35883

2019
VDIXXX10.1177/1040638719835883Spa type and serovar of E. rhusiopathiaeShimoji et al.

Brief Communication

Journal of Veterinary Diagnostic Investigation

Disassociation of Spa type and serovar of an 2019, Vol. 31(3) 488­–491

© 2019 The Author(s)
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DOI: 10.1177/1040638719835883
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isolated from a diseased pig

Yoshihiro Shimoji,1 Makiko Bito, Kazumasa Shiraiwa,

Yohsuke Ogawa, Sayaka Nishikawa, Masahiro Eguchi

Abstract. The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae is an important component in
protecting pigs against swine erysipelas. The Spa protein has been antigenically divided into 3 types: SpaA, SpaB, and SpaC.
Swine erysipelas vaccines are formulated with strains of serovar 1 and/or 2, both of which are SpaA-possessing serovars. The
association of Spa type with E. rhusiopathiae serovar has been reported, and therefore, the determination of the Spa type and
the serovar of clinical isolates are important to assess vaccine efficacy. An E. rhusiopathiae strain, designated Ireland, was
isolated from a diseased pig and identified as serovar 6 by a conventional agar gel precipitation test. Sequence analysis of
the chromosomal locus presumably defining the serovar antigenicity of E. rhusiopathiae revealed that the gene content and
organization of the chromosomal regions of the Ireland strain were identical to those of the serovar 6 reference strain (Tuzok).
Sequence analysis of the spa gene and dot blots using a SpaA-specific monoclonal antibody confirmed that, unlike the Tuzok
strain possessing SpaB, the Ireland strain expressed SpaA, indicating that the Spa type is not associated with the serovar in
this strain. Thus, further investigation into the association between Spa type and serovar of clinical swine isolates is warranted.

Key words: Erysipelothrix rhusiopathiae; serovar 6; Spa type.

Erysipelothrix rhusiopathiae is a gram-positive intracellular
pathogen that causes a variety of diseases, called erysipelas,
in many animals.20 E. rhusiopathiae is ubiquitous in nature
and has been isolated from a wide range of hosts, including
domesticated and wild animals, humans, poultry, wild birds,
aquatic mammals, and on the exterior surfaces of fish and
crustaceans.20
The genus Erysipelothrix comprises at least 4 species, E.
rhusiopathiae, E. tonsillarum, E. inopinata, and E. larvae,
and 3 unnamed species.17 Historically, Erysipelothrix species
have been differentiated based on their serovar, which is
determined by their cell wall peptidoglycan antigens.20 The 2
major Erysipelothrix species are E. rhusiopathiae (serovars
1a, 1b, 2, 4–6, 8, 9, 11, 12, 15–17, 19, 21, 23, and N; serovar
N lacks serovar-specific antigens) and E. tonsillarum
(serovars 3, 7, 10, 14, 20, 22, 24–26), with the latter species
being commonly found in the tonsils of healthy animals.16
Among the Erysipelothrix serovars, 1a, 1b, and 2 are mainly
associated with disease in pigs, chickens, and humans.3,12,18
In 2018, the chromosomal locus essential for the serovar-
specific antigen and virulence of serovar 1 and 2 strains was
identified.10 This genetic region is required to express and
maintain the molecular integrity of the capsule, which is the
most important virulence factor of E. rhusiopathiae.13 It was
further revealed that the serovar N or untypeable strains,
which are often isolated from diseased pigs, had mutation(s)
in the serovar-defining genetic region and had lost the
serovar-specific antigen, suggesting that most, if not all, of
the serovar N or untypeable strains were originally clinically
important serovars, namely, 1a, 1b, or 2.10,15
Swine erysipelas, which is characterized by acute septice-
mia, subacute urticaria, and chronic endocarditis and polyar-
thritis, causes great economic losses in the swine industry
worldwide.20 Live attenuated vaccines and inactivated bac-
terins are available to control swine erysipelas.20 It has been
reported that the Spa (surface protective antigen) protein,
which is the most important vaccine antigen of E. rhusio-
pathiae,4,7,14 shows genetic and antigenic diversity and is
divided into 3 types, SpaA, SpaB, and SpaC.19 The Spa type
has been shown to be associated with the serovar of the
strain. For example, E. rhusiopathiae serovars 1a, 1b, 2, 5, 8,
9, 12, 15–17, 23, and N possess SpaA, and E. rhusiopathiae
National Institute of Animal Health, National Agriculture and Food
Research Organization (NARO), Tsukuba, Ibaraki, Japan (Shimoji,
Shiraiwa, Ogawa, Nishikawa, Eguchi); Research Institute for Biomedical
Sciences, Tokyo University of Science, Noda, Chiba, Japan (Shimoji);
Animal Quarantine Service Haneda Airport Branch, Ota-Ku, Tokyo, Japan
(Bito).
1
Corresponding author: Yoshihiro Shimoji, National Institute of
Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
shimoji@affrc.go.jp
 Spa type and serovar of E. rhusiopathiae
serovars 4, 6, 11, 19, and 21 possess SpaB.19 Additionally, E.
tonsillarum strains do not possess the Spa protein, and
serovar 18, a yet unnamed species, possesses SpaC.19 It has
been reported that the Spa type is more variable in fish and
cetacean strains.5 It was also observed that a fish isolate of
serovar 8, which was previously reported as a SpaA-possess-
ing serovar, possessed SpaB.11 These findings suggest that
the Spa type and E. rhusiopathiae serovar are not strictly
associated in strains of aquatic origin. Swine erysipelas vac-
cines are produced from E. rhusiopathiae serovar 1 and/or 2
strains, and therefore, the current SpaA-based vaccines do
not confer full protection against the strains possessing a dif-
ferent Spa type.19 In our study reported herein, we identified
a serovar 6 swine isolate possessing the SpaA protein, sug-
gesting that the SpaA type and serovar are also not strictly
associated in certain strains of terrestrial origin.
We used the E. rhusiopathiae reference strains Fujisawa
(serovar 1a) and Tuzok (serovar 6). As well, we used 3 iso-
lates, one named Ireland, obtained from different urticarial
skin lesions of a diseased pig, known to have been given an
erysipelas vaccine. The bacteria were grown at 37°C for 16 h
in brain-heart infusion broth (Becton, Dickinson, Baltimore,
MD) supplemented with 0.1% Tween 80 and 0.3% Tris–HCl
(pH 8.0). Serotyping was carried out by agar gel precipita-
tion tests with autoclaved cell extracts and rabbit antisera
raised against formalin-killed cells of the relevant reference
strain as described previously.10
Genomic DNA of the E. rhusiopathiae strains was pre-
pared as described previously,9 with the following modifica-
tions: after cell lysis using 10% sodium dodecyl sulfate, the
samples were mixed with an equal volume of phenol–chloro-
form solution, centrifuged (21,880 × g, 15 min), and then
DNA was recovered by ethanol precipitation. Based on the
whole genome sequence of the Fujisawa strain, primers
spa-F (5’-ACCGTTTATCGCGAGAGTCA-3’) and spa-R
(5’-CCTCGCATTAAAGATGTTTC-3’) were designed to
amplify a 2.3-kbp DNA fragment including the whole spa
gene. PCR was performed in a 50-µL reaction mixture con-
taining 50 ng of template DNA, 0.3 µM of each primer,
0.4 mM of each dNTP, PCR buffer, and 1.0 U of KOD FX
DNA polymerase (Toyobo, Osaka, Japan). PCR amplifica-
tion was performed (T-100 thermal cycler; Bio-Rad, Hercu-
les, CA) with the following conditions: initial denaturation,
95°C for 2 min; and 3 steps of amplification (35 cycles),
98°C for 10 s, 60°C for 30 s, and 68°C for 2 min.
Sequencing of the chromosomal region defining serovar-
specific antigenicity was performed as described previ-
ously.10 Briefly, the chromosomal region between ERH_1438
and ERH_1451 was divided into 2 genetic regions, and each
region was PCR-amplified with the following primer sets:
seq1F (5’-TGACGATTTCCTGGGCAATTCCCGCG-3’)
and 6R (5’-TTCATGGCATGGTGGTGGCG-3’); 6F
(5’-CAAGGCTTGCGCGTTTGGAC-3’) and seq3R
(5’-TGGCATTTATCCTTAACGGC-3’). PCR was per-
formed as described above with the following conditions:
489
initial denaturation, 94°C for 2 min; and 3 amplification steps
(35 cycles) consisting of 98°C for 10 s, 60°C for 30 s, and
68°C for 6 min. The PCR products were directly sequenced
(BigDye Terminator v.3.1 cycle sequencing kit; ABI PRISM
3130xl genetic analyzer; Applied Biosystems, Foster City,
CA) according to the manufacturer’s instructions. Sequence
identity was analyzed (Genetyx software; Genetyx, Tokyo,
Japan).
A SpaA-specific monoclonal antibody (mAb; laboratory
stock, unpublished) was used to detect SpaA expression in E.
rhusiopathiae strains. Briefly, 3 µL of bacterial culture was
spotted onto a nylon membrane (MagnaGraph; Funakoshi,
Tokyo, Japan) and air-dried. The nylon membrane was
blocked with 1% skim milk in phosphate-buffered saline
containing 0.05% Tween 20 and incubated with the SpaA-
specific mAb. The membrane was further treated with horse-
radish peroxidase conjugated with goat anti-mouse
immunoglobulin antibody (IgG, IgM, and IgA; H+L; Zymed
Laboratories, San Francisco, CA). The blots were developed
with 3,3-diaminobenzidine tetrahydrochloride/hydrogen
peroxide (WAKO, Tokyo, Japan).
We deposited the nucleotide sequences obtained in the
DDBJ/GenBank/EMBL database under accessions
LC425606 (spaA from the Ireland strain), LC425605 (spaB
from the Tuzok strain), LC425604 (serovar-defining region
of the Ireland strain), and LC425603 (serovar-defining
region of the Tuzok strain). The whole genome sequence of
the Fujisawa strain (accession AP012027) was retrieved
from the database.
By conventional agar gel precipitation test, the 3 isolates
from the diseased pig produced a precipitation line with
serovar 6 antiserum (data not shown). One selected strain,
designated Ireland, failed to produce a precipitation line with
the other serovar-specific rabbit antisera raised against for-
malin-killed cells of all 26 Erysipelothrix reference strains
(data not shown).
Sequences of the chromosomal region responsible for
serovar antigenicity of the Fujisawa strain and the corre-
sponding regions of Ireland and Tuzok were compared. In
the serovar 6 Ireland and Tuzok strains, there were 7 com-
mon genes between ERH_1438 and ERH_1451. Among
these common genes, the gene “e” was shared with strain
Fujisawa (Fig. 1). The gene content and organization in this
genomic region was identical between the two serovar 6
strains, with >99.4% amino acid sequence identity between
each gene.
The spa genes of the Ireland strain and the serovar 6
reference strain Tuzok were amplified by PCR, sequenced,
and compared to that of the Fujisawa strain. Sequence
comparison revealed that the Ireland strain exhibited
99.2% and 61.2% amino acid sequence identity with the
Fujisawa and Tuzok strains, respectively. The sequence of
the N-terminal protective domain, which comprises amino
acids 12–195 of the SpaA protein of the Fujisawa strain,14
was 99.5% identical to that of the Ireland strain. The Fuji-
490
Figure 1. Schematic representations of the chromosomal region defining the antigenicity of serovar 1a of Erysipelothrix rhusiopathiae
and corresponding regions in serovar 6 strains. Identical genes are indicated by the same letter. Small arrows indicate the orientation and
corresponding locations of the primers used for sequencing. Putative functions of the genes (a–g) are shown.
sawa and Ireland strains reacted with the SpaA-specific
mAb in the immunoblot (data not shown), indicating that
the Ireland strain expresses the SpaA protein on the cell
surface.
A previous study6 observed incomplete protection of a
commercial inactivated swine vaccine (serovar 2) against
dolphin strains of unknown Spa type. It has also been
reported that protection in mice vaccinated with a bacterin
(serovar 2) was incomplete or nonexistent against non–
spaA-type strains of aquatic origin (herring fish, beluga
whale, and dolphin).5 Thus, disassociation of the Spa type
and serovar is problematic if the Spa type of the vaccine
and field isolates are different. The serovar 6 swine isolate
analyzed in our study possessed the SpaA protein. There-
fore, the pig, which had received an erysipelas vaccine,
should have been protected against this strain. The reason
for the lack of protection is unknown. It is possible that
disease may have been the result of inappropriate vaccine
handling or management and/or the effect of transportation
stress.
Our study showed that disassociation of the Spa type
and serovar of E. rhusiopathiae strains can occur in swine
isolates. A serovar 11 strain isolated from a pig carcass at
a slaughterhouse in the United States that possessed SpaA
has been described.1 Although the most common serovars
in diseased pigs are serovars 1 and 2, other serovars are
frequently isolated from clinically affected pigs.8,18 In
2018, we suggested that serovar switching may occur in
Shimoji et al.
E. rhusiopathiae.10 This hypothesis is based on the fol-
lowing observations: 1) several Erysipelothrix serovars
are shared by different Erysipelothrix species,17 2) some
E. rhusiopathiae strains belonging to phylogenetically
distinct clades displayed the same serovars,2 and 3) the
G+C contents of the E. rhusiopathiae serovar-defining
chromosomal region differ from those in other parts of the
genome,10 suggesting that the serovar-defining region
may consist of laterally transferred sequences. It is possi-
ble that less dominant serovar strains in clinical cases,
including the serovar 6 swine strain analyzed in our study,
may have previously been another serovar and then
acquired foreign genes, resulting in a change of serovar.
Importantly, our data suggest that some strains of clini-
cally dominant serovars 1 and 2 may possess a heterolo-
gous Spa type. To determine if the vaccines provide
complete cross-protection against field isolates, investiga-
tions of the Spa type and serovar of the field strains are
required.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, author-
ship, and/or publication of this article.
 Spa type and serovar of E. rhusiopathiae
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