Fish & Shellfish Immunology 36 (2014) 270e275

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Fish & Shellfish Immunology

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Full length article

Vaccination of channel catfish with extracellular products of

Aeromonas hydrophila provides protection against infection by
the pathogen
Dunhua Zhang*, Julia W. Pridgeon, Phillip H. Klesius
Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, USA

a r t i c l e i n f o a b s t r a c t

Article history: Aeromonas hydrophila, a Gram-negative bacterium, is one of the economically-important pathogens in
Received 19 September 2013 modern aquaculture. Among various traits, extracellular products (ECP) secreted by the bacterium are
Received in revised form considered to be essential factors for virulence. Whether vaccination with the ECP could produce im-
19 November 2013
mune protection in catfish against the pathogen was determined in this study. The results showed that
Accepted 22 November 2013
Available online 8 December 2013
fish vaccinated with ECP had 100% of relative percent survival (RPS) when challenged with the pathogen
two weeks post vaccination. The anti-ECP serum from vaccinated fish could aggregate cells of homo-
geneous bacteria as well as other virulent strains (isolates) of A. hydrophila but not an A. veronii isolate
Keywords:
Aeromonas hydrophila
and a low virulent field isolate. The agglutination titers increased from two weeks to four weeks post
Extracellular products immunization and sustained a high level at week seven when the RPS remained at 100%. The anti-ECP
Vaccination serum could also provide naïve fish with immediate protection against A. hydrophila as evidenced by
Agglutination passive immunization. Immunoblotting analysis showed that the anti-ECP serum contained antibodies
Passive immunization that bound to specific targets, including protein and lipopolysaccharide-like molecules, in the ECP. Mass
spectrometric analysis identified following putative proteins that may serve as important immunogens:
chitinase, chitodextrinase, outer membrane protein85, putative metalloprotease, extracellular lipase,
hemolysin and elastase. Findings revealed in this study suggest that, while ECP prepared in a conven-
tional and convenient way could be a vaccine candidate, further characterization of antibody-mediated
targets in the ECP would uncover quintessential antigens for the future development of highly efficacious
vaccines.
Published by Elsevier Ltd.

1. Introduction
Aeromonas hydrophila, a gram-negative bacterium, had long
been implicated as the etiological agent in a wide range of
fresh water fish diseases worldwide [1] and was lately culpable for
some disease outbreaks in aquaculture, causing severe economic
losses [2e4].
To control this emerging disease, research has been carried out
to develop vaccines against the pathogen as a sustainable preven-
tion method. Attempts have been made to use formalin- or heat-
killed whole cells of A. hydrophila (baterins) for Nile tilapia [5],
Indian major carp [6e8], and rainbow trout [9]. Live and attenuated
A. hydrophila cells generated via gene inactivation, transposon
insertion or antibiotic resistance were assessed for protection
* Corresponding author. Tel.: þ1 (334) 887 3741; fax: þ1 (334) 887 2983.
E-mail addresses: dunhua.zhang@ars.usda.gov, dhzhang8@gmail.com
(D. Zhang).
efficacy in rainbow trout [10], swordtail fish [11], and channel
catfish [12], respectively. Alternatively, functional or structural
proteins of A. hydrophila in recombinant form were evaluated for
vaccine candidates, such as adhesin for blue gourami [13], outer
membrane protein for Indian major carp [14] and European eel [15],
S-layer protein for common carp [16], and flagellar protein for
channel catfish [17]. Additionally, complex components from
A. hydrophila cells or cultures were used to vaccinated fish, such as
crude lipopolysaccharide [18] or biofilm [19] for common carp,
whole cell lysate [20] for rainbow trout and formalin-inactivated
extracellular products [7] for Indian major carp. Various degrees
of immune protection against A. hydrophila were achieved among
these studies though variations of performance remained to be
addressed; search for critical immunogens to improve vaccination
efficiency is still needed.
Strains of A. hydrophila have been shown to be heterogeneous
biochemically and serologically [21] and virulence determinants
may also vary among clinical and environmental strains [22].
Additionally, ECP secreted by the bacterium was considered to be

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http://dx.doi.org/10.1016/j.fsi.2013.11.015
 D. Zhang et al. / Fish & Shellfish Immunology 36 (2014) 270e275
associated with not only pathogenicity but also environmental
adaptability [23]. Toxicity of A. hydrophila ECP has been reported to
be lethal in tilapia [24], rohu [25], and catfish [26]. Therefore, to
generate a wide spectrum of protective immune response against
various heterogeneous isolates, use of extracellular products (ECP)
of A. hydrophila would be a better choice among different immu-
nogens studied.
The aim of this study was to assess the protective efficacy of
A. hydrophila ECP-mediated vaccination in channel catfish and
evaluate activities of anti-ECP serum in agglutination, passive im-
munization and immunoblotting.
2. Materials and methods
2.1. Bacterial culture and ECP preparation
A virulent strain of A. hydrophila, ML-10-51K [26,27], was used in
this study. The bacterium was regularly cultured from glycerol stock
at 80  C and routinely maintained in tryptic soy agar (TSA) at 24e
26  C. To prepare ECP, the bacterium was inoculated in liquid me-
dium, tryptic soy broth (TSB), and shake-incubated at 26  C for 16e
18 h when optical density at 600 nm (OD600) reached to about 5e6.
The bacterial suspension was centrifuged at 5000 rpm for 30 min
and the supernatant recovered was filtered through a 0.22 mm PES
membrane (Millipore, Billerica, MA, USA). The filtered supernatant
was, then, concentrated to approximately 1000 times using 20K-
MWCO centrifugal concentrator (Pierce/Thermo Scientific, Rock-
ford, IL, USA). The concentrated supernatant was referred to ECP
hereafter and kept at 4  C (for less one week before use) or 80  C
(for more than one week storage). For control, uncultured TSB was
processed in the same way and had the similar concentration factor.
Protein quantities in the concentrated ECP and TSB were estimated
with Bradford Reagent (SigmaeAldrich, St. Louis, MO, USA) using
bovine serum albumin as the standard protein.
2.2. Immunization, challenge of pathogen and serum preparation
Catfish fingerlings, weighing 10.8  1.5 g, were maintained in
aquaria tanks (57 L water) with 20 fish per tank. Protocols estab-
lished for fish rearing and treatment were followed as described
previously [28]. Immunogens were prepared by emulsification of
equal volumes of Freund’s complete adjuvant (Sigma) and ECP
solution (16 mg ml1 of total protein) in sterile phosphate-buffer
saline (PBS; Sigma). For control, the concentrated TSB with the
same volume of the ECP applied was diluted in sterile PBS and
emulsified in the same way. One hundred twenty fish were vacci-
nated by intraperitoneal (IP) injection with 100 ml of the emulsified
ECP and other 120 were sham-immunized with the same amount
of emulsified TSB. Two weeks after immunization, 30 fish were
randomly chosen from the ECP-treated pool, IP-injected with 100 ml
of fresh-cultured A. hydrophila in TSB [with 2  108 cfu (colony
forming units) ml1; the same bacterial concentration was used
when mentioned challenge of pathogen below], and equally
divided into 3 tanks. The same challenge of pathogen was carried
out at weeks 4 and 7 with 3  10 fish from the ECP-treated pool
each time. For sham-immunized fish, the exact same challenging
procedure was followed. Mortality of fish was recorded daily for 1
week for each individual challenge. The protective efficacy of ECP
vaccination was expressed as relative percent survival (RPS) of fish,
comparing with the sham-immunized, after challenge of pathogen
{RPS ¼ [1  (% mortality in immunized group/% mortality in control
group)]  100}. Additionally, 5 fish were randomly chosen and
subjected to blood collection from both ECP- and sham-immunized
fish (all unchallenged) weekly (from 2 weeks to 7 weeks post im-
munization). Blood samples from each set of 5 fish were pooled and
271
allowed to clot at 4  C for 2 h. Sera were collected after centrifu-
gation at 4  C (7000 rpm) for 20 min and stored at 80  C until use.
All of above experiments were repeated once and partial of them
were twice.
To assess the effect of repeated challenges of pathogen on fish
survival and serum response, a group of the same size fish (30
fish  2 replicates), which had RPS equal to 100% after challenge of
pathogen two weeks post immunization with ECP, was subjected to
2 more challenges at two-week intervals. In the meanwhile, five
fish from each replicate were sampled for serum collection before
challenge and one week after each challenge.
2.3. Passive immunization and challenge of pathogen
The ECP-immunized sera with high bacterial cell agglutination
titers (see below) and those of the sham-immunized were sepa-
rately pooled and used for passive immunization as described
previously [29]. Briefly, 108 fish (12.5  1.7 g) were equally divided
into 9 aquaria tanks (57 L water) and subdivided into 3 groups (36
fish in 3 replicate tanks per group). Fish in each of the groups were
IP-injected 100 ml of either ECP-immunized serum, heat-treated
ECP-immunized serum (incubated at 56  C for 1 h) or sham-
immunized serum. After taking 2 fish from each tank for serum
collection 2 days post the passive immunization, all fish were
challenged by IP-injection of 100 ml of A. hydrophila cell suspension
in TSB. Mortality was recorded daily for 2 weeks. This experiment
was repeated once.
2.4. Bacterial cell agglutination and titration
Serum collected from each set sample was serially diluted in PBS
in wells of a 96-well microtiter plate. Cells of A. hydrophila cultured
on TSA for 24e48 h were suspended in PBS. The cell suspension was
centrifuged at RT (5000 rpm) for 5 min. The cell pellet was re-
suspended in PBS and precipitated by centrifugation for two
times. After re-suspended in PBS, bacterial cells were adjusted to
7.5  0.2  108 cells ml1 in PBS by reading at OD600. Aliquots of
50 ml of this bacterial cell suspension were, then, mixed into indi-
vidual wells containing 50 ml of undiluted or diluted serum. After
sealed with a 3 M Scotch Pad sealer, the microtiter plate was kept
still at RT for at least 4 h. The titer of a serum was determined by the
reciprocal of the highest dilution factor that resulted in visible
clumping of bacterial cells. For each serum sample, the agglutina-
tion test was repeated twice.
With ML-10-51K, used in this study, as a reference strain of
A. hydrophila, a serum sample with known agglutination titer was
also used to test relative titration for other five strains (isolates) of
Aeromonas species which were characterized or partially charac-
terized (Table 3).
2.5. SDS-PAGE, immunoblotting and mass spectrometric analysis
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) was conducted to separate samples of ECP and
concentrated TSB for protein profiling and western analysis, using
NuPAGE 4e12% Bis-Tris precast gel, MES/SDS running buffer and
associated protocols (Invitrogen, Carlsbad, CA, USA). The SeeBlue
Plus2 Prestained Standard (Invitrogen) was used as reference mo-
lecular weight marker. After electrophoresis, the gel was either
stained using SimpleBlue Safestain (Invitrogen) or subjected to
western blot using Novex Mini-cell apparatus and associated pro-
tocols (Invitrogen). For western analysis, the blot was first probed
using either the ECP-immunized serum or the sham (TSB)-immu-
nized serum and, then, incubated with peroxidase-conjugated goat
anti-catfish-Ig M IgG (produced by Rockland, Gilbertsville, PA, USA),
272 D. Zhang et al. / Fish & Shellfish Immunology 36 (2014) 270e275

followed by colorimetrical development using 4-chloro-1-

Agglutination titer (log2 scale)

128
7
naphthol/H2O2 (Bio-RAD, Hercules, CA, USA). Additionally, protein
bands in gel after SDS-PAGE, approximately corresponding to pro- 64
6
teins revealed by immunoblotting, were excised and subjected to
LC-MS/MS (liquid chromatography with tandem mass spectrom- 32
5
etry) analysis and identification (Scafford program, Proteome
16
4
Software, Portland, OR, USA).
38
2.6. Statistic analysis
24
Differences of mortality between different vaccination treat-
12
ments were analyzed using one-way analysis of variance with
Turkey’s method. 0
0 21 32 43 54 65 76 7
3. Results Weeks post immunization
Fig. 1. Agglutination titer changes following challenge of A. hydrophila. Arrows above
3.1. Relative percent survival of fish immunized with ECP
the line indicated challenge times (The RPS was 100% in the first challenge and no
mortality occurred in subsequent two challenges); diamond dots indicated serum
Two weeks post immunization with ECP, all experimental fish collecting times. Numbers of fish subject to serum collection were shown in paren-
survived the challenge of pathogen at dosage of 2  107 cfu fish1, at theses and numbers of fish subject to challenge were in square brackets.
which all sham-immunized fish died within 4e5 h (Table 1). No
mortality occurred for those survived the challenge in following
3.3. Agglutination titer changes following challenge of
two weeks and thereafter (at least seven weeks later as observed).
A. hydrophila
Likewise, those challenged 4 and 7 weeks post immunization had
RPS equal to 100% (Table 1).
ECP-immunized and challenged fish, with RPS equal to 100%
two weeks post immunization, were subjected to two more chal-
3.2. Agglutination of A. hydrophila cells and the serum titration
lenges of pathogen; no more mortality was observed for these
repeated challenged fish; and serum agglutination titers changed
All serum samples collected from ECP-immunized fish were able
differentially following each challenge (Fig. 1). One week after the
to cause aggregates of A. hydrophila cells, resulting in visible
first challenge, the titer increased from 8 to 32; following the sec-
clumping (i. e. agglutination), while no agglutination (<2) was
ond challenge, the titer reached to 64; and the same level of titer
observed for the sham-immunized serum (Supplemental Fig. 1). By
remained after the third challenge.
serial dilution of individual anti-ECP sera, the maximal dilution that
gave agglutination (i. e. the titer) was different among the samples
3.4. Passive immunization and challenge of pathogen
(the far right column of Table 1). The serum two weeks post im-
munization had a titer of 8, at which the RPS of experimental fish
The serum used for passive immunization had an agglutination
was equal to 100%. The agglutination titers increased 8 fold (i. e. 64)
titer of 64 and, after heat treatment, the titer decreased to 8. Two days
at week 3 and reached to the highest (i. e. 128) at week 4. After that,
post passive immunization, serum titers were 4, 2 and <2 for fish
the titers decreased steadily in the following three weeks at levels
received untreated anti-ECP serum, heat-treated anti-ECP serum and
of 64, 64, and 32, respectively.
sham-immunized serum, respectively. The cumulative percentage of
mortality for the three corresponding treatments were 15.5  3.9
Table 1 (mean  SD), 37.8  3.9 and 100, respectively, after challenge of
Relative percent survival (RPS) of catfish immunized with ECP and serum aggluti- pathogen (Table 2). In terms of RPS, the untreated anti-ECP serum
nation titer. could provide about 85% protection against infection while the heated
Immunizationa Fish challengedb Mortality (%)c RPSd Agglutination one did about 62%. And, notably, all fish received control serum died
titere within 4 h after challenge of pathogen while mortality of those
ECP @wk2 (3  10) ^3 0 100 8 received anti-ECP serum was delayed for 1e3 days.
Sham @wk2 (3  10) ^3 100 e <2
ECP @wk3 64 3.5. Cross agglutination of different strains (isolates) of
Sham @wk3 <2
A. hydrophila and related species
ECP @wk4 (3  10) ^2 0 100 128
Sham @wk4 (3  10) ^2 100 e <2
ECP @wk5 64 Serum samples generated from fish immunized with ECP of
Sham @wk5 <2 strain ML-10-51K (used in this study) were able to cross-
ECP @wk6 64 agglutinate cells of other 3 strains (isolates) of A. hydrophila but
Sham @wk6 <2
ECP @wk7 (3  10) ^2 0 100 32
not those of 2 related species (Table 3). The agglutination titers for
Sham @wk7 (3  10) ^2 100 e <2 ML-09-73, ML-10-208K and ML-10-20S were the same as that of
a ML-10-51K. The ML-09-73 and the ML-10-208K had been positively
Indicating that challenge of A. hydrophila or serum collection was carried out
two (2) to seven (7) weeks (wk) post immunization (@wk2e@wk7). identified as virulent strains of A. hydrophila while the ML-10-205
b
Ten experimental fish per group with three replicates (3  10) were challenged was also virulent but partially characterized to be an isolate of
per trial and, when the test was repeated two or three times, a mark “^2” or “^3” was A. hydrophila (unpublished data). For those that could not be
followed. agglutinated, the ALG-10-89 was identified as A. veronii, a species
c
Overall mortality (percentage) from two or three repeats.
d
Overall RPS ¼ [1  (% mortality in immunized group/% mortality in control
closely related to A. hydrophila, based on partial genomic DNA se-
group)]  100. quences (unpublished data) while the AL-98-C1B was a very low
e
Sera were collected and pooled from 5 unchallenged fish. virulent isolate of A. hydrophila.
 D. Zhang et al. / Fish & Shellfish Immunology 36 (2014) 270e275 273

Table 2
Passive immunization of catfish with anti-ECP serum.a

Fish treatment Cumulative percentage of mortality (mean  SD) RPS Agglutination titer before challenge

4h 24 h 2 day 3 days 14 daysc

Anti-ECP serum 0 0 8.9  3.9 15.5  3.9 15.5  3.9 84.5 4

Heated anti-ECP serumb 4.5  3.9 17.8  3.9 37.8  3.9 37.8  3.9 37.8  3.9 62.2 2
Shamed serum 100 100 100 100 100 e <2
a
Mixed anti-ECP serum with agglutination titer of 64 was IP-injected to catfish fingerlings at volume of 100 ml fish1. Control fish received the same volume of serum from
the sham-immunized fish. At 48 h post passive immunization, fish were challenged with 100 ml of A. hydrophila cell suspension in TSB at dosage of 2  107 cfu fish1.
b
The serum was heated at 56  C for 1 h and cooled to room temperature before use.
c
The overall mortality was significantly different (p < 0.0001) between treatments.

3.6. SDS-PAGE, immunoblotting and mass spectrometry
Profiles of samples of ECP and concentrated TSB used for im-
munization of fish were shown in Fig. 2A after SDS-PAGE and
staining. The ECP had complex stainable bands ranging mostly from
3 to 98 kDa (Lane 1 of Fig. 2A) while no distinct band was seen in
concentrated TSB (Lane 2 of Fig. 2A). The same samples, upon
blotted to PVDF membrane, showed different profiles recognized
by fish serum. There was no visible band shown for both ECP and
TSB samples which were probed by the sham-immunized fish
serum (1 antibody source; Fig. 2B). A cluster of prominent bands
mostly ranging from 38 to 98 kDa was seen in ECP but not TSB
sample when the blot was probed by ECP-immunized fish serum
(Fig. 2C). Among these visible bands, some were intensely stained
while others were in light shade. While the lightly stained bands
were apparently arranged in an orderly, ladder-like pattern with
resemblance to lipopolysaccharide profile (Supplemental Fig. 2),
the intensely-stained bands (indicated by arrows in Fig. 2C) were
most likely protein molecules. Mass spectrometric analyses of the
corresponding bands excised from the protein gel (Fig. 2A) revealed
that they were chitinase (accession #A0KGX3) & chitodextrinase
(#A0KNS8); outer membrane protein85 (#A0KHH1) & putative
metalloprotease (#A0KGX4); extracellular lipase (#A5A359);
and hemolysin (#D2XPP9) & elastase (#A0KGK2), respectively
(arrow-pointed bands from top to bottom; protein identification
probability ¼ 100%).
4. Discussion
A. hydrophila secreted an array of extracellular products [23,30],
including not only well-known virulence factors (such as metal-
loproteases, glycerophospholipid-cholesterol acyltransferase, he-
molysin, aerolysin, and lipases) but also proteins for digestion (such
as amylases and chitinases) and host cell attachment (such as S-
layer, and flagella), which are considered to contribute to their wide
distribution, great adaptability and pathogenicity. The ECP pre-
pared in this study showed a complex profile (Fig. 2A) and may
represent majority of products the bacterium secreted in milieu.
Vaccination of fish with this ECP preparation apparently provided
immune protection against fatal challenge of A. hydrophila.
The success of 100% of RPS of vaccinated fish may be attributed
to the representation of antigens in the ECP preparation and,
Table 3
Cross agglutination of Aeromonas species by anti-ECP serum.
Strain (isolate) Agglutination titer Identification Reference
ML-10-51K 64 A. hydrophila This study
ML-09-73 64 A. hydrophila [3]
ML-10-208K 64 A. hydrophila [26]
ML-10-205 64 A. hydrophila Unpublished
ALG-10-089 <2 A. veronii Unpublished
AL-98-C1B <2 Aeromonas sp. [3]
therefore, the development of critical humoral immune responses
against the establishment of pathogenesis. This is evidenced by the
bacterial cell agglutination activity of the anti-ECP serum from
vaccinated fish. By a rough analogical calculation, about 12 ml of
serum in fish two weeks post immunization could aggregate about
3.5  107 A. hydrophila cells, which are 1.75  more than the
challenge dosage (2  107 cfu) at which all control fish were killed.
The agglutination titer could actually increase 16 times more from
week 2 to week 4 post immunization and sustained relatively high
level at week 7 (Table 1). Similar strong humoral immune response
was also reported in Indian major carp vaccinated with formalin-
inactivated ECP and the high RPS (80e90%), similar to those ob-
tained from vaccination with heat-killed bacterins, was correlated
with high antibody titers [7]. In addition, the agglutination titer
could keep increasing and/or remain high level following challenge
of A. hydrophila (Fig. 1), suggesting that a one-time vaccination
would protect fish from re-infestation of the pathogen although the
protection period beyond 7 weeks has yet to be determined.
The humoral antibody-mediated immunity was manifested by
the passive immunization, which provided fish with passively-
transferred antibody (immunity) against A. hydrophila (Table 2).
The relatively lower RPS (about 85%) was probably due to dilution
of the transferred antibody in the blood circulation, as it is observed
that, 48 h post passive immunization, the serum of the recipient
fish had only agglutination titer of 4 (16 fold lower than the donor
serum). Additionally, mortality of passively-immunized fish
occurred 2e3 days later than that of control fish, further suggesting
reduced level of specific antibody. The RPS obtained from heat-
treated antiserum was even significantly lower than that of the
untreated. This is probably because the heating not only inactivated
complement or cytokines but also caused certain degrees of ag-
gregation of immunoglobulins [31]. The observed lower aggluti-
nation titer of heated-treated serum (from 64 to 16) would be
therefore due to antibody aggregation, which resulted in lower RPS.
The antibody titer associated passive immunization would be
further addressed using hyper-immune fish serum [20].
The agglutination activity of anti-ECP serum was further shown
to be able to cross-agglutinate cells of other virulent strains (isolates)
of A. hydrophila besides the homogeneous ML-10-51K, but not
A. veronii and a likely weak virulent isolate of A. hydrophila (Table 3).
More assays will be surely conducted in the future to test 1) if the
ECP-immunized fish would have immunity against virulent isolates
from diverse sources and 2) if the anti-ECP serum would have
application in diagnosis of field isolates which are complicated in
many common traits shared among species of Aeromonas [32].
The results of western analysis showed that the anti-ECP serum
selectively recognized some components in the complex ECP
(Fig. 2A and C) and the antibody-binding bands may include pro-
teinous and non-proteinous molecules. Those bands (apparent
molecular weights ranging from 38 to > 62 kDa; Lane 1 in Fig. 2C)
with distinctive ladder-like pattern [33] were most probably lipo-
polysaccharides (LPS). This was supported by the preliminary
274 D. Zhang et al. / Fish & Shellfish Immunology 36 (2014) 270e275

A. B. C.

188 188 188

98 98 98

62 62 62

49 49 49

38 38 38

28 28 28

17 17 17
14
14 14

6 6 6

kDa kDa kDa

Fig. 2. SDS-PAGE and western analysis of A. hydrophila ECP and catfish anti-ECP sera. Approximately 9 mg of ECP or equivalent volumes of concentrated TSB medium were loaded to
individual lanes in 4e12% NuPAGE Bis-Tris gel (Invitrogen). After electrophoresis, the gel was stained (Panel A) using SimpleBlue SafeStain (Invitrogen) or subjected to western blot
to PVDF membrane (Panels B & C). Panel B: the blot was probed using sham-immunized serum. Panel C: the blot was probed using ECP-immunized serum. All samples in lanes
labeled 1 were ECP and those in lanes labeled 2 were concentrated TSB medium while lanes in M were molecular weight markers (Invitrogen). Bands indicated by arrows were
analyzed by mass spectrometry (detailed in Section 3.6).

observation that the similar ladder-like banding pattern was
revealed by immunoblotting of purified A. hydrophila LPS using
anti-ECP serum as primary antibody (Supplemental Fig. 2). LPS was
known as one of the major structural molecules of the outer
membrane and as an endotoxin associated with pathogenesis [34];
extracellular LPS was, however, rarely examined except for the
report by Horisberger and Dentan [35], in which cellular LPS and
extracellular LPS of Moraxella glucidolytica were found to have
minor structural differences but were similar in animal toxicity. The
extracellular LPS in A. hydrophila was not reported before. Earlier
study showed that vaccination of common carp with the LPS,
extracted from A. hydrophila cells, could provide some protection
against A. hydrophila but no antibody in vaccinated fish serum was
found in agglutination assay [18]. The presence of anti-LPS antibody
in the anti-ECP serum, observed in this study, would probably
result from combination with other ECP components [36] and have
conferred the fish with better immunity against A. hydrophila.
Protein identities revealed by mass spectrometry (Fig. 2A and C)
were likely the antibody’s targets and may serve as important
immunogens although individual roles has yet to be determined
using recombinant proteins. In addition, the possible synergic ef-
fects between protein antigens and LPS merit further investigation.
In conclusion, fish could develop immunity against fatal chal-
lenge of A. hydrophila when vaccinated with ECP secreted by the
pathogen and the immunity could be transferred via serum to
provide naïve fish with immediate protection. The anti-ECP serum
could have differential agglutination activities toward different
virulent isolates within A. hydrophila and different species in genus
Aeromonas. Fish immunized with the ECP of ML-10-51K could have
immunity against various virulent isolates of A. hydrophila in the
fields. Antibodies in the anti-ECP serum were shown to target
specific components in the complex ECP. Further characterization
of these antibody’s targets would help uncover quintessential an-
tigens for the development of higher efficient vaccines.
Acknowledgements
We thank Drs. Perng-Kuang Chang and Victor Panangala for
critical review of this manuscript, and Mrs. Ning Qin and Beth
Peterman for their technical support. This study was funded by
USDA-ARS CRIS project #6420-32000-024-00D.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.fsi.2013.11.015.
 D. Zhang et al. / Fish & Shellfish Immunology 36 (2014) 270e275 275

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