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Vaccine 22 (2004) 3411–3418

A conserved 37 kDa outer membrane protein of Edwardsiella tarda


is an effective vaccine candidate
Kenji Kawai, Ying Liu, Kouhei Ohnishi, Syun-ichirou Oshima∗
Department of Aquaculture, Fish Disease Laboratory, Faculty of Agriculture, Research Institute of Molecular Genetics,
Kochi University, Nankoku, Kochi 783-8502, Japan
Received 22 October 2003; received in revised form 24 February 2004; accepted 25 February 2004

Available online 17 March 2004

Abstract
An effective vaccine against Edwardsiella tarda has not been reported, one of main reasons is the variation in its serotypes. This study
aimed to develop an effective vaccine against different serotypes of E. tarda. A conserved 37 kDa outer membrane protein (OMP) of E.
tarda was obtained by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). It showed comprising several proteins
by two-dimensional (2D) PAGE analysis and showed a high similarity to glyceraldehyde-3-phosphate dehydrogenase by N-terminal amino
acid sequence analysis. Japanese flounder (Paralichthys olivaceus) vaccinated with the 37 kDa OMP was significantly protected against
the infections by different serotypes of E. tarda. A specific antibody was also detected by using ELISA. This study suggests that the 37 kDa
OMP is an effective potent vaccine candidate against different serotypes of E. tarda.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Edwardsiella tarda; Outer membrane protein (OMP); Vaccine candidate

1. Introduction nisms [17], and it has protective antigenicity, because the


components of the outer membrane are easily recognized
Edwardsiella tarda is an enteric Gram-negative bacterium as foreign substances by immunological defence systems of
of the Enterobacteriaceae, first isolated from pond-cultured hosts. Vaccination trials using the lipopolysaccharide of the
eel by Hoshina in 1962 [1]. It is the causative agent of E. tarda outer membrane [18,19] have shown protective anti-
the systemic disease, edwardsiellosis, in many freshwater genicity, but the trials did not encourage the development of
and marine fish worldwide, including many commercially an effective practical vaccine, because the polysaccharide in
important fish, such as eel [2], flounder, plaice [3], tilapia [4], the lipopolysaccharide is the main antigenic structure that
chinook salmon [5], carp [6], channel catfish [7], mullet [8]. includes O-serotype specificity as for other Enterobacteri-
E. tarda is widely distributed in nature, having been isolated aceae species [17].
from reptiles, birds, mammals [9] including humans [10,11] Recent studies emphasized the role of the outer mem-
and environmental water [12,13], and has been found in 14 brane protein (OMP) of pathogenic bacteria in protective
countries and 39 states of the USA [14]. antigenicity [20–23], which is often related to inducing
This broad host range and geographical distribution sug- neutralization antibodies that are bactericidal [20,21], in-
gest variation in E. tarda. Sixty-one different serotypes of hibiting bacterial colonization in hosts [22] and inducing
E. tarda have been differentiated according to the O-antigen cell-mediated immunity [23]. This study aimed to find a
[15,16], and this variation in serotype is possibly one of common OMP that is conserved in different serotypes of E.
main factors that hinder development of a practical vaccine tarda, and to evaluate if it can be used as an effective vaccine
against E. tarda. Current research is focusing on finding a candidate against infection by different serotypes of E. tarda.
common antigen among different serotypes of E. tarda.
The outer membrane of Gram-negative pathogenic bac-
teria has an important role in the interaction with hosts in 2. Materials and methods
the bacterial pathogenicity during adherence, uptake of nu-
trients from the host, and eliminating host-defence mecha- 2.1. Bacterial strains and growth conditions

∗ Corresponding author. Tel.: +81-88-864-5214; fax: +81-88-864-5214. Six E. tarda strains in this study were isolated from
E-mail address: s-oshima@cc.kochi-u.ac.jp (S.-i. Oshima). four fish species in Japan and China (Table 1). Aeromonas

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2004.02.026
3412 K. Kawai et al. / Vaccine 22 (2004) 3411–3418

Table 1
Strains used in this study
Strains Source

Year Area Fish

1401 1970 Kochi, Japan Seriola quinqueradiata (yellow tail)


M-1 1974 Mie, Japan Evynnis japonica (crimson sea bream)
EF-1 1979 Shizuoka, Japan Anguilla japonica (Japanese eel)
F-1 1979 Taiwan, China A. japonica
HH-1 1988 Hiroshima, Japan Paralichthys olivaceus (Japanese flounder)
V-1 – Japan A. japonica
Aeromonas hydrophila 3500 – Shizuoka, Japan A. japonica

hydrophila 3500 was used as reference. All seven bacterial bit. The rabbit antiserum was separated as described in
strains were pre-cultured for 24 h (strain 1401 was cultured Section 2.2.1.
for 48 h because of slow growth) at 30 ◦ C in brain heart in- Anti rabbit IgG(H + L) goat IgG conjugated with perox-
fusion (BHI, Difco) broth and was inoculated into 1000 ml idase (Wako, Japan) was used as the last conjunct antibody
BHI broth, cultured with shaking at 30 ◦ C for 18 h (36 h for for Western blotting and enzyme-linked immunosorbent as-
strain 1401). The cells were harvested by centrifugation at say (ELISA).
4000 × g for 15 min at 4 ◦ C and were stored at −80 ◦ C until
used. 2.3. O-antigen preparation

2.2. Antisera Broth cultures of the six E. tarda strains were heated at
100 ◦ C for 2 h and then centrifuged at 4000 × g for 10 min.
2.2.1. Rabbit antisera against 37 kDa OMP of EF-1 and The pellets were washed with PBS and the cells suspended
whole cells of six E. tarda strains in PBS were used as O-antigen suspension.
37 kDa OMP prepared from E. tarda EF-1 (see Section
2.5) and formalin-killed cells (FKC) of six E. tarda strains 2.4. Serotype differentiation of E. tarda strains by cross
(EF-1, HH-1, F-1, M-1, V-1 and 1401) were used as immu- absorption and agglutination with O-antigens
nizing antigens. The FKC were prepared by inactivating cul-
tured E. tarda cells with formalin (final concentration was
Rabbit antisera raised against the six E. tarda strains were
0.3%) for 48 h at 15 ◦ C. The FKC were collected by centrifu-
absorbed by mixing with an equal volume of each O-antigen
gation and suspended in phosphate buffered saline (PBS).
suspension of all the six strains. The mixtures were incu-
The 37 kDa OMP (150 ␮g per rabbit) of EF-1 and FKC
bated at 37 ◦ C for 2 h and then centrifuged at 4000 × g. The
preparations (5 × 108 CFU per rabbit) were emulsified with
supernatants were absorbed with the same O-antigens again.
Freund’s incomplete adjuvant and injected subcutaneously
The supernatants finally obtained were used as absorbed an-
into rabbits. All rabbits were boosted 4 weeks later with
tisera. The absorbed antiseria were diluted two-fold serially
the same antigen preparations. Four weeks after the booster
with PBS in a 96-well microtiter plate and then equal vol-
injection, the rabbits were bled and the blood was allowed
umes of O-antigen suspension of the six strains were added
to clot at room temperature for 1 h and then stored at 4 ◦ C
to react with each diluted serum. The plate was incubated
overnight. The separated antisera obtained by centrifugation
at 37 ◦ C for 2 h and then incubated at 4 ◦ C overnight. The
were stored individually in aliquots at −80 ◦ C until used.
agglutination titer of each serum is expressed as the highest
dilution of the serum that gave a positive agglutination.
2.2.2. Flounder antiserum against whole cells of EF-1
The FKC preparation (2 × 108 CFU per fish) of EF-1
was injected intraperitoneally into Japanese flounder (Par- 2.5. Preparation of the total OMPs fraction
alichthys olivaceus; BW, 500 g). Four weeks later, the fish
were bled, the antisera were separated and stored in aliquots OMPs of all seven strains were prepared by using the
at −80 ◦ C until used. sodium dodecyl sulfate (SDS) method of Suzuki et al. [24].

2.2.3. Anti-flounder IgM rabbit serum and goat anti-rabbit 2.6. Preparation of a 37 kDa OMP by SDS-polyacrylamide
IgG gel electrophoresis (PAGE) and Western blotting
An antibody (IgM) fraction was obtained from the anti-
EF-1 flounder serum by gel-filtration and ion-exchange chro- The total OMP of each strain was subjected to
matography by monitoring the agglutinating reaction against sodium dodecyl sulfate-polyacrylamide gel electrophoresis
EF-1 cells. The IgM fraction was mixed with Freund’s in- (SDS-PAGE) which containing 4% stacking gel and 14%
complete adjuvant and injected subcutaneously into a rab- separating gel. After staining with Coomassie brilliant blue
K. Kawai et al. / Vaccine 22 (2004) 3411–3418 3413

(CBB) R-250, the protein at the 37 kDa location was cut out fish were reared in aquaria for 4 weeks, and then five fish
and extracted from the gel by horizontal electrophoresis. in each group were bled and the sera were used for specific
After the SDS-PAGE, the total OMPs of each strain antibody titer detection.
were subjected to Western blotting. The proteins were elec- The remaining fish (99 fishes) in each group were then
trophoretically transferred to nitrocellulose paper (0.45 ␮m divided into three sub-groups for challenge test. The fish in
pore size, Bio-Rad) by using a semi-dry apparatus (Bio- each sub-group were challenged by intraperitoneal injection
Rad) as described by Towbin et al. [25]. After blocking with with one of the three live E. tarda strains EF-1, HH-1 and
1% skim milk at 4 ◦ C overnight, the membrane was reacted V-1 (n = 33 in each), which were cultured as described
with rabbit anti-FKC of EF-1 serum or rabbit anti-37 kDa in Section 2.1. Challenge doses of the three strains were
OMP of EF-1 serum as the first antibody, and then goat anti- 2.0 × 106 CFU per fish for EF-1, 1.1 × 104 CFU per fish for
rabbit IgG was used as the secondary antibody. Konica im- HH-1 and 2.0 × 103 CFU per fish for V-1. Mortality was
munostaining HRP-1000 (Konica) was used for coloration. recorded twice a day for 19 days. The number of injected
E. tarda was counted by using the plate count method with
2.7. Two-dimensional (2D) PAGE and Western blotting BHI agar (Difco) from serial dilutions of the original bac-
terial suspension. The significant difference was calculated
The 37 kDa OMPs prepared from all seven strains were by using the Fisher’s exact test.
analyzed by 2D-PAGE that comprising the first dimensional
and the second dimensional electrophoresis as described by 2.10. ELISA
O’Farrell [26] and Garrels [27]. A 70 mm × 3 mm × 0.5 mm
isoelectric focusing (IEF) dry polyacrylamide gel strip with Sera from the five vaccinated and five control fish were
an immobilized pH gradient at 3–10 (Amersham Pharma- used for ELISA detection. The antiserum was diluted at
cia Biotech) was used in this study. The first dimensional 10 times in a 96-well ELISA plate (50 ␮l per well) and
electrophoresis was subjected with a succession of three immobilized by incubation at 4 ◦ C for 12 h. The plate was
steps: 300 V for 1 min, 3500 V for 1.5 h and 3500 V for washed with PBS containing 0.1% Tween-20, blocked
4 h, by using a Multiphor II electophoresis system (Amer- with 10% skim milk containing 0.02% NaN3 for 12 h at
sham Pharmacia Biotech). The whole procedure of the 4 ◦ C and then washed three times with PBS-Tween. The
first dimensional electrophoresis was done at under 15 ◦ C 37 kDa OMP antigens in PBS prepared from EF-1, HH-
by a water-cycle cooling system. The IEF gel strip was 1 and V-1 (30 ␮g/ml, 50 ␮l per well) were added. After
then undergone the second dimensional electrophoresis by 12 h incubation at 4 ◦ C, the plate was washed three times
SDS-PAGE described in Section 2.6. with PBS-Tween and reacted with rabbit anti-37 kDa OMP
After the 2D-PAGE, the gels were transferred to mem- serum and then reacted with goat anti-rabbit IgG. A color-
branes that were reacted with flounder anti-FKC of EF-1 ing reagent (0.25% o-phenylenediamine, citric acid·EH2 O
serum, anti-flounder IgM rabbit serum and then goat anti- 1.02 g, Na2 HPO4 ·E12H2 O 3.69 g/100 ml) was added at
rabbit IgG. 100 ␮l in each well, and incubated for 20 min at 25 ◦ C. 1 N
H2 SO4 was added to stop the reaction. The plate was then
2.8. N-terminal amino acid sequence read at 492 nm by using a micro-plate reader.
In addition to the one control for the ELISA, other con-
After the SDS-PAGE (as described in Section 2.6) and trols were constructed in which each of the 37 kDa OMP,
2D-PAGE (as described in Section 2.7) electrophoresis, flounder anti-37 kDa OMP antibody and rabbit anti-37 kDa
37 kDa proteins of EF-1 were electrophoretically transferred OMP antibody was absent, the 37 kDa OMP and the floun-
to polyviniliden difluoride (PVDF) membrane (0.2 ␮m pore der antibody were serially diluted. The significant difference
size, Bio-Rad) by using a semi-dry apparatus (Bio-Rad). was calculated by using the Student’s t-test.
The PVDF membranes were stained with CBB R-250 and
then the N-terminal amino acid sequences of the 37 kDa
OMP and its component proteins were analyzed by using 3. Results
an ABI-492 protein sequencer [28].
3.1. 37 kDa OMP is a common protein of E. tarda strains
2.9. Vaccination and challenge
Table 2 shows the results of the cross agglutinating re-
Japanese flounder (n = 208) weighing about 18 g from an action with cross-absorbed antisera. Antisera against EF-1
aquaculture farm in Kochi Prefecture, Japan, were divided and HH-1 were completely absorbed by HH-1 and EF-1 O-
into two groups. Group 1 (n = 104) was intraperitoneally antigen, respectively. All other antisera were not absorbed
immunized with the 37 kDa OMP (30 ␮g per fish) in 100 ␮l completely by heterogeneous O-antigens. EF-1 and HH-1
PBS prepared from EF-1, and group 2 (n = 104) was in- were the same serotype, and other strains belonged to differ-
traperitoneally injected with 100 ␮l PBS as a control. The ent serotypes. In the SDS-PAGE of the total OMPs extracted
3414 K. Kawai et al. / Vaccine 22 (2004) 3411–3418

Table 2
Cross absorption test of rabbit antisera against E. tarda EF-1, HH-1, F-1, M-1, V-1 and 1401
Antiserum Titers before and after absorption Serotype
Absorbed with O-antigens of six strains
EF-1 HH-1 F-1 M-1 V-1 1401
EF-1 25600a,b 25600 1600 1600 1600 3200 1
<100c <100 400 400 800 400
HH-1 25600 25600 800 800 1600 1600 1
<100 <100 200 200 400 200
F-1 1600 1600 25600 3200 3200 3200 2
800 800 <100 800 800 800
M-1 1600 1600 3200 25600 3200 1600 3
400 800 400 <100 400 400
V-1 1600 1600 3200 3200 25600 3200 4
800 1600 1600 800 <100 800
1401 3200 3200 3200 1600 3200 25600 5
1600 1600 1600 800 1600 <100
a Figures indicate the highest dilution times of the serum with positive agglutination.
b Upper figures indicate titers with non-absorbed antisera against O-antigens of six strains.
c Lower figures indicate titers with twice-absorbed antisera against O-antigen of the strain used for antiserum production.

from all strains, many proteins were detected in each strain 3.3. N-terminal amino acid sequence of 37 kDa OMP
(Fig. 1A), however, in the Western blotting assay, only the
OMP at 37 kDa location of all E. tarda strains was strongly The 37 kDa OMP of EF-1 from SDS-PAGE and the OMP
reacted with rabbit antiserum against the FKC of EF-1, as components from 2D-PAGE were subjected to N-terminal
a control, no positive band was detected in A. hydrophila amino acid sequence analysis. All these proteins had the
(Fig. 1B). The same result was obtained by Western blotting same amino acid residues (Table 3), suggesting that they
by using the rabbit antiserum against 37 kDa OMP of EF-1 are operated by the same gene and are modified differ-
(Fig. 1C). The 37 kDa OMP of E. tarda had an epitope com- ently, which lead to different PIs. The 20 N-terminal amino
mon among the E. tarda strains. Aeromonas did not react to acid sequences of 37 kDa OMP showed high homology
the antisera against E. tarda (Fig. 1B, lane 7; Fig. 1C, lane to glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
7), indicating that this common epitope is E. tarda-specific. a historically well-known cytoplasmic enzyme in the gly-
colytic pathway.
3.2. 37 kDa OMP contains several proteins that have
different isoelectric points (PIs) 3.4. Vaccination with 37 kDa OMP induces protection
against E. tarda infection
The 37 kDa OMP of seven strains was further analyzed by
2D-PAGE. Fig. 2 shows that the 37 kDa OMP of each strain Fig. 3 shows the fish survival rate after being challenged
was separated into several proteins that had a wide range of with three different E. tarda strains. On the second and third
isoelectric points from about 4.5 to 8.0. By Western blotting, day of the challenge, fish died that led to a sudden decrease
only two or three proteins at PI 5.9–7.0 showed an obvious in the survival rate at 3–5 days. Fish continued to die un-
positive reaction to flounder antiserum against E. tarda. til the 17th day. At the end of the observation in HH-1 and

Table 3
N-terminal amino acid sequences of 37 kDa OMPs of E. tarda EF-1 and comparison with the known GAPDH of other bacteria
Protein Amino acid sequence
37 kDa OMP TI KVGING F GRIGRIVFRAA
37 kDa OMP spot 1a ** ******* *
37 kDa OMP spot 2a ** ******* *
37 kDa OMP spot 3a ** ******* *
GAPDHb of Escherichia coli ** ******* * **********
GAPDH of Vibrio cholerae ** ******* * ****F*****
GAPDH of Salmonella enterica serovar ** ******* * **********
*: Same amino acid residue as 37 kDa OMP.
a Spots 1, 2, and 3 proteins marked in 2D-PAGE of EF-1 in Fig. 2.
b Glyceraldehyde-3-phosphate dehydrogenase.
K. Kawai et al. / Vaccine 22 (2004) 3411–3418 3415

A specific antibody titer against 37 kDa OMP was de-


tected in all five vaccinated flounder sera by using ELISA
(Fig. 4, P < 0.05). The control test showed decrease of ab-
sorbance according to dilution of the antigen and antibody,
non-specific absorbance were below 0.07 (data not shown).

4. Discussion

E. tarda strains EF-1, HH-1, F-1, M-1, V-1 and 1401,


which were originally isolated from different fishes at dif-
ferent locations, were temporally differentiated into five
serotypes EF-1 (or HH-1), F-1, M-1, V-1 and 1401 by a
cross absorption test of the O-antigen, though the present
serotypes were not identified to those reported by Tamura
et al. [16]. A 37 kDa OMP was commonly detected among
these strains, and was strongly recognized by rabbit anti-
serum raised against whole E. tarda cell. The results suggest
that this protein participates in cell antigenicity. It carries
a common epitope specific to the E. tarda species. Further
analysis of 37 kDa OMP showed that it comprises several
proteins with different PIs. The N-terminal amino acid se-
quence of 37 kDa OMP and its components showed the
same amino acid residues, and 20 residues of 37 kDa OMP
showed a high similarity to GAPDH. The components are
possibly operated by the same gene and were modified dif-
ferently after being synthesized, which led to different PIs
as shown in this study. In the Western blotting assay, these
components seemed to have different contribution levels
qualitatively or quantitatively in the reaction of the flounder
antibody against E. tarda.
Isocyanate formed by decomposition of urea might result
in carbamylation of protein. In this study, precautions were
taken to prevent carbamylation in 2D-PAGE by preparing
fresh urea solutions and doing, the whole procedure of the
first dimensional electrophoresis at under 15 ◦ C. Carbamy-
lation does not explain multiple spots separated by 2D-
PAGE in tests in the absence of generalized modification
Fig. 1. SDS-PAGE and Western blotting profiles of total OMPs of all of 2D-PAGE [26]. Different PIs may be caused by different
strains. (A) SDS-PAGE gel stained with Coomassie brilliant blue R-250. phosphonium modification. However, we believed that gly-
(B) Western blotting with rabbit antiserum against formalin-killed cells cosyl modification leads to different PIs because the 37 kDa
of the EF-1 strain. (C) Western blotting with rabbit antiserum against OMP of E. tarda is a glycoprotein [29]. To clarify these
37 kDa OMP of EF-1. Lanes 1-6, E. tarda strains: 1, EF-1; 2, HH-1; 3,
hypotheseses in the future, the full-length amino acid se-
F-1; 4, M-1; 5, V-1; 6, 1401; 7, A. hydrophila 3500; M, molecular weight
markers. quence of the 37 kDa OMP and its modification analysis are
needed.
Development of a vaccine against E. tarda has been un-
V-1 challenge groups, almost all unvaccinated fish had died dergoing for many years, but a practical and commercially
(12 and 9% survival rates, respectively). However, the fish available vaccine against E. tarda has not been developed for
vaccinated with 37 kDa OMP had a significantly higher sur- an important reason of the variation in serotypes of E. tarda.
vival rate than the unvaccinated fish (Fig. 3B, P < 0.0005; This study showed that the 37 kDa OMP extracted from
C, P < 0.0005). In the groups of EF-1 challenge, relatively strain EF-1 has protective antigenicity in a challenge with
high survival rates were recorded in both vaccinated and E. tarda strains EF-1, HH-1 and V-1, where the serotype
unvaccinated fish because of its week virulence, significant was different between EF-1 (or HH-1) and V-1 strains. The
difference was not obtained (P = 0.7), however, the vacci- results strongly suggest the 37 kDa OMP is an effective vac-
nated fish still kept a higher survival rate than the unvacci- cine candidate against infection caused by different serotype
nated fish (Fig. 3A). strains of E. tarda.
3416 K. Kawai et al. / Vaccine 22 (2004) 3411–3418

Fig. 2. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting of the separated 37 kDa OMP of all strains. The gels were
stained with silver. The OMP were mixed with 2D-PAGE markers (numbers 1–7 in each photograph). Western blotting with flounder antiserum against
formalin-killed cells of EF-1, rabbit antiserum against flounder IgM and goat antiserum against rabbit IgG. IEF, isoelectric focusing electrophoresis. SDS,
SDS-PAGE. pI, isoelectric point.

The specific antibody against 37 kDa OMP after immu- classical glycolytic protein but a multifunctional protein
nization was also detected by ELISA. The result showed [30–32,34,35] including contribution to vaccine develop-
correlating well with the protection of the 37 kDa OMP. The ment. A 37 kDa surface GAPDH of Schistosoma mansoni
ELISA system we used was thought to be responsible for is highly antigenic in schistosome infection in humans
the detection of the specific antibody by comparing it with [35]. Our results also suggest 37 kDa OMP of E. tarda
the absorbances of the controls. can be a candidate vaccine antigen against E. tarda infec-
Surprising was that the 20 N-terminal amino acid residues tion. GAPDH is a common enzyme of organisms, and is
of 37 kDa OMP showed a high similarity to GAPDH that well conserved in many pathogenic bacterial species, such
was historically thought to be a cytoplasmic enzyme in the as Salmonella, Vibrio, Streptococcus, Mycobacterium (the
glycolytic pathway. Recent reports indicated that GAPDH homologies of GAPDH among these bacteria are >50%
binds to a membrane [30–32] and exists on the cell surface when comparing their protein sequences by using the DDBJ
[33,34]. As the surface GAPDH of Streptococcus agalac- Homology Search System). Therefore, if the antigenic pep-
tiae has no N-terminal signal peptide [33], the mechanism tides of GAPDH are conserved by different pathogenic
of attachment of such proteins to the cell membrane is bacterial species, it will be a useful vaccine effective not
unclear. The membrane-bound GAPDH is not a simple only against E. tarda, but against other pathogenic bacterial
K. Kawai et al. / Vaccine 22 (2004) 3411–3418 3417

100 0.6
90 RPS=50 0.5
80 0.4
70
0.3
60
0.2
50
40 0.1

30 0
20 (A) 1 2 3 4 5 1 2 3 4 5
10
0 0.6
(A) 0 1 2 3 4 5 6 7 8 9 10 1112131415161718 19
Absorbed at 492 nm

0.5
0.4
100
Survival rate (%)

90 0.3
80 0.2
70 RPS=65.5 0.1
60
0
50 (B) 1 2 3 4 5 1 2 3 4 5
40
30
0.6
20
0.5
10
0 0.4
(B) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0.3
0.2
100
90 0.1
80 0
70 RPS=70 1 2 3 4 5 1 2 3 4 5
(C)
60 Vaccinated Unvaccinated
50
40
Fig. 4. Specific antibody titer by ELISA in the flounder serum 4 weeks
after vaccination with 37 kDa OMP of EF-1. Reactive antigens were
30
37 kDa OMPs of (A) EF-1, (B) HH-1 and (C) V-1. Five left columns,
20
Serum from fish vaccinated with 37 kDa OMP; Five right columns, Serum
10
from fish unvaccinated with 37 kDa OMP; 1–5, individual fish number.
0 The results were from duplicate experiments. Significant differences from
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 the control groups were P < 0.05 (A, B and C).
(C) Days after challenge
ies against this protein may inhibit GAPDH activity associ-
Fig. 3. Survival rate of flounder after a challenge with E. tarda strains (A)
EF-1 (2.0 × 106 CFU per fish), (B) HH-1 (1.1 × 104 CFU per fish) and
ated with the outer membrane of E. tarda as hypothesized
(C) V-1 (2.0 × 103 CFU per fish). (䊉), Vaccinated with 37 kDa OMP of by Argiro et al. [35]. Studies on parasites have shown that
EF-1; (䊊), Unvaccinated with 37 kDa OMP. Significant differences from drugs inhibiting GAPDH enzyme activity decrease parasite
the control groups were obtained in B (P < 0.0005) and C (P < 0.0005). survival [36,37].
RPS = {1 − (mortality of vaccinated fish/mortality of control fish)} × In conclusion, this study characterized a 37 kDa OMP that
100%.
is conserved in different serotype strains of E. tarda and
showed a similarity to GAPDH. This 37 kDa OMP has a
infections. An antigenic peptide of S. mansoni GAPDH, protective antigenicity against E. tarda infection in Japanese
Sm37-5 is well conserved between schistosome species flounder. For these reasons, the 37 kDa OMP should be se-
but is markedly different from the corresponding human riously considered as a vaccine candidate that would con-
sequence, and is highly antigenic in schistosome infections tribute to the development of an effective vaccine against
in humans [35]. The antigenic peptide of E. tarda GAPDH infection by different serotypes of E. tarda.
and its homology to the corresponding human sequence
must be analyzed to find a vaccine against E. tarda in
humans. Acknowledgements
Research on a protection mechanism in the immunization
with 37 kDa OMP of E. tarda will be an interesting project. The authors are grateful for friendly help made by Mr. M.
If the 37 kDa OMP is a GAPDH, we believe this study is the Watanabe and Mr. K. Kurohara, Kochi Prefectural Fisheries
first to show that GAPDH of bacteria is antigenic. Antibod- Experimental Station, Japan.
3418 K. Kawai et al. / Vaccine 22 (2004) 3411–3418

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