Fish & Shellfish Immunology 26 (2009) 619–624
Contents lists available at ScienceDirect
Fish & Shellfish Immunology
journal homepage: www.elsevier.com/locate/fsi
Specific and natural antibody response of cod juveniles vaccinated against
Vibrio anguillarum
S. Gudmundsdóttir*, B. Magnadóttir, B. Björnsdóttir, H. Árnadóttir, B.K. Gudmundsdóttir
Department of Fish Diseases, Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavı́k, Iceland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2008
Received in revised form
19 September 2008
Accepted 26 September 2008
Available online 14 October 2008
The purpose of the present study was to study specific and natural antibody levels in individual cod
juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and
vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and
the performance of different groups to bath challenge by the bacterium tested. Antibody responses to
V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses
were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant
antibody response. Both groups showed increased levels of natural antibodies while levels were low in
other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while
fish that received a second vaccination showed the best protection. It was not ascertained whether there
is a long term difference between the effects of immersion versus i.p. injection as a booster method.
Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest
mortalities show that neither could have been used to predict protection given by the vaccines tested.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cod
Fry
Vaccinations
Vibrio anguillarum
Antibody response
1. Introduction
Cod has for centuries been one of the most important species for
fishing communities around the North Atlantic [1]. Today, with
catches rapidly declining, cod is probably the marine species with
the highest potential for sustainable cold water aquaculture [2].
Intensive rearing of fish, suffers from outbreaks of diseases. In
cultivated cod, vibriosis, caused by Vibrio anguillarum, is one of the
major bacterial diseases [3,4]. Vibriosis in cod is often associated
with serotypes O2a and O2b [5,6] while serotypes O1 and O2a are
most commonly isolated from salmonids. Vaccinations are the
most effective way to avoid bacterial diseases and different fish
species may need different vaccines, vaccination methods and
vaccination schemes [7,8]. Cod has been successfully vaccinated
against vibriosis and recently, vaccines adopted for use in cod,
containing serotypes O1, O2a and O2b, have become available
commercially. Vaccine trials have included cod of various sizes,
using bathing, immersion and injection and different challenge
methods [9–12].
Several studies of antibodies and antibody responses in cod
have been carried out during the last two decades. Cod blood has
high concentration of IgM in serum that increases by influence of
* Corresponding author. Tel.: þ354 5855100; fax: þ354 5673914.
E-mail address: [email protected] (S. Gudmundsdóttir).
1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2008.09.017
environmental factors such as temperature [13] and size [14]. Cod
mounts specific response to a limited number of antigens following
infections and immunizations and typically individuals in a group
show a wide range of responses [10,15,16]. Studies on cod immunoglobulins were reviewed recently and the authors concluded
that the explanation for the lack of specificity does not appear to be
due to deficiencies in their structure, organization, diversity or
expression [17].
Natural antibodies are important in the first line of defence
against infections [18]. They react to common antigens of pathogens and various self-antigens. They are present in sera of normal,
non-immunized individuals, where they are proposed to form
a link between innate and adaptive immunity [19]. Studies on
natural antibodies have been carried out in a number of fish species
[20] and high levels of natural or non-specific antibodies have been
reported in cod [13,14]. These antibodies reacted to a variety of
antigens and the strongest response was towards haptenated
bovine serum albumin or TNP–BSA. Subsequently it was shown
that i.p. injections with Freund’s complete adjuvant (FCA) led to
increased activity against this antigen [16]. In a vaccination trial
with V. anguillarum in adult cod some of the pre-immune sera
contained antibodies that bound to antigens in the bacterial
membrane [9] and all non-immunized control fish had antibodies
that reacted with V. anguillarum in another trial [10]. A study in
goldfish has shown that natural antibodies can influence results of
vaccination [21].
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S. Gudmundsdóttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624
The aim of the present study was to study the levels of specific
and natural antibody responses in individual cod juveniles before
and after being vaccinated against V. anguillarum. Two different
vaccine preparations and six vaccination regimes were employed.
Further, the aim was to compare the performance of different
groups to bath challenge by the bacterium.
Table 1
Experimental set up. Vaccinations by bathing, dipping, intra-peritoneal injection or
combinations thereof (V), kidney samples for bacteriology (K) blood samples (S), and
bath challenge (X).
Vaccine administration
Weeks from start of experiment. Vaccinations (V),
sampling (K, S) and challenge (X)
0 weeks
2. Material and methods
2.1. Fish
The cod, of wild parentage from Icelandic waters, was hatched
and reared at the Marine Institute’s Experimental farm, Stadur,
Grindavı́k [22]. Mean weight standard deviation was 1.2 0.4 g at
the time of bath vaccination, 10.3 1.1 g at dipping, 25.7 7.2 g at
i.p. injection and 61.5 12.7 g when challenged. From the start of
the experiment and until i.p. vaccination, different groups were
kept separately in 350 l tanks, 120 individuals in each. After i.p.
injection and individual marking of all fish along fin margins with
Visible Implant Fluorescent Elastomer dye (Northwest Marine
Technology, Inc. (Salisbury, Great Britain)), all groups were transferred to one 3500 l tank. Tricaine methanesulphonate, 50 mg ml1
seawater, was used for light anaesthetization during tagging, i.p.
immunization, weighing and blood sampling. Nine weeks post-i.p.
vaccination, the fish were transported to another facility (Sandgerdi
Research Center). There, 30 fish from each group to be bath challenged were evenly distributed between six 350 l tanks, i.e. two
tanks for each bath challenge and acclimatized for two weeks
before challenge. Aerated borehole seawater of temperature
9 2 C and salinity 32& was used in both facilities and the fish
were fed commercial dry pellets.
C: PBS control
B: bath
D: dip
BD: bath and dip
A: AJ-oil adjuvant i.p.
VA: adjuvated vaccine i.p.
B and VA
V
V
6 weeks
15 weeks (I)a
24 weeks (II)b
K, S
K
V
V
K,
K,
K,
K,
V
V
V
K,
K,
K,
K,
K,
K,
K,
V
S
S
S
S
S,
S,
S,
S,
S,
S,
S,
X
X
X
X
X
X
X
Mean weight of fish at bathing 1.2 g; dipping 10.3 g; i.p. injection 25.7 g; bath
challenge 61.5 g.
a
I: blood sampling before i.p. vaccination.
b
II: blood sampling before bath challenge.
2.4. Bath challenge
Sixty fish from each group were divided between 6 tanks. Two
tanks were used for each of three bacterial concentrations tested by
bath challenge, 2 105 CFU ml1, 2 106 CFU ml1 and
2 107 CFU ml1. The fish were bathed in 200 l seawater containing
bacteria in the concentrations as stated above. After 1 h, 150 l of
fresh seawater were added to each tank and bating continued for an
additional hour. The seawater was oxygenated during these 2 h and
subsequently the flow rate was restored to normal. The fish were
observed for four weeks, hand fed twice daily, deaths recorded and
dead individuals frozen.
2.2. Bacterium for challenge
2.5. Kidney samples for bacteriology
A frozen stock, of an Icelandic isolate of V. anguillarum (F-13903) serotype O2b recently passaged three times through cod, was
inoculated onto blood agar with 1.5% NaCl (BA-NaCl) and incubated
at 16 C for 3 days. Growth was verified with BioNor agglutinating
antibodies against V. anguillarum. A loopful of the growth was
suspended in 1.5 ml1 phosphate buffered saline with 1.1% peptone
and 1.5% NaCl (PBS-P-S) and distributed evenly on 150 mm BA-NaCl
agar plates and incubated at 16 C for 19 h. Then, 4.5 ml of ice cold
PBS-P-S was poured over each agar plate, the growth scraped into
the buffer, harvested and placed on ice. Density measured 2.0 at
590 nm in spectrophotometer, or close to 1011 colony forming units
(CFUs) according to previous measurements. The solution was
diluted by tenfold steps to the concentrations needed to do the bath
challenge. Doses were chosen according to results from previous
challenge tests. CFUs were confirmed by plate counting after
incubation for 3 days at 16 C.
A total of 130 kidney samples for bacterial isolation on BA-NaCl
agar were collected. Sixty samples were collected during the
vaccination period at the indicated times and a total of 70 samples,
10 from each group before challenge, see Table 1. Fish that died
during challenge were frozen on the day of death and kidney
samples inoculated on BA-NaCl agar at the end of the experiment.
2.3. Vaccines and immunizations
The vaccines tested were a generous gift from Pharmaq in
Norway, where they were already licensed. A special permit was
issued by The Veterinary Officer for Fish Diseases in Iceland for the
use of these vaccines in the current experiment. The vaccines were
V. anguillarum bacterins for use in cod, containing serotypes O1a,
O2a and O2b. A water-based preparation was used for bathing or
dipping and a preparation containing mineral oil adjuvant formula
(AJ-oil) for injection. The immunizations were carried out as
described by the manufacturers, i.e. bathing for 30 min in vaccine
diluted 1:200 in seawater, dipping for 30 s into vaccine diluted 1:10,
injection with 0.1 ml i.p. or a combination thereof, see Table 1.
Control fish were subjected to bathing, dipping and injection with
PBS-P-S and one group received AJ-oil only.
2.6. Blood sampling
On three occasions, as shown in Table 1, a total of 120 blood
samples were taken from the caudal vein of 10 fish in each experimental group. The blood was allowed to clot overnight at 4 C,
spun and the serum harvested and kept at 20 C until tested.
2.7. Serology
ELISA tests. Cod sera were diluted 1:100 and added to 96 well
microtrays (Maxisorp, Nunc) coated with sonicated preparation of
V. anguillarum, protein concentration 10 mgml1, followed by mouse
antibodies to cod IgM, alkaline phosphatase conjugated goat antimouse Ig and the color developed [23]. When measuring natural
antibodies, the plates were coated with 5 mgml1 TNP–BSA containing 15.8 residues per BSA molecule. In both ELISAs the value of
the blank was detracted from the reading and the final value for
each sample was the mean of OD readings from two wells.
2.8. Statistics
The Kruskal–Wallis test was used to test for normality of the
distribution of ELISA values and a t-test for comparisons of means
between groups. The programmes used were InStat and StatView.
S. Gudmundsdóttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624
3. Results
Table 2
Accumulated death in % following bath challenge and antibody responses against
V. anguillarum and TNP–BSA shown as mean ELISA values in vaccinated groups and
controls before challenge.
3.1. Challenge
Two of three bath challenge doses induced very little death. For
fish challenged with the highest dose, 2 107 CFU ml1, accumulated death in the PBS control group was 20%, Fig. 1. In groups either
bathed or dipped there was 50% accumulated death, with bathed
fish dying off faster than dipped. Accumulated death was 15% or
less in other groups, Fig. 1. Means of antibody responses to
V. anguillarum and TNP–BSA before challenge are shown in Table 2
for comparison to challenge results. Evidently neither antibody
responses against V. anguillarum nor TNP–BSA ensured survival
after challenge since group BD with 5% mortality showed negligible
antibody responses while in group B–VA with no mortality the
highest antibody responses were observed.
3.2. Bacterial isolations
V. anguillarum was not isolated from any of the 130 samples
taken at various times before challenge, but the bacterium was
isolated from 83% of samples from fish that died during challenge.
Vaccine-groups
Accumulated death %
C: PBS control
B: bath
D: dip
BD: bath and dip
A: AJ-oil adjuvant i.p.
VA: adjuvated vaccine i.p.
B and VA
20
50
50
5
10
15
0
Mean ELISA values of
antibodies against
V. anguillarum
TNP–BSA
0.126
0.131
0.094
0.058
0.270
0.474
0.846
0.281
0.203
0.252
0.173
0.221
0.454
0.499
receiving i.p. vaccination showed moderate responses (OD405
0.5–1.0) to V. anguillarum antigen preparation, while the same
number of fish bathed and vaccinated i.p. showed high responses
(OD405 > 1.0). Order of samples on the X-axis is always the same so
individual responses to either antigen can be compared within each
group. Antibodies to TNP–BSA in majority of individuals in these
two groups showed raised levels.
4. Discussion
1
,5
2
Control
II-B-VA
II-VA
II-A
II-D
II-BD
II-B
II-C
I-BD
I-C
0
I-BD
Bath and -vacc. i.p.
1,5
I-D
Bath and dip
Adjuvant i.p.
a
I-D
Vaccine i.p.
Dip
2
I-B
Bath
In the present study, levels of specific and natural antibody
responses in individual cod fry from groups subjected to different
I-B
Antibody responses to V. anguillarum antigens in controls and
groups receiving bathing, dipping, or both treatments were negligible both at 15 or 24 weeks after the start of the experiment
(Fig. 2a). Fish receiving vaccination i.p. showed raised antibody
levels but comparison with controls did not show significant
differences. On the other hand samples from fish receiving i.p.
injection in addition to bathing, II-B–VA, showed significantly
raised mean, p < 0.05, when compared to controls before challenge.
Adjuvant alone raised the antibody mean slightly but insignificantly compared to controls. Groups receiving i.p. vaccination
showed raised antibody responses to TNP–BSA while other groups
showed negligible changes in their response to TNP–BSA during the
time of the experiment (Fig. 2b).
In Fig. 3, responses to V. anguillarum and TNP–BSA are shown for
every individual in four groups. There are no examples of high
responses in controls and bathed individuals. Four out of 10 fish
OD
405
3.3. Antibody responses following vaccinations
70
621
b
1,5
50
40
OD
405
1
30
,5
20
10
0
5
10
15
20
25
30
Days
Fig. 1. Accumulated death percentage in seven groups of vaccinated cod juveniles and
controls receiving PBS, subjected to bath challenge with 2 107 CFU ml1 V. anguillarum of serotype O2b for 2 h and observed for four weeks.
II-B-VA
II-VA
II-A
II-BD
II-D
II-B
0
II-C
0
I-C
Accumulated death in %
60
Fig. 2. Antibodies against a) V. anguillarum and b) TNP–BSA in groups of cod following
one or two vaccinations against V. anguillarum, showing median and range of ELISA
readings of cod sera, 10 fish in each group. The line within each box is the median the
boxes show 25–75% percentiles, the whiskers 10–90% percentiles and circles the
extreme values. I and II represent sampling times as shown in Table 1; C: PBS control
group; B: bathed group; D: dipped group; BD: group bathed and dipped; A: adjuvant
administered i.p.; VA: adjuvated vaccine administered i.p.
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S. Gudmundsdóttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624
Antibodies to V.anguillarum
a
Antibodies to TNP-BSA
Control group
1,5
1,5
1
1
,5
,5
0
b
0
Serum no. 1-10
Serum no. 1-10
Bath vaccinated group
1,5
1,5
1
1
,5
,5
0
c
0
Serum no. 11-20
Serum no. 11-20
I.p. vaccinated group
1,5
1,5
1
1
,5
0
d
,5
0
Serum no. 21-30
Serum no. 21-30
Bath and i.p. vaccinated group
1,5
1,5
II-B-VA
1
,5
1
,5
0
0
Serum no. 31-40
Serum no. 31-40
Fig. 3. Antibodies against V. anguillarum and TNP–BSA in individual cod sera, 10 sera in each of four groups, sampled before challenge: a) control group; b) bath vaccinated; c) i.p.
vaccinated; d) bath and i.p. vaccinated.
vaccination schemes were measured. This is the first study
addressing antibody profiles of cod groups where vaccination starts
with small fry. Further, performance of the different groups when
subjected to bath challenge was assessed.
In terms of survival, the strongest bath challenge dose used in
the present study was suboptimal, since accumulated death in the
control group was only 20%. Interestingly the groups bathed or
dipped showed less survival than the control group with bathed
individuals dying faster than dipped and with both groups reaching
50% accumulated death on day 21 after challenge. The cod juveniles
in the current study were 1.2 0.4 g at the time of bathing which is
too small for a long time protection to be established according to
a recent paper [11]. In that same paper dipping at 5 g resulted in
long-term protection in an experiment where 95% of controls died,
which is in contrast with the results reported here. Difficulties in
the establishment of bath-challenge models are well known.
Sometimes observed death of controls is too high and benefits of
vaccinations are in danger of being underestimated while too low
accumulated death of untreated controls can obscure a real difference between types of vaccines and vaccination modes [24]. The
manufacturers of the vaccines tested recommended two vaccinations and according to our results, bathing followed by either
dipping or injection, can give good results. AJ-oil alone did not
induce significant protection in the current experiment. This is in
agreement with another study where frys receiving AJ-oil only
were not protected against bath challenge while i.p. challenge of
the same group resulted in significant protection [12]. Due to the
set up of the experiment, there were 24 weeks between bathing
and challenge, 18 weeks between dipping and challenge while 9
weeks passed between injection and challenge.
Antibody responses in the vaccinated groups demonstrate that
only individuals receiving adjuvated vaccine i.p. show raised antibodies to V. anguillarum. There were higher individual readings as
well as higher mean in the B–VA group (bathed prior to i.p.
S. Gudmundsdóttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624
vaccination) than in the group receiving single i.p. injection (VA)
suggesting secondary response. In a study where frys of 55–60 g
were inoculated with bacterins, suspended either in FIA or AJ-oil
[10], only the latter group showed specific antibody response in
a pooled serum sample. A closer look revealed that the titer of the
pool was mainly due to serum from one individual. In that trial i.p.
vaccinated groups also showed high levels of protection. Western
blots done on selected sera in the present study (results not shown)
showed antibodies directed mainly at LPS antigens of sizes ca. 43
and 34 kDa. In a study on specificity and durability of antibodies to
different V. anguillarum antigens in two adult cod immunized with
a bacterin in AJ-oil, antibodies against LPS were prominent and
lasted at least 44 weeks post-vaccination [25]. In that study, preimmune sera and controls did not show elevated levels of antibodies to V. anguillarum. In another study [9], bacterins in FCA were
administered to 1–3 kg cod, inducing moderate antibody response,
mainly to LPS antigens and there was no increase in antibody levels
following second immunization. In that study some of the nonimmune sera reacted with bacterial antigens possibly because the
cod studied was taken from a sea cage and may previously have
come into contact with V. anguillarum or antigens from related
bacteria. From these studies it can be suggested that bacterins
administered i.p. in AJ-oil adjuvant evoke more antibody responses
in cod than bacterins administered in FCA or FIA.
In comparison to fish of wild origin, all test groups showed low
levels of natural antibodies [13,14]. The explanation is probably
both the young age and clean environment of the test groups. Fish
that received V. anguillarum antigens i.p. were the only ones to
show some rise in antibodies against TNP–BSA during the trial
period. Antibodies to TNP–BSA have not been measured previously
in sera of cod vaccinated against V. anguillarum but FCA, regardless
of the antigen used, induced increase in antibodies to TNP–BSA in
cod [16]. In the present study, the group receiving AJ-oil only,
showed lower response towards TNP–BSA than fish in the groups
receiving bacterin in AJ-oil. This indicates an influence of the
bacterial antigens in raising antibodies reacting with TNP–BSA as
was the case with FCA, an oil adjuvant containing bacterial antigens. In the current study natural antibody levels were very low
when the vaccinations started and hence less likely to influence the
results than could have been the case in older fish with higher
levels of natural antibodies as reported recently for vaccination trial
in goldfish [21].
The present results suggest that antibody measurements are
not the way to assess protection by vaccines against V. anguillarum in cod. Other possibilities are already being contemplated
and a recent paper reports that there was enhanced serummediated antibacterial activity in samples from cod, 3 days after
i.p. vaccination [26]. Other methods of vaccination were not
investigated. In the same study, some upregulation in expression
of immune response genes was demonstrated 3–7 days after
immunization. The same was true for some of the PBS injected
control fish. Looking at the present results it can be inferred that
methods for studies on cellular immunity are needed in order to
reveal the mechanisms explaining the observed susceptibility of
fish either bath or dip vaccinated as well as the protection
obtained in fish receiving both treatments. Further information on
the cod immune responses is needed in order to develop
successful vaccines and answer the question on a method other
than challenge that could be used to study efficacy of vaccines/
vaccination schedules in cod.
In conclusion, antibodies against V. anguillarum antigens or
against TNP–BSA could not have been used to predict protection in
a bath challenge. A booster vaccination had positive effects under
the conditions of the experiment, but it was not ascertained
whether there is a long-term difference between the impact of
immersion versus i.p. injection as a booster method.
623
Acknowledgements
Thanks are due to the staff at the Marine Institute’s Experimental farm at Stadur and at Sandgerdi Research Center for help
during vaccination and challenge periods. Further to Gudmundur
Logi Norddahl and Harpa Lind Björnsdóttir for technical help and
finally to Kjersti Gravningen and her colleagues at Pharmaq for
providing the vaccines. The work was funded by The Icelandic
Research Council, grant no.: 0308000003.
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