Journal of Clinical Microbiology, December 1999, p. 4093-4098, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Direct and Rapid Detection by PCR of
Erysipelothrix sp. DNAs Prepared from Bacterial
Strains and Animal Tissues
Kouichi
Takeshi,1,*
Souichi
Makino,2
Tetsuya
Ikeda,1
Noriko
Takada,3
Atsushi
Nakashiro,4
Kazunori
Nakanishi,4
Keiji
Oguma,5
Yoshinobu
Katoh,1
Hiroyuki
Sunagawa,1 and
Tohru
Ohyama1
Department of Food Science, Hokkaido
Institute of Public Health, Sapporo 060,1
Department of Veterinary Microbiology, Obihiro University of
Agriculture and Veterinary Medicine, Obihiro
080,2 Kurume Health Center of
Fukuoka Prefectural Government, Kurume 830,3
Meat Inspection Office of Hokkaido Prefectural Government,
Sapporo 060,4 and Department of
Bacteriology, Okayama University Medical School, Okayama
700,5 Japan
Received 19 March 1999/Returned for modification 13 July
1999/Accepted 24 August 1999
 |
ABSTRACT |
A PCR method for rapid screening of Erysipelothrix spp.
in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the
primer set MO101-MO102, which amplifies the 16S rRNA sequences of all
four Erysipelothrix species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes of
Erysipelothrix rhusiopathiae serovar 2 (DNA Data Bank of
Japan accession no. AB019247), E. tonsillarum serovar 7 (accession no. AB019248), E. rhusiopathiae serovar 13 (accession no. AB019249), and E. rhusiopathiae serovar 18 (accession no. AB019250) ranged from 96.0 to 98.4%. The PCR
amplifications were specific and were able to distinguish the DNAs from
each of the four Erysipelothrix species. The results of PCR
tests performed directly with tissue specimens from diseased animals
were compared with the results of cultivation tests, and the PCR tests
were completed within 5 h. The test with this species-specific
system based on PCR amplification with the DNA sequences coding for the
rRNA gene cluster was an accurate, easy-to-read screening method for
rapid diagnosis of Erysipelothrix sp. infection in the slaughterhouse.
| |
INTRODUCTION |
Erysipelothrix
rhusiopathiae is a causative agent of swine erysipelas and human
erysipeloid (14), a disease that occurs in acute and chronic
forms and that causes development of arthritis and endocarditis
(9, 10); this agent causes economic loss and remains an
animal hygiene problem in swine production areas of the world. Pigs in
which the pathogen is detected must be disused in Japan
(13); therefore, methods of rapid diagnosis and appropriate treatment are needed.
Generally, routine bacteriological culture methods require a minimum of
3 days to detect Erysipelothrix cells and about 10 days to
identify their serovars (3, 5).
Recently, several methods that can replace the time-consuming classical
methods for detection of bacteria have been proposed, for example,
DNA-DNA hybridization with bacterium-specific probes and PCR
(8). Makino et al. (7) established a PCR system using highly specific primers, MO101 and MO102, for detection of
Erysipelothrix species. On the basis of studies with DNA-DNA hybridization and with MO101 and MO102, the genus
Erysipelothrix was reported to be divided into four species,
E. rhusiopathiae (serovars 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21, and N), E. tonsillarum (serovars 3, 7, 10, 14, 20, 22, and 23), E. rhusiopathiae serovar 13, and
E. rhusiopathiae serovar 18; and those four species could
not be distinguished from each other by PCR (4, 11, 12).
In this study, the DNA sequences of the rRNA gene clusters in each of
the four Erysipelothrix species were determined, and consequently, we improved the PCR method so that it can be used to specifically identify each of the four Erysipelothrix
species and applied the system to inspections in slaughterhouses.
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MATERIALS AND METHODS |
Bacterial strains and DNA preparation.
All standard
bacterial strains used in this study are listed in Table
1. Lesions caused by
Erysipelothrix infections were tested microbiologically
immediately after the lesions were isolated by staff in the
slaughterhouse. Total DNA from bacterial cells was prepared by using
previously published methods (7). DNA from tissue samples
was prepared for PCR as described previously (6) or by using
a DNA preparation kit for gram-positive bacteria (GenTLE; TaKaRa Ltd.,
Kyoto, Japan).
PCR primers and PCR.
The five universal primer sets used to
amplify the 23S rRNA gene of Erysipelothrix were designed on
the basis of the alignment of the DNA sequences of the 23S rRNA gene in
15 different bacteria (Fig. 1), whose
species and GenBank accession numbers are as follows: Bacillus
subtilis, D11460; Bacillus stearothermophilus, X01387; Clostridium botulinum, M94259; Listeria
monocytogenes, X92951; Staphylococcus aureus, X68425;
Staphylococcus carnosus, X68419; Streptococcus
oralis, X68427; Streptococcus parauberis, S60368; Micrococcus luteus, X06484; Rhodopseudomonas
capsulata; X06485; Pseudomonas marina, X07408;
Escherichia coli, V00331; Pseudomonas aeruginosa,
Y00432; Pseudomonas cepacia, X16368; and Leptospira interrogans, X14249. To amplify the remaining region of the 23S
rRNA gene, four kinds of primer sets were designed (Fig. 1). PCR was
performed in a 50-µl reaction mixture containing 10 ng of template
DNA, each primer at a concentration of 25 mM, 50 mM KCl, 10 mM Tris-HCl
(pH 8.3), 1.5 mM MgCl2, each deoxynucleoside triphosphate
at a concentration of 200 µM, and 1.25 U of AmpliTaq DNA polymerase.
The PCR was carried out for 35 cycles consisting of denaturation for 1 min at 94°C, annealing for 1 min at 58°C, and extension for 1 min
at 72°C by using a Gene-Amp thermal cycler (model 2400; Perkin-Elmer
Co., Foster City, Calif.). The PCR products were subjected to
electrophoresis in 1.5% agarose gels for 30 min. After running for 30 min, the gels were stained with ethidium bromide solution and were then
photographed under transillumination.
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FIG. 1.
Designation of species-specific primer sets for
detection of Erysipelothrix sp. DNA by PCR. (A) DNA
sequences of 23S rRNA genes of gram-positive bacteria and positions of
four primer sets for amplification of the remaining region of the 23S
rRNA gene. (B) Region of DNA sequences determined after amplification
by using the five universal primer sets and the remaining regions to be
amplified by using the four kinds of primer sets. (C) In vitro cloning
by combination of PCR with cassettes and cassette primers.
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|
In vitro cloning by combination of PCR with cassettes and
cassette primers.
The in vitro cloning (1, 2) and
determination of the entire nucleotide sequence of the 23S rRNA genes
of four strains, E. rhusiopathiae ATCC 19414, Pécs 56, and 715 and E. tonsillarum ATCC 43339, were performed with
TaKaRa LA PCR in vitro cloning kits (TaKaRa Ltd.) according to the
manufacturer's instructions. The purified genomic DNAs from each of
the four strains were digested with EcoRI, the
digested fragments were ligated to a synthetic EcoRI
cassette; and then the first PCR was performed with the EcoRI cassette primer (primer C1;
5'-GTACATATTGTCGTTAGAACGCG-3'), known sequence-specific
primers ER2 and ER9 (Table 2), and
cassette-ligated DNA as a template. Nested PCR was then performed with
nested primer sets ER-S1 and ER-S2 (Fig. 1) and with C2
(5'-TAATACGACTCACTATAGGGAGA-3'), and DNA was amplified with
the product of the first PCR as the template.
Direct sequencing of DNA.
Each of the PCR products was
extracted from the agarose gel and was purified with glass powder
(TaKaRa EasyTrap; TaKaRa Ltd.) according to the manufacturer's
instructions. The DNA sequences of both strands of the DNA fragments
were then determined by use of a dye termination kit and a model 377 DNA sequencer (Perkin-Elmer Co.). The primers digested for PCR
amplification were used for the cycle sequencing reaction (Table 2;
Fig. 1). The Gene Works sequence analysis program from Teijin
(Yokohama, Japan) was used to determine the complete sequence and to
compare the sequences.
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RESULTS |
Nucleotide sequence of rRNA gene cluster.
The PCR was
performed with the DNAs from four strains of Erysipelothrix
by using five universal primer sets specific for the 23S rRNA gene
(Fig. 1A; Table 2). The sizes of the PCR products, as determined by
their mobilities on the agarose gel, were 500, 550, 350, 240, and 310 bp, which corresponded to the sizes of the products generated from the
primer sets ER-1F-ER-2R, ER-3F-ER-4R, ER-5F-ER-6R, ER-7F-ER-8R, and
ER-9F-ER-10R, respectively. Both DNA strands of the PCR products were
then directly sequenced with the same 10 primers.
Further PCR was performed with the DNAs from the four strains by using
four primer sets designed from the PCR products sequenced (Fig. 1B;
Table 2), and then these PCR products were directly sequenced with the
same eight primers, resulting in final nucleotide sequences of
approximately 2,600 bp, corresponding to about 75% of the 23S rRNA
gene sequence (Fig. 1B).
To clone the 5' upstream and 3' downstream regions of the 23S rRNA
genes, cassette-mediated PCR cloning was adopted (Fig. 1C). The method
consists of (i) digestion of genomic DNA with EcoRI, (ii)
ligation of cleavage products to double-stranded DNA cassettes
possessing an EcoRI site, and (iii) amplification of cassette-ligated restriction fragments containing known sequences by PCR with specific and cassette primers. The specific primers (primers ER2, ER9, ERS1, and ERS2) were designed to prime synthesis from the known sequences of the DNA, whereas the cassette primers (cassette primers C1 and C2) anneal to one strand of cassette DNA.
The amplified DNA fragments contained a 1,280-bp sequence upstream of
the 23S rRNA genes of the four strains, a 1,400-bp sequence downstream
of the 23S rRNA genes of E. rhusiopathiae ATCC 19414 and
E. tonsillarum ATCC 43339, and a 1,200-bp sequence
downstream of the 23S rRNA genes of the other two strains. Then,
sequencing from the cassette primers and specific primer sets provided
information for designing a new primer for the next "walking" step,
which permitted direct sequencing without the need to synthesize
internal sequencing primers. Finally, by using seven primers (primers
ER-S3, ER-S4, ER-S5, ER-S6, ER-S7, ER-S8, and ER-S9), the entire
sequences of approximately 4.5-kb genomic segments containing the rRNA
genes of the four strains were determined.
The resulting nucleotide sequences of the four strains included the
complete regions coding for 23S rRNA and 5S rRNA genes and major parts
of the 16S rRNA gene and the 3'-end noncoding region (Fig.
2). The coding regions were deduced on
the basis of alignments with other bacterial species whose sequences
had high degrees of homology to those of the four strains examined. A
comparative alignment of the sequence established in this study with the sequences of the four strains revealed that E. rhusiopathiae ATCC 19414 exhibited 98.2% (4,867 bp) identity with
E. tonsillarum ATCC 43339, 96.0% (4,865 bp) identity with
E. rhusiopathiae Pécs 56, and 98.4% (4,543 bp)
identity with E. rhusiopathiae 715 (4,566 bp), respectively.
It is apparent that the rRNA genes of the four strains exhibit a high
degree of identity with each other.
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FIG. 2.
Putative rRNA gene cluster localization and location of
species-specific primers for each species. Primer sets specific for
detection of E. rhusiopathiae, E. tonsillarum,
and E. rhusiopathiae Pécs 56 were designed with a
sequence from within the noncoding region near nucleotides 4110 and
4500; in contrast, the specific primer set for detection of strain 715 was designed with a sequence from within the 23S rRNA gene.
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|
Design of primers specific for each species.
Specific primer
sets for detection of each species of Erysipelothrix were
designed on the basis of the rRNA sequences determined, as shown in
Table 2 and Fig. 2. Specific primer sets for detection of E. rhusiopathiae, E. tonsillarum, and E. rhusiopathiae Pécs 56 were designed from sequences within
the noncoding region near nucleotides 4110 and 4500 (Table 2), because
the nucleotide sequences of those strains exhibited weak homology with
each other. In contrast, a specific primer set for detection of
E. rhusiopathiae 715 was designed from a sequence within the
23S rRNA gene (Fig. 2).
PCR was performed with the DNAs from different strains by using the
four primer sets. PCR products were obtained only when the DNAs
extracted from the isolates and the matching specific primer sets were
used, as shown in Fig. 3. The sizes of
the amplified products, based on the mobilities in agarose gels of
E. rhusiopathiae ATCC 19414, E. tonsillarum ATCC
43339, E. rhusiopathiae Pécs 56, and E. rhusiopathiae 715, were 399, 384, 288, and 387 bp, respectively, which correspond to the sizes predicted from the nucleotide sequences.
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FIG. 3.
Amplification of rRNA gene fragments by PCR. PCR was
carried out with DNAs extracted from four species and corresponding
species-specific primer sets. Lane M, DNA size marker (100-bp DNA
ladder); lane 1, strain ATCC 19414 DNA amplified with ER1-F-ER1-R (399 bp) but negative for amplification with ER1-F-ER1-R from ATCC 13339 (lane 2), Pécs 56 (lane 3), and 715 (lane 4) DNAs; lane 6, strain
ATCC 43339 DNA amplified with ER2-F-ER2-R (384 bp) but negative for
amplification with ER2-F-ER2-R from ATCC 19414 (lane 5), Pécs 56 (lane 7), and 715 (lane 8) DNAs; lane 11, strain Pécs 56 DNA
amplified with ER3-F-ER3-R (289 bp) but negative for amplification
with ER3-F-ER3-R from ATCC 19414 (lane 9), ATCC 43339 (lane 10), and
715 (lane 12) DNAs; lane 16, strain 715 DNA amplified with ER4-F-ERF-R
(387 bp) but negative for amplification with ER4-F-ERF-R from ATCC
19414 (lane 13), ATCC 43339 (lane 14), and Pécs 56 (lane 15)
DNAs.
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|
PCR was then performed with Erysipelothrix strains,
including strains of 26 serotypes which were classified into one of the four species. PCR products were obtained when DNA sequences from the
particular strains matched those of the species-specific primer sets,
as indicated in Table 3. The results
demonstrated that the four species of Erysipelothrix could
be distinguished from each other by PCR with the species-specific
primer sets with specificity (Table 1).
Furthermore, PCR was performed with 17 swine specimens obtained at the
slaughterhouse from animals with endocarditis, arthritis, and
dermatitis (Fig. 4). Amplified DNA
products were obtained with the E. rhusiopathiae-specific
primer set from nine of the specimens, which were confirmed to be
infected with E. rhusiopathiae by the cultivation tests. No
PCR product was obtained with any of the four kinds of primer sets from
eight of the specimens from which Actinomyces pyogenes,
Staphylococcus aureus, and group C Streptococcus
were isolated in cultivation tests. The sequences of the 399-bp
products amplified from E. rhusiopathiae isolates from
slaughterhouses were determined to be identical to the sequences from
E. rhusiopathiae ATCC 19414 determined in this study (data not shown).
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FIG. 4.
Specific amplification of E. rhusiopathiae
DNA by PCR from clinical isolates. Template DNAs were prepared from the
diseased tissues as described in the text, and then PCR was carried out
with one genus-specific primer set and four species-specific primer
sets. Lane M, DNA size marker (100-bp DNA ladder); lanes 1 and 2, 407- and 399-bp DNA fragments amplified from sample 1 (from an animal with
dermatitis), respectively; lanes 3 to 5, negative for amplification
with ER2-F-ER2-R (lane 3), ER3-F-ER3-R (lane 4), and ER4-F-ER4-R
(lane 5); lanes 6 and 7, 407- and 399-bp DNA fragments amplified from
sample 2 (from an animal with endocarditis), respectively; lanes 8 to
10, negative for amplification with ER2-F-ER2-R (lane 8), ER3-F-ER3-R
(lane 9), and ER4-F-ER4-R (lane 10); lanes 11 to 15, negative for
amplification with one genus-specific and four species-specific primer
sets and DNA from sample 3 (from an animal with endocarditis), which
was negative for Erysipelothrix spp. in cultivation tests.
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DISCUSSION |
It has been thought that the genus Erysipelothrix
consists of a single species, E. rhusiopathiae. However,
Takahashi et al. (12) reported that the genus
Erysipelothrix contains two main species, E. rhusiopathiae and E. tonsillarum, and two other species represented by E. rhusiopathiae serovars 13 and 18, respectively, on the basis of DNA-DNA hybridization experiments. As to
the detection of Erysipelothrix species, Makino et al.
(7) reported that the PCR system with primer set MO101-MO102
corresponding to E. rhusiopathiae DNA coding for 16S rRNA
could produce amplified products from the DNA samples from 35 Erysipelothrix strains, including those of the 4 species
used in this study. This is compatible with the fact that the
homologies of the DNA sequences of the 16S rRNAs of the four
species ranged from 98.5 to 98.8%. Accordingly, the four species of
Erysipelothrix are indistinguishable from each other with
the PCR system based on the MO101-MO102 primer set. Generally, the
results from pathogenicity tests (5) have confirmed that
E. rhusiopathiae strains are pathogenic for swine but
E. tonsillarum strains are not. When
Erysipelothrix is suspected in joints and/or other organs of
swine on the basis of a clinical diagnosis, it is important for
detection purposes to distinguish among the four species of
Erysipelothrix as well as other causative bacterial pathogens.
In this study, we have established a method for the direct detection of
Erysipelothrix sp. DNA from tissue samples from
diseased animals without cultivation by PCR using oligonucleotide
primers complementary to the DNA sequences of the rRNA gene cluster
(including 16S, 23S, and 5S rRNAs and the noncoding region) of the four
species by cassette cloning and primer-walking methods. This PCR system is able to distinguish the four species of Erysipelothrix
from each other, giving different sizes of PCR products corresponding to each species.
The usefulness of this PCR method of diagnosis was confirmed with
tissue specimens derived from animals with endocarditis, arthritis, and
dermatitis which were suspected to be caused by Erysipelothrix. The rRNA gene fragments were amplified by
PCR from diseased tissue specimens, and the species of
Erysipelothrix identified on the basis of the fragments
amplified with the species-specific primer sets was identical to that
identified by the cultivation tests. From these data, it is concluded
that the PCR system developed in this study is rapid, specific, and
reliable for identification of the 23S rRNA genes of the four
Erysipelothrix species and that it can be used for the rapid
detection of Erysipelothrix in tissues from diseased swine
obtained in slaughterhouses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science, Hokkaido Institute of Public Health, Kita 19, Nishi 12, Kita-ku, Sapporo, Hokkaido 060-0819, Japan. Phone: 001-81-11-747-2211. Fax: 001-81-11-736-9476. E-mail:
takeshi{at}iph.pref.hokkaido.jp.
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Journal of Clinical Microbiology, December 1999, p. 4093-4098, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
