i DETECTION AND IDENTIFICATION OF ACTINOBACILLUS PLEUROPNEUMONIAE SEROTYPE 5 BY MULTIPLEX POLYMERASE CHAIN REACTION by Terry M. Lo Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree MASTER OF SCIENCE in Veterinary Medical Sciences APPROVED: ________________________ Thomas J. Inzana, Chair ________________________ _______________________ Nammalwar Sriranganathan Eric A. Wong July 1997 Blacksburg, Virginia
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DETECTION AND IDENTIFICATION OFACTINOBACILLUS PLEUROPNEUMONIAE SEROTYPE 5
BY MULTIPLEX POLYMERASE CHAIN REACTION
by
Terry M. Lo
Thesis submitted to the Faculty of theVirginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree
MASTER OF SCIENCE
in
Veterinary Medical Sciences
APPROVED:
________________________Thomas J. Inzana, Chair
________________________ _______________________Nammalwar Sriranganathan Eric A. Wong
July 1997Blacksburg, Virginia
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DETECTION AND IDENTIFICATION OFACTINOBACILLUS PLEUROPNEUMONIAE SEROTYPE 5
BY MULTIPLEX POLYMERASE CHAIN REACTION
by
Terry M. Lo
Dr. Thomas J. Inzana, Chair
Department of Veterinary Medical Sciences
(ABSTRACT)
Traditional serologic assays of Actinobacillus pleuropneumoniae often have
problems with cross-reactivity. To avoid the complications of antibody-antigen
reactions, a PCR assay was developed to detect Actinobacillus pleuropneumoniae and
identify serotype 5 strains. Primers specific to the conserved capsular export region of A.
pleuropneumoniae amplified a 0.7 kb DNA band in all strains with the exception of
serotype 4. A second set of primers specific to the unique capsular biosynthesis region of
serotype 5 amplified a unique 1.1 kb band for serotype 5 only. The sensitivity of this
assay was determined to be less than 102 colony forming units. This PCR assay enables
us to detect A. pleuropneumoniae and definitively distinguishes serotype 5 strains from
other serotypes.
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This thesis is dedicated to my parents,
Julie and Christopher Lo, a.k.a. Mom and Dad.
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ACKNOWLEDGMENTS
It has been my good fortune to have worked with a number of great people that
have helped make my graduate experience worthwhile and enjoyable. I would like to
thank Dr. Thomas Inzana, my graduate advisor, for all of his guidance, support and
patience. .My committee members, Dr. Eric Wong and Dr. Nammalwar Sriranganathan,
for their excellent suggestions and advice. I greatly appreciate the time and effort they
have put forth. I would also like to thank Christine Ward for helping me get on track
when I first started, Gretchen Glindemann for all of her continuing help, and John
McQuiston for allowing me to pester him with millions of questions. Thanks to Mark
Lawrence, Todd Pack, and Rhonda Wright, who have all showed me great kindness as
well as technical advice in the lab. Thanks to Dr. John Lee and Dr. Lud Eng for their
financial support, and Linda Price, Sherrie Settle, Kim Stowers, and Tracie Sweeny for
their help with all of the administrative details. And let’s not forget about my friends and
co-workers: Mike Howard, Maureen Fallon, Jennifer Hensley, Jane Lee, Sergio Harding,
Noel Hikes, Dave Copeland, everyone at the CMMID, the media lab, and all my fans.
Thanks.
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TABLE OF CONTENTSPage
Abstract ..................................................................................................................... ii
Dedication................................................................................................................. iii
Acknowledgments .................................................................................................... iv
List of Figures .......................................................................................................... vii
List of Tables ............................................................................................................viii
The Polymerase Chain Reaction and ItsUses in Microbial Detection
History.................................................................. 32Principle of the Polymerase Chain Reaction......... 34PCR and Bacteria.................................................. 41Summary............................................................... 43
Chapter 2 Detection and Identification of Actinobacilluspleuropneumoniae Serotype 5 by MultiplexPolymerase Chain Reaction
Materials and MethodsStrains and cell cultures.....................................................50Tissue and nasal swab samples..........................................52DNA isolation....................................................................52Multiplex PCR...................................................................52
The same cpx primers that were used for the multiplex PCR assay were used to produce
an identical 0.7 kb fragment. The PCR reaction consisted of the following quantities of
reagents: 48.5 µl of sterile H2O, 10 µl of Taq 10X buffer B (Fisher Scientific, Atlanta,
GA), 2 µl of 10mM dATP, 2 µl of 10mM dCTP, 2 µl of 10 mM dGTP, 1.4 µl of 10mM
dTTP, 6 µl of DIG-dUTP, 15 µl of 25 mM MgCl2, 1.5 µl of 20 µM of cpx forward
primer, 1.5 µl of 20 µM of cpx reverse primer, and 10 µl of A. pleuropneumoniae
template DNA. The PCR product was verified by gel electrophoresis. A cps probe was
manufactured by random primed labeling. A PCR fragment was produced with the cps
primer and using similar conditions as the cpx probe with the exception of the Dig-dUTP.
The product was then purified through Promega (Madison, WI) spin columns and labeled
according to the procedure in the GeniusTM System manual.
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Southern Blotting. A Southern blot was performed on organisms that produced non-
specific products from the cpx and cps primers. Transfer of DNA from agarose gels to
MagnaGraph nylon membranes (Micron Separations Inc., Westboro, MA) were carried
out by the reverse blot method (ref Current Protocols in Molecular Biology). DNA was
covalently linked to the nylon membranes by ultraviolet irradiation using a UV
Stratalinker (Stratagene, La Jolla, CA). The hybridizations were performed under high
stringency at a temperature of 68o C and at 5X SSC. All other hybridization procedures
were done following the manufacturer’s recommendation. Ten µl of the cps probe were
added to 25 ml of the prehybridization solution to be used as the hybridization solution
for cps products, and 5 µl of the cpx probe was added to 25 ml of prehybridization buffer
for use as the hybridization solution for cpx products. The membranes were washed and
developed as recommended by the manufacturer.
RESULTS
Assay of serotype 5 genomic DNA
Two primers pairs were designed from the sequenced DNA of the A.
pleuropneumoniae serotype 5 capsular region. The four primers are listed in Table 2.2.
Primers A and B were designed to target the serotype-specific cps loci, while primers C
and D targeted the serotype-conserved cpx loci. PCR products that are 0.7 kb in size and
are consistent with expected product of primers C and D will be referred to as the cpx
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product; products that are 1.1 kb in size and are consistent with expected product of
primers A and B will be referred to as the cps product.
Amplification of purified A. pleuropneumoniae J45 genomic DNA resulted in a
single product when using the cps primers A and B. The product was detected as a 1.1
kb band by electrophoresis of the DNA through an ethidium-stained agarose gel.
Amplification of purified J45 genomic DNA also resulted in a single product when using
the cpx primers C and D. This product resulted in a band that was determined to be 0.7
kb in size. When both the cpx and cps sets of primers were added to a single PCR tube,
products of 1.1 kb and 0.7 kb could be detected (Fig. 2.2). Both products were consistent
with the sizes predicted from the sequenced data of those regions.
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Figure 2.2. Agarose gel electrophoresis of PCR products from A. pleuropneumoniaeserotype 5 genomic DNA. Lane 1, 1-kb DNA ladder; lane 2, cps primers A and B; lane 3,cpx primers C and D; lane 4, primers A, B, C, and D.
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Assay optimization
The multiplex PCR assay was optimized using bacterial samples of serotype 5.
The magnesium concentrations used for the assay were varied from 1 mM to 7.5 mM;
annealing temperatures used for the assay were varied from 49o C to 55o C. These
conditions did not seem to affect the amplification of the 1.1 kb cps or 0.7 kb cpx bands
as no differences in the intensity of the bands was observed when the conditions were
altered. Initially, the optimum conditions for assaying serotype 5 samples were
performed at 5 mM MgCl2 and at an annealing temperature of 54o C.
The multiplex PCR assay was then applied to whole cells of the type strains of all
12 A. pleuropneumoniae serotypes. The following parameters were adjusted to optimize
the multiplex PCR assay for all serotypes: annealing temperature, primer concentration,
Taq polymerase concentration, and MgCl2 concentration. Non-specific products were
observed from some serotypes under the conditions used for serotype 5. For serotypes
2, 3, 6, and 7, a band slightly larger than the 1.1 kb cps band was produced.
Amplification of serotype 4 DNA produced a band that was approximately 1.3 kb in
size, but did not amplify the 0.7 kb cpx product. In addition, non-specific bands of
various sizes were often amplified in the background of some serotypes. An example of
this is shown in Figure 2.3 for serotypes 1-6 at a MgCl2 concentration of 5 mM.
Serotypes 5, 9, 11, and 12 appeared to produce more intense bands than the other
serotypes. Lowering the concentration of MgCl2 resulted in progressively fainter to non-
detectable cpx bands in serotypes 1, 2, 3, 6, 7, 8, and 10, whereas the effects on serotypes
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9, 11, and 12 were less noticeable. No cpx band was detected with serotype 4 under any
conditions.
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Figure 2.3. Agarose gel electrophoresis of PCR products of amplified from whole cells ofserotypes 1-6 at a MgCl2 concentration of 5 mM. Lane 1, 1-kb DNA ladder; lanes 2through 7, the PCR products from serotypes 1-6, respectively, amplified with primers A,B, C, and D.
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Annealing temperatures were varied from 49o C to 56o C. Increasing the annealing
temperature to eliminate the non-specific products also resulted in the loss of specific
PCR products, and therefore did not help to optimize the assay.
In order to eliminate the non-specific products similar in size to the cps product
bands observed in serotypes 2, 3, 6, and 7, without eliminating the 0.7 kb cpx band, the
concentration of the cps primers A and B was systematically reduced from a starting
concentration of 480 µM to a final concentration of 25 µM. Figure 2.4 depicts the results
of reducing the cps primers to 25 µM for serotypes 1-6. Although the non-specific bands
in 2, 3, and 6 showed a marked reduction of amplification, the intensity of 1.1 kb band
from serotype 5 was also substantially reduced.
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Figure 2.4. Agarose gel electrophoresis of PCR products of bacterial samples ofserotypes 1-6 with primers A and B reduced to 25 µM and primers C and D at 480 µM.Lane 1, 1-kb DNA ladder; lanes 2 through 7 contain the PCR products from serotypes 1-6, respectively.
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The inability to eliminate the non-specific bands by reducing the cps primer concentration
suggested that the non-specific bands may have been amplified other primers. To
determine which primers were responsible for generating the non-specific bands, bacterial
samples of serotypes 2 and 4 were amplified with only a single pair of primers at a time.
Figure 2.5 and Figure 2.6 illustrate the results using the following combination of primers
for serotypes 2 and 4, respectively: A and B; C and D; A and D; or C and B. A band
similar in size to the 1.1 kb cpx product was amplilfied from serotype 2 with primers A
and B, C and D, and primers 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. This
indicated that the non-specific band amplified from the DNA of serotypes 2 and 4 could
not be eliminated by reducing the cps primers because the non-specific band was
amplified from cpx primers as well as cps primers.
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Figure 2.5. Agarose gel electrophoresis of serotype 2 PCR products amplified fromvarious combinations of primer pairs. Lane 1, 1-kb DNA ladder; lanes 2-5, productsamplified from primers A and B; primers C and D; primers A and D; and primers C andB, respectively.
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Figure 2.6. Agarose gel electrophoresis of serotype 4 PCR products amplified fromvarious combinations of primer pairs. Lane 1, 1-kb DNA ladder; lanes 2-5, productsamplified from primers A and B; primers C and D; primers A and D; and primers C andB, respectively.
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The effect of the Taq polymerase concentration was examined on PCR products
in volumes of 50 µl and 100 µl. Figure 2.7 depicts serotype 2 products and the effect of
varying the Taq polymerase concentrations when the MgCl2 concentration was 2 mM. At
2.5 U of Taq polymerase in 100 µl reaction volumes, no amplified products were visible.
However, at 7.5 U of Taq polymerase in 100 µl reactions, the cpx band is visible as well
as a faint non-specific band similar in size to the 1.1 kb cps product. In the 50 µl reaction
volumes, 2.5 U of Taq polymerase resulted in the amplification of the cpx band without
the amplification of non-specific products. At 7.5 U of Taq polymerase, both the cpx
band and the non-specific band are clearly visible. This indicated the concentration of Taq
polymerase had a pronounced effect on the amplification of PCR products.
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Figure 2.7. Agarose gel electrophoresis of amplified PCR products when theconcentration of Taq polymerase is varied using serotype 2 template DNA. Lane 1, 1-kbDNA ladder; lane 2, 2.5 U Taq polymerase in 50 µl; lane 3, 5 U Taq polymerase in 50 µl;lane 4, 7.5 U Taq polymerase in 50 µl; lane 5, 2.5 U Taq polymerase in 100 µl; lane 6, 5U Taq polymerase in 100 µl; lane 7, 7.5 U Taq polymerase in 100 µl.
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Increasing the MgCl2 concentration also resulted in an increase of non-specific
products for serotypes 2 and 4. Figure 2.8 shows the effect of varying the MgCl2
concentration from 2 mM to 7.5 mM for serotype 2. Using 2 mM MgCl2 only the cpx
band was visible. However, as MgCl2 was increased from 2 mM to 5 mM, there was a
noticeable increase in non-specific banding. When the concentration of MgCl2 was greater
than 5mM,the amount of non-specific bands began to decrease. Thus, the optimum
specificity of the multiplex PCR assay with bacterial samples required 2 mM MgCl2,
while maximum sensitivity required 5 mM MgCl2.
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Figure 2.8. Agarose gel electrophoresis of DNA products from bacterial samples ofserotype 2 at varying MgCl2 concentrations. Lane 1, 1-kb DNA ladder; lane 2, 2 mMMgCl2; lane 3, 3 mM MgCl2; lane 4, 4 mM MgCl2; lane 5, 5 mM MgCl2; lane 6, 6 mMMgCl2; lane 7, 7.5 mM MgCl2.
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The sensitivity and specificity of the multiplex PCR assay was also influenced by
the sample type. In order to visualize the differences in DNA amplification for different
sample types under the same conditions, the multiplex PCR assay was run at 5 mM
MgCl2 for the following serotype 2 sample types: Purified genomic DNA; rapidly
Figure 2.14 shows that nonspecific bands at higher molecular weights were produced from
the genomic DNA samples, while products that are more intense at the lower molecular
weights were obtained from the boiled bacterial sample. The boiled lung tissue sample
produced a very faint 0.7 kb cpx band, and the nasal swab sample did not produce any
visible bands. While specificity increased from “pure” samples to “dirty” samples, the
sensitivity was decreased.
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Figure 2.9. Agarose gel electrophoresis of PCR products of various serotype 2 sampletypes amplified with primers A-D at a MgCl2 concentration of 5 mM. Lane 1, 1-kb DNAladder, lane 2, purified genomic DNA; lane 3, rapidly prepared genomic DNA; lane 4,bacterial sample; lane 5, lung tissue sample; lane 6, Qiagen prepared nasal swab sample,lane 7, boiled nasal swab sample.
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The optimum conditions for the multiplex PCR assay using samples of bacterial
cells were determined to be: 2.5 U of Taq DNA Polymerase, 10 mM Tris HCl, 50 mM
KCl, 2 mM MgCl2, 400 µM dNTP’s, 480 µM of each primer, and 5 µl of bacterial
sample, all mixed in a final volume of 50 µl. Figure 2.10 shows serotypes 1-12 assayed at
these optimum conditions. With the exception of serotype 4, all of the other 11
serotypes produced a band of 0.7 kb in size. In addition, only serotype 5 had a PCR
product of 1.1 kb .
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Figure 2.10. Agarose gel electrophoresis of PCR products from bacterial samples ofserotypes 1-12 at a MgCl2 concentration of 2 mM. Lane 1, 1-kb DNA ladder; lanes 2through 14, the PCR products from serotypes 1-12, respectively, amplified with primersA, B, C, and D; lane 15, 1-kb DNA ladder.
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Assay sensitivity
The sensitivity of the multiplex PCR assay was determined for strain J45 whole
bacterial cells. The cells were grown in broth to mid-log phase, and harvested at this time
to minimize the number of dead bacteria present in the sample. Both the 0.7 kb cpx and
1.1 kb cps PCR products could be visualized by gel electrophoresis for at least 1 x 102
CFU’s (Fig. 2.11). The use of reagents such as SDS or lysozyme prior to the crude
preparation of cells had no substantial effect. There was no noticeable increase or
decrease in sensitivity or quantity of amplified product when these reagents were used.
Centrifugation of the crude extract after the cells were boiled was important to avoid
decreased sensitivity for PCR preparation. 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.
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Figure 2.11. Sensitivity of the PCR assay for serotype 5 samples of bacterial cells. Lane1, 1 x 106 cells; lane 2, 1 x 105 cells; lane 3, 1 x 104 cells; lane 4, 1 x 103 cells; lane 5, 1 x102 cells; lane 6, 1 x 10 cells; lane 7, 1-kb DNA ladder.
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Assay specificity
PCR with the cps and cpx primers was also performed on six swine respiratory
pathogens (Fig. 2.12). No amplified products were made from three species by the
multiplex PCR assay: A. suis, B. bronchiseptica, or S. choleraesuis. However, PCR
products of various sizes were made from P. multocida, H. parasuis , and S. suis. A band
of approximately 1.5 kb was made from P. multocida. Two sets of double bands were
made from H. parasuis . The first set of bands appeared to have one band slightly larger
than the 0.7 kb cpx band and one band slightly smaller; the second set of bands were of
undetermined smaller molecular size. A band of similar size to the 0.7 kb cpx band was
made from the S. suis genome.
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Figure. 2.12. Agarose gel electrophoresis of the PCR products of bacterial samples ofrespiratory swine pathogens amplified with primers A-D. Lane 1, A. pleuropneumoniaeserotype 5; lane 2, A. suis; lane 3, B. bronchiseptica; lane 4, H. parasuis; lane 5, P.multocida; lane 6, S. choleraesuis; lane 7, S. suis; lane 8, 1-kb DNA ladder.
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Hybridization analysis was performed on the three non-A. pleuropneumoniae
species from which DNA products were amplified, as well as A. pleuropneumoniae
serotypes 2 and 4. The multiplex PCR assay was performed at a MgCl2 concentration of
5 mM in order to amplify all possible PCR products (Fig. 2.13a). The bands were first
probed with a labeled PCR product generated by the cpx primers C and D from A.
pleuropneumoniae serotype 5. Hybridization of the probe to the other PCR products
was performed at a high stringency of 68o C and 5X SSC. There was no hybridization of
the probe to any of the bands produced by serotype 4 or the three non-A.
pleuropneumoniae species (Fig. 2.13b). The cpx probe only hybridized to the 0.7 kb cpx
bands produced by serotype 2 and the serotype 5 positive control. A second Southern
hybridization was performed with the labeled product of cps primers A and. The cps
probe hybridized to a band slightly less than 1.0 kb in serotypes 2 and 4 as well as the
1.1 cps band of serotype 5. The cps probe did not hybridize to any products from the
other respiratory pathogens.
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Figure 2.13a. Agarose gel electrophoresis of the PCR products from bacterial samplesused for Southern hybridizations. Lane 1, serotype 2; lane 2, serotype 4; lane 3, serotype5; lane 4, S. suis; lane 5, P. multocida; lane 6, H. parasuis .
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Figure 2.13b. Southern blot of the PCR products from bacterial samples hybridized witha cpx probe. Lane 1, serotype 2; lane 2, serotype 4; lane 3, serotype 5; lane 4, S. suis; lane5, P. multocida; lane 6, H. parasuis .
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Assay of clinical specimens
The multiplex PCR was used to determine if A. pleuropneumoniae serotypes 1, 2,
and 5 DNA could be detected from lung tissue of infected swine (Fig. 2.14). The assay
was unable to detect any amplified product from serotype 1 samples. Serotype 2 samples
either produced a very faint 0.7 kb band (lane 4 of Figure 14) or no visible product.
Serotype 5 samples gave two distinct products consistent with the 0.7 kb cpx band and
the 1.1 kb cps band. The PCR assay on lung tissue was performed at a MgCl2
concentration of 5 mM to enhance sensitivity.
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Figure 2.14. Agarose gel electrophoresis of PCR products from lung tissue samples takenfrom swine that had been infected with serotypes 1, 2, or 5. Lane 1, 1-kb DNA ladder;lane 2, serotype 1; lane 3, serotype 2, sample 1; lane 4, serotype 2, sample 2; lane 5,serotype 5.
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Attempts to detect A. pleuropneumoniae serotypes 2 and 5 using the multiplex
PCR assay were unsuccessful from nasal swabs. No PCR products were detected from
any of the swabs tested when using the boiling protocol for sample preparation or when
prepared using the Qiagen tissue preparation kit. However, serologic assays (latex
agglutination) and culture did not confirm that any bacteria were present in these
specimens.
To determine if the samples themselves were inhibiting the assay, genomic DNA
from serotypes 2 or 4 were added to the corresponding nasal swab sample. Figure 2.15
shows the bands produced from the Qiagen preparations, boiled preparations, and
samples taken directly from the nasal swabs. PCR amplification of the Qiagen prepared
samples appeared to amplify the genomic DNA without noticeable differences from the
standard conditions of genomic amplification. However, the other 2 samples gave no
products for serotype 2 and reduced amplification for serotype 5.
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Figure 2.15. Agarose gel electrophoresis of the PCR products from genomic DNA of theappropriate serotype added to nasal swab samples. Lane 1, 1-kb DNA ladder; lane 2,serotype 2 Qiagen sample; lane 3, serotype 5 Qiagen sample; lane 4, serotype 2 boiledsample; lane 5, serotype 5 boiled sample; lane 6, serotype 2 sample; lane 7, serotype 5sample.
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DISCUSSION
A. pleuropneumoniae serotypes are distinguished by their unique capsular
polysaccharide (Inzana and Mathison., 1987a). Because cross-reactions often occur from
serotyping with traditional serologic assays, PCR offers a practical alternative that does
not employ antigens or antibodies. This attribute makes capsule genes an ideal target for
typing by PCR. Falla et al. (1994) previously reported using primers from the capsular
DNA region as a reliable method for the typing of H. influenzae by PCR. To date, a
reliable method for serotyping A. pleuropneumoniae by PCR has not yet been
established. Hennessy et al. (1993) have proposed an arbitrarily primed PCR assay for
serotyping A. pleuropneumoniae, but this method can only be used with pure A.
pleuropneumoniae template. The current describes the first use of primers to target
capsular DNA regions to simultaneously identify the species and serotype of A.
pleuropneumoniae.
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
together 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 1 x 102 CFU’s.
The presence of non-specific bands amplified by some serotypes was initially
problematic, particularly from serotypes 2, 3, and 6 while maintaining the cpx band in all
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serotypes. 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 region 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 non-specific bands.
The successful application of the PCR assay to bacterial colonies provided an
effective method of identifying A. pleuropneumoniae. With the exception of the rare
serotype 4, all serotypes amplified a distinct 0.7 kb band. Because both the preparation
and detection of DNA was relatively simple, the entire assay could be performed in under
five hours. Among the common swine respiratory pathogens tested, only S. suis
produced a band of similar size. However, this band was determined to be non-specific as
it did not hybridize with the cpx probe and was distinguished from the A.
pleuropneumoniae cpx band by Southern blotting. In addition, S. suis presents a distinct
clinical picture from that of A. pleuropneumoniae and can, therefore, be distinguished
clinically.
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 the A. pleuropneumoniae serotypes (Ward, 1995). The results of the
Southern blot also indicated that the 0.7 kb band of serotype 2 contained 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
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of non-homology. Of interest, however, was that cps primers A and B did not produce a
1.1 kb band from serotypes other than serotype 5, but the cps probe hybridized to a band
from both serotype 2 and serotype 4. This was surprising since the cps region of A.
pleuropneumoniae is antigenically serotype-specific (Ward, 1995). However, it is
possible that the cps loci may contain some genes that are involved with capsular
synthesis that are conserved by all serotypes. For instance, serotypes 1 and 5 both
conatain N-acetylglucosamine (Perry et al., 1990). Sequencing the cps loci of other
serotypes would reveal any homologous genes responsible for encoding similar proteins.
The potential for using this assay with clinical specimens was also investigated.
A. pleuropneumoniae serotype 5 DNA from lung tissue samples of infected swine was
successfully amplified by PCR. PCR assays have been reported to have greater
sensitivity in detecting bacteria than by the culture of clinical samples (Rodu, 1990).
Therefore, this PCR assay may prove to be faster and a more sensitive method for
identifying and serotyping A. pleuropneumoniae. Although this assay was designed to
specifically detect serotype 5 strains, future development of the protocol may result in
the detection of all serotypes.
Although the identification and serotyping of A. pleuropneumoniae from nasal
swabs of live pigs would be advantageous, attempts to develop a reliable method have
been largely unsuccessful (Kume et al., 1984; Sirois et al., 1991). Isolation of A.
pleuropneumoniae is difficult because of the overgrowth of normal flora. Nasal swab
specimens have also been reported to inhibit PCR (Wadowsky et al., 1994). Evidence of
91
this was shown in Figure 2.15 when genomic DNA added to nasal swab specimens was
not amplified as well as genomic DNA alone.
In conclusion, the PCR assay described was effective in detecting A.
pleuropneumoniae and identifying serotype 5 from whole bacterial cells and infected lung
tissue. This assay can be done both quickly and easily. 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. This work has described a potentially
useful method for detecting and serotyping A. pleuropneumoniae with both high
specificity and sensitivity while avoiding the problems that are associated with serologic
assays.
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