 50 mM KCl; 2 mM MgCl2;
400 µM (each) dATP, dCTP, dGTP, and dTTP; and 480 µM (each) primers
A and B or primers C and D. Five microliters of template DNA thawed at room temperature was added to 45 µl of the master mix for each reaction. The samples were then overlaid with 50 µl of mineral oil to
prevent evaporation.
The PCR assays were performed in an Omnigene thermal cycler (Hybaid
Unlimited). The template DNA was denatured at 94°C for
2 min, and
then 2.5 U of
Taq polymerase (Fisher Scientific, Atlanta,
Ga.) was added. A total of 30 cycles of PCR were performed, with
each
cycle consisting of 1 min of denaturation at 94°C, 2 min
of annealing
at 54°C, and 2 min of extension at 72°C. Twelve microliters
of each
PCR mixture was then loaded into a 0.7% agarose gel containing
0.5%
ethidium bromide. Following electrophoresis the products
were
visualized by exposure to UV light.
PCR sensitivity.
Ten microliters of strain J45 log-phase
broth culture (harvested at 109 CFU/ml) was added to 990 µl of sterile water and vortexed. Log10 serial dilutions
of bacteria were made to obtain dilutions containing 106 to
102 CFU/ml. Bacterial concentrations were confirmed by
viable plate counts. The diluted samples were then boiled for 10 min,
and 5 µl of each dilution was used as a DNA template for PCR
amplification.
Southern blotting.
Southern blotting was done as described
previously (26). DNA was covalently linked to the nylon
membranes by UV irradiation with a UV Stratalinker (Stratagene, La
Jolla, Calif.). The hybridizations were performed under high stringency
at 68°C and in the presence of 5× SSC (1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate). Five microliters of a cpx probe was
added to 25 ml of prehybridization buffer for use as the hybridization
solution for cpx products. The cpx probe was
manufactured by PCR and labeled with digoxigenin-11-UTP (Genius System;
Boehringer Mannheim, Indianapolis, Ind.). The same cpx
primers that were used for the multiplex PCR assay were used to produce
an identical 0.7-kb fragment. PCR conditions were as described above,
except that 1.5 µl of 20 µM cpx forward primer, 1.5 µl
of 20 µM cpx reverse primer, and 10 µl of A. pleuropneumoniae template DNA were used. The PCR product was
verified by gel electrophoresis. Following Southern transfer, the
membranes were washed and developed as recommended by the manufacturer.
Latex agglutination test.
The latex agglutination test was
used to identify serotypes 1, 5, and 7 from A. pleuropneumoniae field isolates, as described by Inzana
(8).
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RESULTS |
PCR standardization and optimization.
Two pairs of primers
were designed from the sequenced DNA of the A. pleuropneumoniae serotype 5 cpx and cps
capsule locus. The four primers are listed in Table 2. Primers A and B
were designed to amplify a portion of the serotype-specific
cps region, while primers C and D amplified part of the
serotype-conserved cpx region. Amplification of purified
A. pleuropneumoniae J45 genomic DNA with cps
primers A and B or with cpx primers C and D resulted in a
single 1.1-kb band and a 0.7-kb band, respectively. When J45 genomic
DNA was amplified with both the cpx and cps sets of primers, both the 1.1- and 0.7-kb bands were detected (Fig. 1). Both products were consistent with
the sizes predicted from the sequenced data of those regions.
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FIG. 1.
Agarose gel electrophoresis of PCR products from
A. pleuropneumoniae serotype 5 genomic DNA. Lanes:
1, 1-kb DNA ladder; 2, cps primers A and B; 3, cpx primers C and D; 4, primers A, B, C, and D.
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The multiplex PCR assay was optimized by using extracts of serotype 5 bacteria. The magnesium concentrations used for the
assay varied from 1 to 7.5 mM; annealing temperatures varied from
49 to 55°C. These
conditions did not seem to affect the amplification
of the 1.1-kb
cps or 0.7-kb
cpx band, as no differences in the
intensity of the bands were observed when these parameters were
varied.
Initially, the assay conditions for amplifying serotype
5 DNA were 5 mM
MgCl
2 and an annealing temperature of 54°C.
The multiplex PCR assay was then used to amplify DNA from whole cells
of the type strains of all 12
A. pleuropneumoniae
serotypes.
The following parameters were examined to optimize the
multiplex
PCR assay for all serotypes: annealing temperature, primer
concentration,
Taq polymerase concentration, and
MgCl
2 concentration. Nonspecific
products were observed
from some serotypes when the conditions
described above for serotype 5 were used. For serotypes 2, 3,
and 6, a prominent band of about 1.2 kb
was produced. Amplification
of serotype 4 DNA produced a prominent band
that was approximately
1.3 kb in size, but the 0.7-kb
cpx
product was not amplified (Fig.
2). In
addition, faint nonspecific bands of various sizes were
often amplified
from these and some other serotypes. Serotypes
5, 9, 11, and 12 appeared to produce more intense bands than the
other serotypes.
Lowering the concentration of MgCl
2 resulted
in
progressively fainter to nondetectable
cpx bands for
serotypes
1, 2, 3, 6, 7, 8, and 10, whereas the effects of lowering the
MgCl
2 concentration on serotypes 5, 9, 11, and 12 were less
noticeable
(data not shown). The 0.7-kb
cpx band was not
amplified from serotype
4 under any conditions. Annealing temperatures
varied from 49
to 56°C. Increasing the annealing temperature to
eliminate the
nonspecific products also resulted in the loss of
specific PCR
products and therefore did not help to optimize the assay.
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FIG. 2.
Agarose gel electrophoresis of PCR products amplified
from whole cells of serotypes 1 through 6 at a MgCl2
concentration of 5 mM. Lanes: 1, 1-kb ladder; 2 through 7, PCR products
from serotypes 1 through 6, respectively, amplified with primers A, B,
C, and D.
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In order to eliminate the nonspecific bands amplified from serotypes 2, 3, 4, 6, and others without eliminating the 0.7-kb
cpx band,
the concentrations of
cps primers A and B were
systematically
reduced from starting concentrations of 480 µM (each)
to final
concentrations of 25 µM (each). Although the 1.2-kb band in
serotypes
2, 3, and 6 showed a marked reduction of amplification, the
intensity
of the 1.1-kb band from serotype 5 was also substantially
reduced
(data not shown).
The inability to eliminate the nonspecific bands by reducing the
cps primer concentration suggested that the nonspecific
bands
may have been amplified by the
cpx primers. To
determine which
primers were responsible for generating the nonspecific
bands,
DNA from serotypes 2 and 4 were amplified with the following
combinations
of primers: A and B, C and D, A and D, and C and B. A band
similar
in size to the 1.1-kb
cps product was amplified from
serotype
2 with primer combinations A and B, C and D, and C and B. Similarly,
the 1.3-kb band produced from serotype 4 was amplified from
one
cps primer, primer A, and one
cpx
primer, primer D (data not shown).
This indicated that the nonspecific
band amplified from serotypes
2 and 4 DNA was being amplified from
cpx primers as well as
cps primers.
The effect of the
Taq polymerase concentration on PCR
products was examined in volumes of 50 and 100 µl. At 2.5 U of
Taq polymerase/100
µl of reaction volume, no amplified
products were visible. However,
at 7.5 U of
Taq
polymerase/100 µl of reaction volume, the
cpx band was
visible as well as a faint nonspecific band similar in
size to the
1.1-kb
cps product. When 2.5 U of
Taq
polymerase/50
µl of reaction volume was used, the
cpx band
was amplified without
amplification of nonspecific products. At 7.5 U
of
Taq polymerase/50
µl of reaction volume, both the
cpx band and nonspecific bands
were clearly visible (data
not shown). These results indicate
that the concentration of
Taq polymerase had a pronounced effect
on nonspecific
amplification of PCR products.
Increasing the MgCl
2 concentration also resulted in an
increase of nonspecific products for serotypes 2 and 4. At a
concentration
of 2 mM MgCl
2, only the
cpx band
was visible. However, as the
MgCl
2 concentration was
increased from 2 to 5 mM, there was a
noticeable increase in
nonspecific banding. When the concentration
of MgCl
2 was
greater than 5 mM, the amount of nonspecific bands
began to decrease.
Thus, the optimum specificity of the multiplex
PCR assay with bacterial
samples required 2 mM MgCl
2, while maximum
sensitivity
required 5 mM MgCl
2.
The optimum conditions for the multiplex PCR assay with samples of
bacterial cells were therefore determined to be a mixture
of 2.5 U of
Taq DNA polymerase, 10 mM Tris-HCl, 50 mM KCl, 2 mM
MgCl
2, 400 µM (each) deoxynucleoside triphosphates, 480 µM (each)
primer, and 5 µl of bacterial sample in a final volume of
50 µl.
Assay sensitivity.
The sensitivity of the multiplex PCR was
determined for strain J45 whole bacterial cells. The bacteria
were harvested in broth at mid-log phase to minimize the
number of dead cells present in the sample. Both the 0.7-kb
cpx and 1.1-kb cps PCR products were visualized
by gel electrophoresis from at least 102 CFU/reaction (data
not shown). There was not any detectable increase or decrease in
sensitivity or quantity of amplified product when reagents such as
sodium dodecyl sulfate or lysozyme were used in the extraction process.
Centrifugation of the crude extract after the cells were boiled was
important to avoid decreased sensitivity in this PCR assay. In
addition, detection of PCR products was diminished from cells that had
been grown in BHI broth unless they were washed prior to preparation.
Other methods used to improve the specificity of the primers, such as
varying the annealing temperature, were found to be ineffective.
Serotype and species specificity.
Amplification of serotype 5 DNA from strains J45, J45-C, K17, and K17-C by multiplex PCR produced
both cps and cpx bands. However, only the
cpx band was amplified from DNA of J45-100, a
nonencapsulated knockout mutant lacking part of the cps
region (28), confirming that the cps primers were
amplifying DNA from the cps locus (Fig.
3). The multiplex PCR assay was used to
amplify DNA from the reference strains of 12 A. pleuropneumoniae serotypes (Fig. 4).
With the exception of serotype 4, a band of 0.7 kb, consistent with the
cpx product, was amplified from all serotypes. However, the
1.1-kb band was amplified only from serotype 5 DNA. No amplified
products were observed for serotype 4 under optimum conditions.
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FIG. 3.
Agarose gel electrophoresis of PCR products amplified
from whole cells of encapsulated and nonencapsulated serotype 5 strains. Lanes: 1, 1-kb ladder; 2, encapsulated strain K17; 3, chemically induced nonencapsulated mutant K17-C; 4, nonencapsulated
recombinant mutant J45-100 containing a large deletion in
cps; 5, chemically induced nonencapsulated mutant J45-C; 6, encapsulated strain J45.
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FIG. 4.
Agarose gel electrophoresis of bacterial samples of
serotypes 1 through 12 at a MgCl2 concentration of 2 mM.
Lanes: 1, 1-kb DNA ladder; 2 through 7, PCR products from serotypes 1 through 6, respectively; 8 through 14, PCR products from serotypes 5 and 7 through 12, respectively; 15, 1-kb DNA ladder. All products were
amplified with primers A, B, C, and D.
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Previously untyped field isolates were assayed by both PCR and
the latex agglutination test (Table
3).
All of the eight strains
typed as serotype 5 were also identified by
multiplex PCR as serotype
5 strains, determined by amplification
of the distinct
cps and
cpx products. PCR assays
were also performed on six strains that
were typed as serotype 1 and
six strains that were typed as serotype
7 by latex agglutination. The
cpx product was amplified by all
12 strains, identifying
them as
A. pleuropneumoniae, but the
cps product was not amplified from any of these strains, confirming
they
were not serotype 5. In addition, one strain that was not
typed as
serotype 1, 5, or 7 by latex agglutination was determined
to be
A. pleuropneumoniae by amplification of only the
cpx product
by multiplex PCR.
Species specificity of the multiplex PCR was examined by applying
the assay to five other swine respiratory pathogens (Fig.
5):
Actinobacillus suis,
Bordetella bronchiseptica,
Haemophilus parasuis,
Pasteurella multocida, and
Streptococcus suis. No amplified
products were made from DNA
of any of these species when the optimum
conditions for specificity
were used. However, when 5 mM MgCl
2 was used in the assay,
nonspecific bands were amplified from
H. parasuis,
P. multocida, and
S. suis DNA.
These bands were distinct
in size from the 0.7-kb
cpx and
1.1-kb
cps products (data not
shown).
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FIG. 5.
Agarose gel electrophoresis of PCR products of bacterial
samples of respiratory swine pathogens amplified with primers A, B, C,
and D. Lanes: 1, A. pleuropneumoniae serotype 5; 2, A. suis; 3, B. bronchiseptica; 4, H. parasuis; 5, P. multocida; 6, S. suis; 7, 1-kb DNA ladder.
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Southern hybridization was performed on amplified products from
H. parasuis,
P. multocida, and
S. suis
DNA, as well as from
A. pleuropneumoniae serotypes 2 and 4 DNA, to further examine
the specificity of products
amplified when 5 mM MgCl
2 was used
in the multiplex PCR
assay (Fig.
6a). The bands were
probed under
high-stringency conditions with a labeled PCR product
generated
by
cpx primers C and D from
A. pleuropneumoniae serotype 5. The
probe did not hybridize to any of
the bands produced by serotype
4 or the three non-
A.
pleuropneumoniae species (Fig.
6b), but
it did hybridize to the
0.7-kb
cpx bands produced by serotypes
2 and 5.
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FIG. 6.
(a) Agarose gel electrophoresis of PCR products from
bacterial samples used for Southern hybridizations. Lanes: 1, 1-kb
ladder; 2, serotype 2; 3, serotype 4; 4, serotype 5; 5, S. suis; 6, P. multocida; 7, H. parasuis. (b)
Southern blot of PCR products from bacterial samples hybridized with a
cpx probe. Lanes: 1, serotype 2; 2, serotype 4; 3, serotype
5; 4, S. suis; 5, P. multocida; 6, H. parasuis.
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Amplification of DNA from clinical specimens.
The multiplex
PCR was used on lung tissue from swine infected with A. pleuropneumoniae serotype 5 to determine if the assay could be
used for rapid diagnosis of clinical disease. Both the cpx
and the cps products were amplified from lung tissue samples of two pigs that had recently been infected with strain J45 (data not
shown). Frozen lung tissue samples from eleven serotype 5-challenged pigs that had been used for an immunization study 6 years earlier (11) were also assayed by PCR. The cpx product
was amplified from each of these samples, indicating that A. pleuropneumoniae DNA was present. However, the cpx band
from five of these samples was weak, and the cps product was
not amplified from four samples (Fig. 7).
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FIG. 7.
Agarose gel electrophoresis of PCR products from lung
tissue samples taken from swine that had been infected with serotype 5. Lanes and strain numbers: 1, 204; 2, 205; 3, 207; 4, 209; 5, 211; 6, 215; 7, 216; 8, 220; 9, 221; 10, 223; 11, 228. Lane 12, 1-kb DNA
ladder.
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DISCUSSION |
Serotypes of A. pleuropneumoniae are distinguished
by their unique capsular polysaccharide (10, 18). Because
cross-reactions often occur in serotyping with traditional serologic
assays (9, 13, 14). PCR offers a practical alternative that
does not employ antigens or antibodies. This attribute makes capsule
genes an ideal target for typing by PCR. Furthermore, culture of
A. pleuropneumoniae may be successful for 50% or fewer
of the specimens submitted. When pigs die before specimens can be
collected, contamination usually prevents any isolation of
A. pleuropneumoniae (27). PCR,
however, is more sensitive than culture for this bacterium (5, 6), and the multiplex PCR described here is specific enough not to be affected by the presence of contaminants.
Failing to isolate the agent for susceptibility testing should not be of concern because A. pleuropneumoniae is susceptible
to most broad-spectrum antibiotics (3). Falla et al.
(2) previously reported using primers from the capsular DNA
region as a reliable method for typing of Haemophilus
influenzae by PCR. To date, a reliable method for serotyping
of A. pleuropneumoniae by PCR has not yet been
established. Hennessy et al. (7) have reported using an
arbitrarily primed PCR assay for serotyping of A. pleuropneumoniae, but this method has the disadvantage of
requiring pure bacterial samples for testing and is highly susceptible
to contamination. Sirois et al. (25) described the use of
uncharacterized primers for amplification of A. pleuropneumoniae DNA. Although this assay was specific for
swine pathogens, it could not discriminate between A. pleuropneumoniae biotype 1, biotype 2, and
Actinobacillus lignieresii, and the sensitivity of the assay
was not determined. The same primers were later used to detect
A. pleuropneumoniae from tonsils, with a sensitivity of
103 CFU/reaction tube (5). Gram et al.
(6) recently described a species-specific PCR
assay for amplification of an A. pleuropneumoniae outer
membrane lipoprotein that has improved specificity. However, culture is
still required with either assay to identify the serotype, which is
needed for epidemiology, herd management, and monitoring of the spread
or introduction of new strains. The present study describes the first
use of primers to amplify conserved and serotype-specific capsular DNA
regions to simultaneously identify A. pleuropneumoniae and the serotype.
Both the cps and cpx regions of serotype 5 DNA
were successfully amplified with samples of purified DNA, bacterial
colonies, and lung tissue. Combining the primers did not require any
change in PCR conditions. The use of multiplex PCR provided the
advantage of using multiple primer sets in a single reaction and
simultaneously determining both the species and the serotype, in this
case A. pleuropneumoniae and serotype 5. The detectable
limit of the PCR products of serotype 5 by agarose gel electrophoresis
was less than 102 CFU/reaction.
The presence of nonspecific bands amplified from some serotypes,
particularly serotypes 2, 3, and 6, while maintaining the cpx band in all serotypes, was initially problematic.
Although the cpx region appears to be highly conserved,
the primers selected from that region were designed from the
sequence of serotype 5 capsular DNA. Because there is no sequence
data available on the capsular export regions of other serotypes, the
amount of homology of these primers to other serotypes is
unknown. The MgCl2 concentration was the single most
important parameter involved in the specificity of PCR amplification
and was successfully used to control the presence of nonspecific bands.
The successful application of the multiplex PCR assay to bacterial
colonies provided an effective method of identifying and serotyping
A. pleuropneumoniae. With the exception of the rare serotype 4, a distinct 0.7-kb band was amplified from all serotypes. This PCR assay also confirmed results obtained by latex agglutination. Of 21 field isolates serotyped by latex agglutination, all 21 isolates
were identified as A. pleuropneumoniae by the multiplex PCR assay. In addition, all strains that were identified as serotype 5 by latex agglutination were also identified as serotype 5 by multiplex PCR. Because results from agglutination tests can often be inconclusive or subject to interpretation, PCR can potentially be
used to definitively type strains that are difficult to assay by
agglutination. Furthermore, the multiplex PCR can confirm that nontypeable isolates are A. pleuropneumoniae. No
amplification of DNA under conditions for optimal specificity was
observed from any of the other swine respiratory pathogens tested,
indicating that the multiplex PCR assay can be used to determine
whether an infection is due to A. pleuropneumoniae. PCR
is also relatively simple and can be performed in under 5 h. The
amplification of the cpx product in all of the serotypes,
with the exception of serotype 4, supports the existing evidence that
the capsular export region is highly conserved among A. pleuropneumoniae serotypes (29). Southern blotting also
indicated that the 0.7-kb band of serotype 2 contains homology to the
0.7-kb band of serotype 5. Although the 0.7-kb product was not
amplified from serotype 4, it is not surprising that even within a
highly conserved region there may be some areas of nonhomology.
However, cps primers A and B were very specific in
amplifying a 1.1-kb band from only serotype 5 isolates.
The multiplex PCR was also applied to testing of clinical specimens.
The 1.1-kb cps and 0.7-kb cpx bands were
amplified from lung tissue samples of swine infected with A. pleuropneumoniae serotype 5. The cpx product was
amplified from all samples, although much less product was made from
five of the samples. However, the cps product was not
amplified from 4 of 11 samples. It is not clear why the 1.1-kb
cps product was not amplified from these samples, but it may
have been due to the samples being 6 years old. The DNA in this region
may have degraded over time to the point that it could not be
amplified. Alternatively, it should be noted that these samples were
taken from a vaccination study. It is likely that efficacious
vaccination enabled the host to clear the challenge infection quickly,
substantially lowering the number of bacteria present in the lung.
In conclusion, the multiplex PCR assay was effective in detecting
A. pleuropneumoniae and identifying serotype 5 from
whole bacterial cells and infected lung tissues. At present,
auto-agglutinating, cross-reacting, and nontypeable strains
are difficult to identify by serologic assays. The use of multiplex PCR
with primers directed to the capsular DNA regions can potentially be
used for detection and serotyping of A. pleuropneumoniae with both high specificity and high sensitivity,
avoiding the problems associated with serologic assays. Once the
sequences for the capsular regions of other serotypes have been
determined, the assay can be expanded to serotype any strain of
A. pleuropneumoniae.
| |
ACKNOWLEDGMENTS |
We thank Eric Wong and N. Sriranganathan for helpful suggestions
and advice; K. Mittal, Martha Mulks, Karen Post, Jacques Nicolet, and
Brad Fenwick for providing bacterial strains; and Gretchen Glindemann,
Mark Lawrence, and John McQuiston for technical assistance.
This work was supported, in part, by grants from Solvay Animal Health
and by HATCH formula funds to the Virginia State Agricultural Experiment Station.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 1410 Prices Fork
Rd., CMMID, College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061-0342. Phone: (540) 231-4692. Fax: (540) 231-3426. E-mail: tinzana{at}vt.edu.
| |
REFERENCES |
| 1.
|
Brandreth, S. R., and I. M. Smith.
1985.
Prevalence of pig herds affected by pleuropneumonia associated with Haemophilus pleuropneumoniae in eastern England.
Vet. Rec.
117:143-147[Abstract].
|
| 2.
|
Falla, T. J.,
D. M. Crook,
L. N. Brophy,
D. Maskell,
J. S. Kroll, and E. R. Moxon.
1994.
PCR for capsular typing of Haemophilus influenzae.
J. Clin. Microbiol.
32:2382-2386[Abstract/Free Full Text].
|
| 3.
|
Fedorka-Cray, P. J.,
L. Hoffman,
W. C. Cray,
J. T. Gray,
S. A. Breish, and G. A. Anderson.
1993.
Actinobacillus (Haemophilus) pleuropneumoniae. Part I. History, epidemiology, serotyping, and treatment.
Compend. Contin. Ed. Practic. Vet.
15:1447-1455.
|
| 4.
|
Frosch, M.,
U. Edwards,
K. Bousset,
B. Krause, and C. Weisgerber.
1991.
Evidence for a common molecular origin of the capsule gene loci in gram-negative bacteria expressing group II capsular polysaccharides.
Mol. Microbiol.
5:1251-1263[Medline].
|
| 5.
|
Gram, T.,
P. Ahrens, and J. P. Nielsen.
1996.
Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils.
Vet. Microbiol.
51:95-104[Medline].
|
| 6.
|
Gram, T.,
P. Ahrens, and J. P. Nielsen.
1998.
Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein.
J. Clin. Microbiol.
36:443-448[Abstract/Free Full Text].
|
| 7.
|
Hennessy, K. J.,
J. J. Iandolo, and B. W. Fenwick.
1993.
Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.
J. Clin. Microbiol.
31:1155-1159[Abstract/Free Full Text].
|
| 8.
|
Inzana, T. J.
1995.
Simplified procedure for preparation of sensitized latex particles to detect capsular polysaccharides: application to typing and diagnosis of Actinobacillus pleuropneumoniae.
J. Clin. Microbiol.
33:2297-2303[Abstract].
|
| 9.
|
Inzana, T. J.,
G. F. Clark, and J. Todd.
1990.
Detection of serotype-specific antibodies or capsular antigen of Actinobacillus pleuropneumoniae by a double-label radioimmunoassay.
J. Clin. Microbiol.
28:312-318[Abstract/Free Full Text].
|
| 10.
|
Inzana, T. J., and B. Mathison.
1987.
Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae.
Infect. Immun.
55:1580-1587[Abstract/Free Full Text].
|
| 11.
|
Inzana, T. J.,
J. Todd, and H. P. Veit.
1993.
Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines.
Infect. Immun.
61:1682-1686[Abstract/Free Full Text].
|
| 12.
|
MacInnes, J. I., and S. Rosendal.
1988.
Prevention and control of Actinobacillus (Haemophilus) pleuropneumoniae infection in swine: a review.
Can. Vet. J.
29:572-573[Medline].
|
| 13.
|
Mittal, K. R.
1990.
Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotypes 1 and 9.
J. Clin. Microbiol.
28:535-539[Abstract/Free Full Text].
|
| 14.
|
Mittal, K. R.,
R. Higgins, and S. Lavriviere.
1987.
An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates.
Am. J. Vet. Res.
48:219-226[Medline].
|
| 15.
|
Nicolet, J.
1988.
Taxonomy and serological identification of Actinobacillus pleuropneumoniae.
Can. Vet. J.
29:578-579[Medline].
|
| 16.
|
Nicolet, J.
1992.
Actinobacillus pleuropneumoniae, p. 401-408.
In
A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor (ed.), Diseases of swine. Iowa State University Press, Ames.
|
| 17.
|
Niven, D. F., and M. Lévesque.
1988.
V-factor-dependent growth of Actinobacillus pleuropneumoniae biotype 2 (Bertschinger 2008/76).
Int. J. Syst. Bacteriol.
38:319-320[Abstract/Free Full Text].
|
| 18.
|
Perry, M. B.,
E. Altman,
J.-R. Brisson,
L. M. Beynon, and J. C. Richards.
1990.
Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus pleuropneumoniae strains. Serodiagn.
Immun. Infect. Dis.
4:299-308.
|
| 19.
|
Rodu, B.
1990.
Molecular biology in medicine. The polymerase chain reaction: the revolution within.
Am. J. Med. Sci.
299:210-216[Medline].
|
| 20.
|
Rogers, R. J.,
L. E. Eaves,
P. J. Blackall, and K. F. Truman.
1990.
The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae.
Aust. Vet. J.
67:9-12[Medline].
|
| 21.
|
Rosendal, S.,
D. Boyd, and K. A. Gilbride.
1985.
Comparative virulence of porcine Haemophilus bacteria.
Can. J. Comp. Med.
49:68-74[Medline].
|
| 22.
|
Saiki, R. K.,
D. H. Gelfand,
S. Stoffel,
S. J. Scharf,
R. Higuchi,
G. T. Horn,
K. B. Mullis, and H. A. Erlich.
1988.
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science
239:487-491[Abstract/Free Full Text].
|
| 23.
|
Sebunya, T. K., and J. R. Saunders.
1983.
Haemophilus pleuropneumoniae infections in swine: a review.
J. Am. Vet. Med. Assoc.
182:1331-1337[Medline].
|
| 24.
|
Shope, R. R.
1964.
Porcine contagious pleuropneumonia. I. Experimental transmission, etiology and pathology.
J. Exp. Med.
1119:357-358.
|
| 25.
|
Sirois, M.,
E. G. Lemire, and R. C. Levesque.
1991.
Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction.
J. Clin. Microbiol.
29:1183-1187[Abstract/Free Full Text].
|
| 26.
|
Straus, W. M.
1993.
Hybridization with radioactive probes, p. 6.3.1-6.3.6.
In
F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 1. John Wiley & Sons, New York, N.Y.
|
| 27.
|
Torrelorell, M.,
C. Pijoan,
K. J. Janni,
R. Walker, and H. S. Joo.
1997.
Airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and repiratory syndrome virus in nursery pigs.
Am. J. Vet. Res.
58:828-832[Medline].
|
| 28.
|
Ward, C. K., and T. J. Inzana.
1996.
Mutations in capsular polysaccharide biosynthesis render Actinobacillus pleuropneumoniae avirulent, abstr. 25, p. 34.
In
Haemophilus, Actinobacillus, Pasteurella 96 International Conference, Acapulco, Mexico.
|
| 29.
|
Ward, C. K., and T. J. Inzana.
1997.
Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.
Infect. Immun.
65:2491-2496[Abstract].
|
| 30.
|
Willson, P. J.,
G. Falk, and S. Klashinsky.
1987.
Detection of Actinobacillus pleuropneumoniae infection in pigs.
Can. Vet. J.
28:111-116.
|
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Abstract
Introduction
