Fish & Shellsh Immunology 25 (2008) 533541

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Fish & Shellsh Immunology

journal homepage: www.elsevier.com/locate/fsi

Development of adaptive immunity in rainbow trout, Oncorhynchus mykiss

(Walbaum) surviving an infection with Yersinia ruckeri
Martin K. Raida*, Kurt Buchmann
Department of Veterinary Pathobiology, Section of Fish Diseases, Faculty of Life Sciences, The University of Copenhagen, Stigbjlen 7, DK-1870 Frederiksberg C, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 26 March 2008
Received in revised form 2 July 2008
Accepted 15 July 2008
Available online 23 July 2008

Development of adaptive immunity in rainbow trout (Oncorhynchus mykiss) surviving a primary infection with 5  105 CFU Yersinia ruckeri O1 (LD50 dose) was investigated by transcriptome analysis of spleen
tissue. These sh surviving a primary infection showed also a signicantly increased survival following
a secondary infection (same dose) when compared to nave trout. The weight of the rainbow trout spleen
doubled during the rst 14 days of the primary infection but the affected organs subsequently recovered
normal weight which remained constant during the re-infection period. Gene transcription in the spleen
was measured using Quantitative real-time RT-PCR (qPCR). Samples taken 8 h.p.i., 1, 3, 7, 14 and 28 d.p.i.
were compared to PBS-injected control sh sampled at the same time points. The investigated cytokines
and chemokines comprised interleukin (IL)-1b, IL-1 receptor antagonist (Ra), IL-6, IL-8, IL-10, IL-11 and
IFN-g, IL-1 receptor I and II (IL-RI and IL-RII). Transcript levels of genes encoding cytokines and receptors
were increased during the primary infection but not during the secondary infection. Changes of T cell
occurrence or activity in the spleen during the infections were inferred from the transcript level of T cell
receptor (TCR), CD4 and CD8a genes. No alteration in the expression of MHC class ll and immunoglobulin
(Ig)M and IgT was detected during the experiment. The amount of Y. ruckeri O1 in the spleen was
measured with a Y. ruckeri 16S ribosomal RNA specic qPCR and this parameter was correlated to the
expression of IL-1b, IL-8 and IL-10 genes with a peak expression at 3 d.p.i. (rst infection). The low
transcript levels of the bacterial gene and the hosts immune genes during the re-infection can be
interpreted as a result of development of adaptive immunity. This would explain the relatively fast
elimination of the bacteria during the secondary infection whereby the activation of cytokines becomes
less pronounced.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Immunity
Rainbow trout
Yersinia ruckeri
qPCR
Hostpathogen interaction
Immune response
Adaptive immunity

1. Introduction
Yersinia ruckeri is the aetiological agent of enteric redmouth
(ERM) disease or yersiniosis, affecting mainly salmonids [1,2].
Although generally well controlled by means of vaccination and
antibiotic treatment, this disease has kept on causing outbreaks,
especially in endemic areas. In some cases the losses due to this
disease can be as high as 3070% of the stock [3]. Protective
immunity in rainbow trout against ERM has been known since the
rst commercial sh vaccines based on formalin killed bacteria
were introduced [4]. Recently, transcription of genes encoding both
innate and adaptive immune parameters in the spleen following
vaccination has been described [5].
The spleen seems to represent a major secondary lymphoid
organ in sh during bacterial infections. Thus, it has been reported
that antigens are captured by immunocompetent cells at

* Corresponding author. Tel.: 45 35332701; fax: 45 5282742.

E-mail address: mkr@life.ku.dk (M.K. Raida).
1050-4648/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2008.07.008

inammatory foci and then transported to the spleen for the

initiation of adaptive immune responses [6]. In vaccinated rainbow
trout, antigen trapping takes place in the walls of the splenic
ellipsoids, which suggests a specic role for these cell clusters
during development of immunity [7]. Further, during Y. ruckeri
infection in rainbow trout a dramatic increase in spleen weight (up
to threefold) has been observed and interpreted as a result of inux
of cells recruited by inammatory cytokines [8]. This complies with
the fact that Y. ruckeri counts increase in spleen tissue after challenge [8,9], which is associated with migration of leukocytes from
the anterior kidney to the blood and the spleen in rainbow trout
during Y. ruckeri infection [9]. Likewise, expression of cytokines and
chemokines was increased in the spleen during Y. ruckeri infection,
indicating that the spleen is actively involved in rainbow trout
immune responses against this pathogen [8,9]
It is generally agreed that regulation of inammation results
from a balance between pro- and anti-inammatory cytokines,
which also will minimize the negative effects of the inammatory
processes [10]. However, the immediate activation of the innate
immune response is an important event during induction of the

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M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

adaptive response eventually leading to specic long-term

protection [11]. Also in sh a strict regulation of these two immune
branches is likely to produce an optimal immune response.
The present work contributes to our understanding of the
complex interactions between humoral and cellular factors in
rainbow trout responding to primary and secondary Y. ruckeri
infections. Due to the central role of the spleen in this process the
investigation is based on a description of the expression of immune
relevant genes in spleen tissue. The work emphasizes genes
encoding cytokines, their antagonists, immunoglobulins and T cell
markers and adds to the notion that these are central elements of
the adaptive immune response in rainbow trout.
2. Materials and methods
2.1. Fish and rearing conditions
Juvenile rainbow trout (Skinderup strain from Jutland, Denmark), hatched and reared under pathogen-free conditions (Danish
Centre for Wild Salmon, Randers, Denmark), were brought to the
experimental sh keeping facility at the University of Copenhagen
when reaching a body weight of 46 g. The pathogen-free status of
the sh was conrmed upon their arrival in the laboratory by
analysis for bacterial, parasitic and viral pathogens. The 600 sh
were kept in three 200 l tanks with bio-lters (Eheim, Germany)
and maintained at a 12 h light and 12 h dark cycle in aerated (100%
oxygen saturation) tap water at 13  C. They were fed a commercial
trout feed (BioMar, Denmark) (2% biomass per day).
2.2. Bacterial strain
Y. ruckeri serovar I (strain 392/2003), isolated from diseased
rainbow trout in Spain [12], was used for the challenge experiments. The bacteria were grown in LB-medium (Oxoid LP0042,
Tryptone 10 g, Oxoid LP0021Yeast-extract 5 g, NaCl 5 g, H2O to
1000 ml, pH 7.4) at 20  C for 36 h and enumerated as colony
forming units (CFUs) by the spread plate method on blood agar
(Blood agar base CM55 [Oxoid] supplemented with 5% bovine
blood).
2.3. Primary and secondary challenge experiments
Primary infection trials were conducted using a total of 400
rainbow trout, half of them were used as non-infected control sh.
All sh were anaesthetized by immersion in 40 mg/l tricaine
methane sulfonate (MS-222, SigmaAldrich, Denmark). Two
hundred trout were infected by intra-peritoneal (ip.) injection
(5  105 CFU/sh in 50 ml PBS) corresponding to a previously
determined LD50 (data not shown). Two hundred non-infected
control sh were injected with 50 ml sterile PBS. In the primary
challenge experiment the infected sh received an ip. injection and
were observed for 35 days. In the re-challenge experiment a total of
97 surviving sh from the primary challenge received an additional
injection of 5  105 (CFU/sh) bacteria 35 days after the primary
infection. When performing the re-challenge of the survivors,
a group of 200 nave sh was infected as control to conrm virulence of the bacteria. The non-infected control sh were also reinjected with sterile PBS at day 35 post-primary injection. Bacterial
samples from the head kidney from all sh that died were cultured
on blood agar plates to conrm the cause of death. Mortalities were
only considered to be caused by Y. ruckeri if the bacteria were
recovered as pure culture from the head kidney.
Relative percentage survival (RPS) was calculated using the
following equation: RPS (1  (percent immune mortality/percent
control mortality))  100 [13].

2.4. Detection of Y. ruckeri in blood

Counts of Y. ruckeri in the blood of 5 ip. infected nave rainbow
trout were taken 0, 1, 2, 3, 5 and 6 days post-infection. The sh were
killed after blood sampling. Samples (10 ml blood) from each sh
were plated onto blood agar in a 10-fold dilution series (in
triplicate).
2.5. Sampling for gene expression studies
Spleens from ve infected and ve control sh were sampled at
0, 8 h and 1, 3, 7, 14 and 28 d following infection (both primary and
secondary challenges). No moribund sh were sampled for gene
expression experiments. Fish were killed by immersion into an
overdose of MS-222 (100 mg/l). Spleen tissue was sampled aseptically, immediately transferred to RNA-later (SigmaAldrich), prestored for 24 h at 4  C and subsequently stored at 20  C until
isolation of RNA. When comparing groups for immunological
parameters the infected sh and non-infected control sh sampled
at the same time points were compared. A spleen size index was
calculated as the ratio between spleen weight (g): body weight (g),
for individual sh from day 7 in order to describe changes of spleen
weight during infection.
2.6. Expression of Y. ruckeri-specic 16S ribosomal RNA gene in the
spleen of rainbow trout
A primer pair and a TaqMan probe were designed in an
unconserved region of the Y. ruckeri partial 16S ribosomal RNA gene
(Genbank accession number: X75275), which gives a specic
amplication of Y. ruckeri strains only [14]. The amplicon is 70 bp
long. Forward primer: 50 GCGAGGAGGAAGGGTTAAGTG30 , reverse
primer: 50 GTTAGCCGGTGCTTCTTCTG30 , and the TaqMan probe:
50 AATAGCACTGAACATTGACGTTACTCG30 .
2.7. Isolation of total RNA and cDNA synthesis
Homogenisation of tissue was done by sonication on ice (Sonicator Ultrasonic Liquid Processor Model XL 2020, heat Systems,
New York, USA) and total RNA isolated using GenElute total RNA
kit (SigmaAldrich, Denmark). Removal of genomic DNA was conducted with deoxyribonuclease I (SigmaAldrich). RNA quantity
was checked by OD260/280 measurements (SmartSpec 3000,
BIO-RAD, USA). cDNA synthesis was performed on 400 ng total RNA
in a 20 ml setup using TaqMan Reverse Transcription reagents
following the manufacturers instructions (Applied Biosystems,
USA). Random hexamer primers were used in the reverse transcription reactions. RT-reactions lacking reverse transcriptase (RT
minus) but not RNA were also performed to verify that the samples
did not contain genomic DNA. The synthesised cDNA samples were
diluted 1:10 in MilliQ H2O and stored at 20  C.
2.8. Gene expression analysis
Spleen samples were analyzed using qPCR for expression of
genes encoding cytokines (IL-1b1, IL-1Ra, IL-6, IL-10, IL-11 and IFNg), chemokine IL-8, immunoglobulins (IgM, IgT) and cellular
receptors (TCR, CD4, CD8a, MHC II and IL-1 receptor I and II). qPCR
assays were performed using a Stratagene MX3000PTM real-time
PCR system. Based on available GenBank (NCBI) sequences primers
and dual-labelled TaqMan probes conjugated with 50 HEX, FAM or
CY 50 and a 30 BHQ1 or BHQ2 were designed using Primer3 software
(http://frodo.wi.mit.edu/). Primers and probes were analyzed
for hairpin structure, self- and hetero-dimers in OligoAnalyzer
3.0
(http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
Default.aspx?cEU). Primers and probes are listed in Table 1. All

M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

535

Table 1
Quantitative PCR (qPCR) expression of immune relevant genes in rainbow trout
Gene

GenBank
accession no.

Product
size

Forward
primer

Reverse
primer

Probe

qPCR efciency %

Cytokines
IL-1b1
IL-1Ra
IL-6
IL-8
IL-10
IL-11
IFN-g

AJ223954, AJ298294
AJ295296
DQ866150
AJ279069
AB118099
AJ 535687
AY795563

91
65
91
69
70
104
68

acattgccaacctcatcatcg
aaggaggacaaggaggagga
actcccctctgtcacacacc
agaatgtcagccagccttgt
cgactttaaatctcccatcgac
gcaatctcttgcctccactc
aagggctgtgatgtgtttctg

ttgagcaggtccttgtccttg
cactccattgatcgtcagga
ggcagacaggtcctccacta
tctcagactcatcccctcagt
gcattggacgatctctttcttc
ttgtcacgtgctccagtttc
tgtactgagcggcattactcc

catggagaggttaaagggtggc
gccttcgccagtgaaggagaca
ccactgtgctgatagggctgg
ttgtgctcctggccctcctga
catcggaaacatcttccacgagct
tcgcggagtgtgaaaggcaga
ttgatgggctggatgactttagga

99.7
98.7
104.7
104.8
101.7
98.6
102.4

Cell receptors
CD8-a
CD4
TcR
IL-1RI
IL-1RII
MHC-II b

AF178054
AY973028
AF329700
AJ295296
AJ276474
AF115533

74
89
73
70
91
67

acaccaatgaccacaaccatagag
cattagcctgggtggtcaat
tcaccagcagactgagagtcc
atcatcctgtcagcccagag
ctcaatctgctctcggcatt
tgccatgctgatgtgcag

gggtccacctttcccacttt
ccctttctttgacagggaga
aagctgacaatgcaggtgaatc
tctggtgcagtggtaactgg
gcggaggtagtcgtagtcca
gtccctcagccaggtcact

accagctctacaactgccaagtcgtgc
cagaagagagagctggatgtctccg
ccaatgaatggcacaaaccagagaa
tgcatcccctctacaccccaaa
ttcatcgctcgctctgcctg
cgcctatgacttctaccccaaacaaat

104.5
98.6
96.6
116.2
97.6
101.1

Immunoglobulins
IgM
IgT

S63348
AY870265

72
72

cttggcttgttgacgatgag
agcaccagggtgaaacca

ggctagtggtgttgaattgg
gcggtgggttcagagtca

tggagagaacgagcagttcagca
agcaagacgacctccaaaacagaac

98.4
98.5

House-keeping gene
Elongation factor 1a

AF498320

63

accctcctcttggtcgtttc

tgatgacaccaacagcaaca

gctgtgcgtgacatgaggca

100.0

Primers and probe sets including their accession number, product sizes, sequences and qPCR efciency.

primers and probes were HPLC-puried (SigmaGenosys Ltd., UK).

The primers were optimized according to MgCl2 concentrations.
Melting curve analysis of the primers was conducted with an SYBR
Green based qPCR assay, to make sure that the primers did not form
primer dimers. To assess that the primer and probe pairs were
quantitative within the working range, serial dilutions in 10-fold
increment of cDNA were used, and efciency for the primer pairs
was calculated (Table 1). The cycling conditions were one cycle of
initial denaturation at 94  C for 2 min, followed by 40 cycles with
denaturation at 94  C for 30 s and annealing and elongation in one
step at 60  C for 1 min. Wells contained 6.25 ml of 2  JumpStartTM
Taq ReadyMixTM, 36 mM MgCl2 (all chemicals from Sigma
Aldrich, Denmark), 0.5 ml forward and reverse primer (10 mM), 0.5 ml
TaqMan probe (5 mM), 2.5 ml of diluted cDNA (1:10) and autoclaved
MilliQ water to a volume of 12.5 ml. RT minus and negative controls
(MilliQ water without template) were used for every plate setup.
Several reference genes were validated in spleen tissue, namely bactin, Ribosomal protein S20 and Elongating factor 1-a (EF1-a). By
comparing the expression results of the spleen tissues from infected
and non-infected control sh, it was found that EF1-a was the most
stably expressed gene between all individuals. EF1-a primers with
corresponding probe were therefore used as endogenous control
(reference or house-keeping gene) to correlate for potentially
different loading amounts of RNA added to the RT-PCR reaction and
for variation in cDNA synthesis efciencies [15,16]. If the real-time
curve did not reach the threshold within 40 cycles the sample was
not considered for that particular gene. A high Ct value designates
that the gene is expressed at a low level and one Ct value corresponds to a two-fold difference in gene expression.
2.9. Calculations and statistical analysis
Results from the challenge experiments were analysed using the
KaplanMeier test (GraphPad Prism 4, www.graphpad.com/
manuals/Prism4/PrismUsersGuide.pdf), which were used to
analyse for differences in mortality between groups.
2.10. Data analysis of gene expression
The threshold cycle (Ct) was determined at the linear slope in
a log uorescence/Ct plot. The expression results were analyzed

using the 2DDCt method [17]. Expression of all genes in sh

injected with bacteria or PBS was expressed relative to the gene
expression of the ve unhandled sh sampled pre-injection which
were used for calibration (mean expression 1) for each investigated gene. In order to describe the effects of infection on gene
expression the normalized gene expression data (DDCt) for infected
and PBS-injected control sh were compared to each other. Since
data followed a normal distribution (KolmogorovSmirnoffs test),
Students t-test was used for testing differences in relative transcription level between the controls and infected sh at each
sampling time. Correlations between expression of the Y. ruckeri
specic 16S ribosomal RNA gene and expression of immune genes
in the spleen of rainbow trout were analysed using the Spearman
Rank Order correlation test.
A signicance level of 5% was applied in all tests. The data are
presented as the mean value of fold increase/decrease from ve sh
at each sampling point post-injection. All statistical calculations
were performed with GraphPad Prism 4 (GraphPad Software, Inc.,
San Diego, USA).

3. Results
3.1. Challenge experiment
Each sh received an intra-peritoneal (ip.) injection with
5  105 CFU Y. ruckeri in 50 ml PBS. This dose was found to be the LD50 in
a pilot experiment (data not shown) and was used for both the primary
and the secondary challenge experiment. Pure Y. ruckeri cultures were
re-isolated from head kidney of all infected sh which died in the
challenge experiments. Control sh were injected with 50 ml PBS, and
no mortality was observed during the experiment. During the primary
infection, the survival of infected fry was signicantly (p < 0.0001)
(Fig. 1) lower than the non-infected control group (n 200). Survivors
of the primary infection were re-infected ip. with the same dose
(5  105 CFU/sh) on day 35, and a group of nave sh were infected as
virulence control (to conrm that the virulence of the bacteria was the
same as in the primary infection). The survival of re-infected fry was
signicantly (p 0.0009) lower than the non-infected control group.
When comparing the survival in the primary infected versus the reinfected fry, the survival was signicantly higher during the re-infection (p < 0.0001). During the 35 day period following the primary

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M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

Fig. 1. Percent survival of Y. ruckeri infected and non-infected rainbow trout during
primary and secondary infections. During the primary infection, the survival of
infected fry was signicantly (p < 0.0001) lower than the non-infected control group
(n 200). Survivors of the primary infection were re-infected ip. with the same dose
(5  105 CFU/sh) day 35, and a group of nave sh were infected as virulence control
(to conrm the virulence of the bacterial broth). The survival of re-infected fry was
signicantly (p 0.0009) lower than the non-infected control group. When comparing
the survival in the primary infected versus the re-infected fry, the survival was
signicantly higher during the re-infection (p < 0.0001).

infection 34% of the infected sh died. A total of 97 sh survived the

primary infection and were re-challenged at day 35. During the reinfection only 7% of the previously infected sh died. The cumulative
percent mortality (CPM) was determined after 28 days, and the RPS
was 79%.
3.2. Re-isolation of pathogen
Y. ruckeri was re-isolated from the head kidney of all sh which
died during the challenge experiments. Dead sh exhibited external
signs associated with ERM infection including petechial haemorrhages in the mouth, around the anus and at the base of the dorsal ns.
3.3. Detection of Y. ruckeri in the spleen
The Y. ruckeri specic primer pair with corresponding TaqMan
probe detected the presence of Y. ruckeri in the infected sh. The

bacteria were detectable 8 h.p.i. The Y. ruckeri gene transcripts

increased rapidly and peaked on day 3 with more than a 21,000fold increase relative to the detection level. The amount of Y. ruckeri
decreased from day 3 and at day 28 the infection was barely
detectable (Fig. 2). The expression of Y. ruckeri was detectable
8 h.p.i. (re-infection) but decreased rapidly. Thus, Y. ruckeri was
only detected in one out of ve re-infected sh at day 3 and 7. This
was also supported when agar culturing was conducted. By using
this technique Y. ruckeri was re-isolated from the head kidney of
20% of the sh 28 days after the primary challenge, and in only 4% of
the sh 28 days after the re-challenge.
The spleen weight index (spleen weight/body weight) was twice
as high in the infected sh as in the controls 7 and 14 d.p.i. during
the primary infection. During the secondary infection no signicant
weight differences were found (Fig. 3).
3.4. Detection of Y. ruckeri in blood
The presence of Y. ruckeri in the blood of infected trout was
detected from 2 days after the primary infection (Fig. 4). More than
1 106 CFU/ml blood were detected in infected sh 6 d.p.i..
3.5. Expression of investigated immune genes in the spleen of
rainbow trout
Low levels of constitutive expression of all examined genes in
the spleen were detected in unhandled sh, but transcription of
a range of genes was shown to be signicantly regulated due to
infection (Table 2).
3.6. Expression of the IL-1 family genes in the spleen of rainbow
trout
Genes encoding the pro-inammatory cytokine IL-1b1, the antiinammatory antagonist IL-1Ra and the IL-1 receptor type I (IL-1RI)
and the IL-1 decoy receptor type II (IL-1RII) were investigated in the
spleen tissue. A signicantly increased gene expression of all the
measured IL-1 family genes was detected in the infected rainbow

Fig. 2. Expression of a Y. ruckeri specic 16S sequence, in the spleen of rainbow trout (n 5) during primary and secondary ip. infection with 5  105 CFU/sh Y. ruckeri. The Y. ruckeri
16S transcript was signicantly increased relative to controls day 3 and 7 after the primary infection.

M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

537

p < 0.0001). The expression of IL-1RI was also correlated to

expression of IL-1RII (r 0.85, p < 0.0001).
3.7. Expression of other cytokine and chemokine genes in the spleen
of rainbow trout

Fig. 3. The gure shows a spleen weight index (spleen weight/body weight) from uninfected and infected sh (n 5). The weight of the spleen was signicantly increased
7 and 14 days pi. during the primary infection (p 0.03).

trout when compared to un-infected controls. The number of IL-1b

transcripts was signicantly increased in the infected sh
compared to the non-infected control sh at 8 h.p.i., 1 and 3 d.p.i.
The IL-1b expression peaked 3 d.p.i. with a 77.8-fold increase
compared to PBS injected control sh. No differences in expression
of IL-1b1 were seen at later time points. Expression of genes
encoding IL-1RI and IL-1RII was signicantly increased at 1 and
3 d.p.i. in the primary infected sh. Gene transcript numbers of
both genes peaked on day 3 p.i. IL-1RI transcript was elevated 4.2and 6.9-fold, and IL-1RII was 3.4- and 17.4-fold increased at 1 and
3 d.p.i., respectively. No signicant regulations were seen at later
time points (Tables 2 and 3 and Fig. 5).
The transcript of the IL-1 receptor antagonist (Ra) was signicantly increased in all samples from the infected sh from 8 h.p.i. to
7 d.p.i. IL-1Ra expression peaked 3 d.p.i. Expression of IL-1Ra and
the IL-1 receptor expression were correlated with the expression of
IL-1b1 (IL-1Ra: r 0.69, p < 0.0001), (IL-1RI: r 0.69, p < 0.0001),
(IL-1RII: r 0.79, p < 0.0001). Likewise, IL-1Ra also showed correlation to IL-1RI (r 0.50, p < 0.0001) and IL-1RII (r 0.59,

Gene transcripts encoding IL-6 and IL-11 were signicantly upregulated in infected sh 13 d.p.i. (primary infection) (10.2 to 20.7
and 14.7- to 18.8-fold, respectively) (Table 2).
The gene encoding the chemokine IL-8 was signicantly upregulated during the primary infection from 8 h.p.i. to 14 d.p.i.,
peaking at 3 d.p.i. (Table 2).
The number of IL-10 gene transcripts was 396.2-fold increased
in the infected sh 3 d.p.i. (primary infection) (Table 2), and a minor
down-regulation relative to the un-infected sh was seen 14 d.p.i.
(re-infection) (Table 3).
The IFN-g gene transcript level was increased 22.1-fold in the
infected trout 3 d.p.i. (primary infection), and down-regulated
relative to the un-infected control group 3 and 7 d.p.i. during the
re-infection.
The transcript of the T cell receptor gene was stable during the
infections, but an increase in CD4 expression was seen 13 d.p.i. (2to 2.3-fold) (primary infection), and CD8a was 3.4-fold up-regulated 14 d.p.i. (re-infection).
No signicant changes in expression of genes encoding MHC II,
IgM and IgT were seen during the infections (Tables 2 and 3).
4. Discussion
The head kidney of teleosts is considered to be the major organ
for the capture and clearance of bacteria due to the presence of
resident macrophage populations [18], but it has been speculated
that recruitment and activation of lymphocytes following infection
occur in the spleen [6,19]. This complies with the nding that
expression of genes encoding cytokines was higher in the spleen
compared to the head kidney following ip. injection of a Y. ruckeri
bacterin [5]. In the present study, the immune gene activation in
the spleen was found to be extensive during the primary Y. ruckeri
infection in rainbow trout, concomitant with a signicant increase
of spleen weight (Fig. 3), probably due to inux or proliferation of
cells. Further, the early peak of Y. ruckeri in the spleen compared to
the blood could indicate that the spleen actively clears the bacterial
infection.
4.1. Cytokines within the IL-1 family

Fig. 4. Detection of Y. ruckeri in blood of infected rainbow trout (n 5). More than
1 106 CFU/ml blood were detected in infected sh 6 days post-infection.

IL-1b is one of the best described pro-inammatory cytokines in

rainbow trout [2023]. Increased levels of IL-1b transcripts in
rainbow trout tissue have been reported and ascribed to infections
with ectoparasites [2426], virus [27] and injection of killed Y.
ruckeri bacterin [5]. The IL-1 system of ligands and receptors is
a complex system of agonists and antagonists. IL-1-induced activity
occurs as a consequence of binding to its receptor complex (IL-1R)
on the cell surface of target cells [28]. In mammals the IL-1R
transduction system is extraordinarily sensitive and just a few
ligand-occupied receptors initiate biological activity [29]. We found
a signicantly increased expression of the IL-1b1 gene and its
associated receptors in infected sh (Table 2 and Fig. 5). The IL-1b1
expression was signicantly increased from 8 h.p.i. to 3 d.p.i. where
it peaked (77.8-fold) and also was positively correlated to the
abundance of the pathogen (Table 2 and Fig. 2).
The present work indicates that this established path of
immunological events in mammals also occurs in rainbow trout,
involving IL-1b as an important mediator of the early immune
response in rainbow trout.

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M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

Table 2
Quantitative PCR (qPCR) expression of immune relevant genes in the spleen of rainbow trout following primary ip. infection with Y. ruckeri
Gene

Treat-ment

Gene expression in Spleen. Fold increase of target gene relative to elongation factor a (SD)
0h

8h

1 Day

3 Days

7 Days

14 Days

28 Days

IL-1b

Infected
Control

1.0

0.52.2

2.6*
0.6

1.15.9
0.50.9

13.6*
0.4

0.7269.4
0.30.6

77.8***
0.6

46.8129.4
0.21.5

3.1
0.6

0.242.4
0.31.3

1.2
0.7

0.25.5
0.21.8

0.5
0.6

0.21.3
0.21.9

IL-6

Infected
Control

1.0

0.71.5

0.7
0.4

0.31.8
0.30.5

10.2*
0.5

1.569.3
0.30.8

20.7***
0.5

10.042.8
0.21.1

1.4
1.4

0.45.4
0.63.3

1.9
0.4

0.311.3
0.12.5

0.4
0.8

0.20.8
0.23.4

IL-8

Infected
Control

1.0

0.61.7

4.8*
2.2

2.49.5
1.82.9

32.7*
2.3

5.0212.9
1.63.3

58.9***
2.0

29.2118.9
1.13.7

8.4*
0.9

1.546.1
0.51.8

3.1*
1.4

2.24.5
0.72.7

3.4
1.9

1.96.3
0.94.0

IL-10

Infected
Control

1.0

0.52.0

0.8
0.8

0.41.6
0.51.2

6.6
1.0

0.762.3
0.81.4

396.2***
0.8

169.8924.3
0.32.6

8.7
1.5

0.5138.3
0.29.7

3.7106.5
1.348.2

7.3
6.6

1.341.5
1.138.8

IL-11

Infected
Control

1.0

0.52.0

1.0
1.0

0.42.4
0.42.3

14.7*
1.2

3.659.9
0.62.6

18.8*
1.9

11.132.0
1.32.7

2.1
1.5

0.94.9
0.54.7

3.5
2.6

0.913.8
0.87.8

1.6
2.4

0.83.2
1.05.7

IFN-g

Infected
Control

1.0

0.61.8

2.5
1.9

1.63.8
0.84.3

9.4
3.9

1.850.4
2.27.1

22.1*
3.7

16.429.8
1.68.4

8.7
5.8

2.333.5
1.522.7

15.0
9.7

1.9117.4
2.047.6

10.0
6.7

4.124.6
2.023.3

CD4

Infected
Control

1.0

0.71.3

1.1
0.8

0.61.9
0.51.2

2.0*
0.8

1.04.2
0.61.2

2.3*
0.8

1.34.1
0.51.3

1.6
0.9

0.54.6
0.51.6

1.1
1.0

0.71.7
0.51.8

0.8
0.7

0.51.2
0.51.0

CD8

Infected
Control

1.0

0.61.7

0.7
0.8

0.41.4
0.41.5

0.9
1.1

0.42.0
0.81.5

0.5
1.3

0.21.3
0.82.0

0.9
0.8

0.51.8
0.61.1

2.4
1.6

1.34.4
0.93.0

2.1
1.1

1.43.0
0.52.3

TcR

Infected
Control

1.0

0.71.3

0.9
0.8

0.41.9
0.51.3

1.2
0.9

0.62.4
0.71.2

0.4
0.9

0.20.9
0.61.3

0.7
1.1

0.31.5
0.91.4

2.1
1.0

1.33.3
0.52.0

1.1
1.0

0.71.8
0.61.8

IL-1RI

Infected
Control

1.0

0.71.5

0.9
0.5

0.42.5
0.31.0

4.2*
0.6

1.413.1
0.50.8

6.9*
0.7

3.414.0
0.41.2

2.0
1.1

0.75.6
0.62.1

1.8
1.6

0.56.7
0.64.4

1.4
1.2

0.72.9
0.52.5

IL-1RII

Infected
Control

1.0

0.42.3

0.5
0.3

0.21.5
0.20.7

3.4*
0.2

0.433.2
0.20.2

17.4***
0.3

6.744.9
0.10.5

0.9
0.3

0.15.6
0.10.8

0.8
0.6

0.23.0
0.22.1

0.5
0.3

0.21.6
0.11.0

IL-1Ra

Infected
Control

1.0

0.81.3

1.1*
0.5

0.71.8
0.30.7

3.7*
0.7

1.212.1
0.61.0

8.2***
0.6

5.013.6
0.40.8

2.0*
0.6

0.75.5
0.40.8

0.6
0.4

0.40.8
0.30.5

0.7
0.5

0.51.0
0.40.7

MHC II

Infected
Control

1.0

0.71.5

1.1
0.8

0.62.2
0.61.1

1.4
1.0

0.82.5
0.81.2

0.5
1.0

0.20.9
0.61.6

0.7
0.8

0.41.4
0.51.1

1.0
0.9

0.71.4
0.61.3

1.3
1.1

0.91.8
0.81.4

IgM

Infected
Control

1.0

0.71.4

0.7
0.7

0.50.9
0.41.2

0.6
1.2

0.40.8
0.52.7

0.5
0.6

0.30.9
0.40.8

1.0
0.6

0.61.8
0.50.8

0.7
0.7

0.60.9
0.60.9

0.6
0.6

0.40.9
0.50.9

IgT

Infected
Control

1.0

0.61.7

0.4
0.2

0.20.8
0.04.4

0.5
0.8

0.21.6
0.31.7

0.6
1.2

0.21.5
0.72.1

1.6
0.2

0.74.1
0.14.1

0.4
0.9

0.17.9
0.42.2

1.0
0.3

0.52.2
0.13.9

19.9
7.9

Expression was compared to controls injected with PBS, and *indicates signicant up- or down-regulation relative to control (p < 0.05), **(p < 0.01) and ***(p < 0.001).

IL-1Ra is a structural variant of IL-1 that binds to both types of

IL-1 receptors but fails to activate cells. IL-1Ra functions have not
been described in sh, but in mammals it acts as an anti-inammatory protein which blocks the effects of IL-1. The balance
between IL-1 and IL-1Ra in tissue plays an important role in
susceptibility to and severity of many diseases. Thus, IL-1Ra
protects against IL-1-induced leucocyte inammation [3032], and
augments suppression of serum IFN-g, TNF-a, IL-1b, IL-6 and C3
concentrations [33]. In the present work IL-1Ra transcript was
expressed in the spleen of rainbow trout and the level of transcript
was increased from 8 h.p.i. to 7 d.p.i. (Table 2). The antagonist gene
transcription was also positively correlated to the expression of IL1b1, IL-1RI and IL-1RII, suggesting that the gene is involved in
down-regulation of the IL-1b induced inammation.
4.2. IL-1 receptors
Only binding of IL-1b to the IL-1R type I receptor evokes signal
transduction and activation of the nuclear factor (NF)-kB pathway
[34]. IL-1RII binds IL-1b but is unable to transduce a signal due to
the lack of a functional cytoplasmic tail [35,36]. Thus, IL-1R type II
acts as a decoy receptor, functioning by capturing excess IL-1 [34].
In the present study Y. ruckeri infection induced highly signicant
increases of both IL-1b and IL-1R1 in the spleen (Table 2). Expression of the IL-1RII decoy receptor in rainbow trout is known to be
up-regulated during ectoparasitic infection [2426]. The present
work showed that IL-1RII expression was increased during bacterial

infection, and we suggest that the function of this protein in

rainbow trout is also to bind excess IL-1b and in that way act as
a regulating molecule.
4.3. Other cytokines and chemokines
The rainbow trout IL-6 gene was recently cloned and characterized. It was found expressed in trout spleen, gill, gastrointestinal
tract, ovary and brain [37]. The key features of IL-6 appear to be
phylogenetically well conserved within the vertebrates [37] and
from mammalian immunology it is known that expression of IL-6 is
induced by pro-inammatory mediators including IL-1b [24,38
40]. IL-6 is important as the major mediator of acute phase reactions [34]. In rainbow trout IL-6 is known to be up-regulated from
a very low level following both LPS and b-glucan in vivo stimulation
[41] and to be up-regulated due to bath-vaccination with Y. ruckeri
bacterin [42]. In the present work we found that IL-6 expression
was almost silent in control sh, whereas the expression increased
10-fold 1 d.p.i., and 20-fold 3 d.p.i. (Table 2). These events support
its suggested role as a pro-inammatory mediator in this host. IL-8
belongs to the CXC chemokine subfamily, and is considered to have
a chemo-attractive effect on neutrophils in trout [43,44]. Previous
studies have described increased expression of IL-8 during Y. ruckeri
bath-vaccination and challenge [8]. Our results support the
impression of IL-8 as a central part of the inammatory reaction.
Thus, IL-8 was up-regulated in the spleen from 8 h.p.i. to 14 d.p.i.
and the higher amount of IL-8 transcripts is likely to have attracted

M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

539

Table 3
Quantitative PCR (qPCR) expression of immune relevant genes in the spleen of rainbow trout following secondary ip. infection with Y. ruckeri
Gene

Treatment

Gene expression in spleen. Fold increase of target gene relative to elongation factor a (SD)
8h

1 Day

3 Days

7 Days

14 Days

28 Days

IL-1b

Infected
Control

1.0
0.2

0.52.0
0.10.4

0.7
0.5

0.22.7
0.21.6

0.3
0.6

0.30.5
0.31.3

0.2
0.3

0.10.4
0.10.7

0.2
0.9*

0.10.4
0.41.7

0.4
0.8

0.21.1
0.22.8

IL-6

Infected
Control

1.0
0.4

0.52.1
0.11.4

0.5
2.4

0.21.5
0.86.8

0.4
0.8

0.21.0
0.51.4

0.8
0.9

0.61.2
0.51.6

1.3
2.2

0.72.4
1.05.2

0.4
2.4*

0.20.8
1.24.7

IL-8

Infected
Control

3.7
2.0

1.310.5
1.14.0

3.8
3.2

2.85.3
1.85.6

2.1
3.1

1.43.1
1.95.0

2.8
3.6

1.45.5
2.74.7

1.9
3.3

1.23.3
2.25.0

3.7
5.3

2.94.7
3.09.4

IL-10

Infected
Control

1.1
1.4

0.91.4
0.63.5

2.2
0.9

0.95.4
0.32.8

2.0
2.7

0.94.3
1.54.9

1.5
0.7

0.63.6
0.41.4

1.7
14.5*

0.65.4
3.070.2

0.4
0.7

0.20.8
0.41.3

IL-11

Infected
Control

1.7
1.1

0.65.0
0.62.0

1.4
2.7*

0.82.4
2.13.4

2.3
2.2

1.82.9
1.14.4

1.8
3.7

1.03.1
2.26.4

6.5
6.9

3.911.1
3.812.6

2.7
5.8

2.33.1
3.69.4

IFN-g

Infected
Control

6.3
1.7

2.714.9
1.12.8

4.0
5.2

2.013.5
2.013.5

3.8
8.7*

1.78.2
4.417.0

2.7
20.2*

1.35.3
6.562.6

5.4
2.1

1.915.6
1.53.0

2.2
1.7

0.76.8
0.93.3

CD4

Infected
Control

1.4
1.2

0.92.2
0.62.4

0.9
1.0

0.51.5
0.61.9

0.9
1.5

0.71.1
0.82.9

1.2
1.9

0.82.0
1.42.5

1.6
2.0

1.12.2
1.13.6

1.2
1.7

0.82.0
1.12.6

CD8

Infected
Control

1.2
1.6

0.81.9
1.02.7

0.9
1.0

0.42.1
0.33.9

1.7
1.1

0.83.6
0.71.7

2.5
1.9

1.63.8
1.52.6

3.4*
1.8

2.35.0
1.22.7

2.2
2.8

1.53.2
1.94.1

TcR

Infected
Control

0.8
1.0

0.61.2
0.51.9

1.1
1.1

0.52.4
0.71.8

1.3
1.1

0.81.9
0.62.0

2.0
1.9

1.33.2
1.13.3

2.9
2.5

1.55.3
1.83.4

1.7
2.8*

1.32.1
2.23.6

IL-1RI

Infected
Control

0.9
0.8

0.41.9
0.41.4

0.8
1.4

0.41.6
0.73.0

1.0
1.2

0.91.2
0.72.3

0.9
1.5

0.61.4
0.72.9

1.4
2.4

0.72.6
1.24.5

1.0
1.1

0.52.2
0.62.1

IL-1RII

Infected
Control

0.3
0.3

0.10.7
0.10.5

0.2
0.3

0.10.4
0.10.6

0.3
0.3

0.20.4
0.20.5

0.1
0.2

0.10.2
0.10.4

0.3
0.9*

0.20.5
0.42.2

0.2
0.2

0.10.4
0.10.6

IL-1Ra

Infected
Control

0.9
0.6

0.61.4
0.31.0

1.0
1.1

0.61.5
0.81.5

0.9
0.8

0.71.1
0.51.2

0.8
0.9

0.41.5
0.71.2

1.0
0.5

0.81.3
0.21.0

0.7
0.7

0.41.1
0.41.3

MHC II

Infected
Control

1.1
1.2

0.71.9
0.71.9

0.9
1.4

0.61.4
0.82.5

1.1
1.0

0.91.4
0.71.5

1.6
1.6

1.22.3
1.41.8

1.5
1.6

1.12.1
1.41.9

1.5
1.7

1.02.3
1.42.1

IgM

Infected
Control

0.5
0.6

0.30.8
0.40.9

0.6
0.7

0.31.1
0.41.4

0.6
0.7

0.40.9
0.51.2

0.5
0.9

0.31.0
0.61.3

0.6
0.6

0.50.7
0.40.8

0.4
0.6

0.30.7
0.41.1

IgT

Infected
Control

0.9
1.3

0.41.9
1.01.6

0.8
0.2

0.31.8
0.17.8

1.5
1.7

0.54.2
1.03.0

2.5
1.2

1.44.5
0.72.2

1.1
0.9

0.62.0
0.41.8

1.3
0.5

0.53.0
0.14.7

Expression was compared to controls injected with PBS, and * indicates signicant up- or down-regulation relative to control (p < 0.05).

neutrophils to the spleen which could partly explain the weight

increase of this organ during the primary infection (Fig. 3). IL-8 was
(as IL-1b1 and IL-10) positively correlated to the expression of Y.
ruckeri 16S ribosomal RNA gene, which could indicate that IL-8 is
attracting phagocytes to the site of inammation. This result is in
agreement with the nding that Y. ruckeri bacterial counts have
been associated with increased levels of CXCd mRNA expression in
the spleen of infected rainbow trout [8].
It is generally agreed that regulation of inammation results
from a balance between pro- and anti-inammatory cytokines.
Regulation of inammation is a central event in the immune
response reducing the negative effects of the inammatory
processes. Recently, some anti-inammatory factors in teleosts
have been cloned. The cytokine IL-10 belongs to this group, and IL10 homologues have been found in rainbow trout [45], fugu [46],
carp [47] and zebrash [48]. IL-10, initially known as cytokine
synthesis inhibitory factor, is a multifunctional cytokine and
demonstrates immunosuppressive function. The main function of
IL-10 seems to be regulation of the inammatory response, thereby
minimizing damage to the host induced by an excessive response.
Thus, IL-10 blocks chemokine receptors and inhibits the effect of
pro-inammatory cytokines [49] and inhibits the activation of
macrophages/monocytes, whereby it controls cytokine synthesis,
nitric oxide (NO) production and the expression of other costimulatory molecules [50]. The function of IL-10 in teleosts is less
clear. Our study demonstrated high expression of IL-10 3 d.p.i. and
no pro-inammatory cytokines were found up-regulated after the

high IL-10 expression (Table 2). This could indicate that IL-10 serves
an anti-inammatory role also in rainbow trout corresponding to
its action in mammals.
IL-11 is a multifunctional cytokine that in mammals stimulates
haematopoietic progenitor cells and exerts a series of important
immunomodulatory effects. This cytokine is in rainbow trout
modulated by infection and other cytokines, suggesting that IL-11 is
an active player in the cytokine network and the sh immune
response to infection [51]. During our investigation on infection
with Y. ruckeri, IL-11 transcription increased from day 1 to 3.
Therefore, the exact function of this cytokine should be addressed
in future studies.
4.4. Expression of genes involved in the adaptive immunity
In mammals, CD4 T cells differentiate into IFN-g producing
cells following exposure to IL-1b [52]. The present study could
indicate that a similar pathway occurs in rainbow trout. We
detected an increased expression of IL-1b1 followed by a doubling
of the CD4 expression. This was again associated with a highly
increased expression of IFN-g transcripts (22.1-fold). In the present
study no regulation of expression of the genes encoding IgM, IgT
and MHC II was found in the spleen (Tables 2 and 3). It is possible
that the main regulation of immunoglobulin expression takes place
in the head kidney and not in the spleen. It has previously been
indicated from studies on vaccinated trout, showing that the antibody response was mainly caused by Ig secretion from plasma cells

540

M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

Fig. 5. Gene expression of IL-1b, IL-1 receptor antagonist (Ra) and IL-1 receptor I and II, in the spleen of rainbow trout infected with Y. ruckeri (n 5).

in the anterior kidney [5,53]. However, the lack of increases in Ig

gene transcripts during the infections may also be explained by the
fact that Y. ruckeri-specic Ig mRNA merely represents a limited
fraction of the huge amount of mRNA encoding secreted Ig [5].
In mammals CD8a is known as a marker for cytotoxic T cells.
CD8 positive T cells recognize antigens that are displayed as peptide:MHC class I complexes on the cell surface [54]. The gene
encoding CD8a has been identied in rainbow trout [55] and
specic cytotoxicity of T-cells has also been recognized using clonal
rainbow trout [56,57].
There were no difference in the transcript levels of CD8a
between infected and control sh during the primary Y. ruckeri
infection, but during the secondary infection, the CD8a gene was
the only one up-regulated relative to the controls. It is noteworthy
that the gene encoding CD-8a previously was found up-regulated
in rainbow trout bath-vaccinated with Y. ruckeri bacterin [42],
which indicates that activity of cytotoxic T-cells plays a role in the
cellular adaptive protection mechanisms against Y. ruckeri infection. Increased expression of CD-8a in spleen of rainbow trout was
previously reported following exposure to other viral and bacterial
pathogens such as infectious haematopoietic necrosis virus (IHNV)
and Flavobacterium psychrophilum [19] and a similar reaction in
peripheral blood leucocytes after infection with viral haemorrhagic
septicaemia virus (VHSV) [57].
In conclusion, this study on the development of adaptive
immunity in rainbow trout suggests that the immune response is
initiated by cytokines which activate lymphocytes to initiate an
adaptive immune response eventually leading to long-lasting
protective immunity. The amount of Y. ruckeri in the spleen was
increased 21,000-fold during the rst 3 days before the bacterial
infection decreased probably due to innate immune response
factors. During the re-infection with the same dose of Y. ruckeri, the
presence of bacteria was only detectable in three out of ve reinfected sh 8 h.p.i. and in none of the tested sh 1 d.p.i. These data
comply with the viewpoint that the adaptive immunity is much
more efcient than the innate immune response when clearing
a bacterial infection in rainbow trout.
The weak expression of the investigated genes in the well protected rainbow trout following the re-infection was noteworthy. It
corresponds to expression data on the immune response in
rainbow trout reacting to a parasite where, following full recovery

from the primary infection, re-infection did not elicit transcription

levels above those seen in un-infected rainbow trout [25]. One
explanation of the weak gene expression during the re-infection is
that the pathogen is killed very fast, whereby the associated
expression of some pro-inammatory cytokines is kept at
a minimum. The present work has pin-pointed a series of immunological events during this dynamic infection and re-infection
interaction between host and pathogen. Following the initial
regulated cytokine expression (involving IL1b and its antagonists,
receptor and decoy receptor) it is indicated that the protective
effect may comprise regulated activity of T-cells.
Acknowledgments
This work was supported in part by a grant to the project 27407-0354 from the Danish Agency for Science Technology and
Innovation and by the integrated research project IMAQUANIM
sponsored by the European Commission and by a grant to the
project FFS05-7 Welfare in farmed Rainbow trout from the
Danish Ministry for Food.
References
[1] Fernandez L, Mendez J, Guijarro JA. Molecular virulence mechanisms of the
sh pathogen Yersinia ruckeri. Veterinary Microbiology 2007;125:110.
[2] Tobback E, Decostere A, Hermans K, Haesebrouck F, Chiers K. Yersinia ruckeri
infections in salmonid sh. Journal of Fish Diseases 2007;30:25768.
[3] Horne MT, Barnes AC. Enteric redmouth disease (Yersinia ruckeri). In:
Woo PTK, Bruno DW, editors. Fish diseases and disorders. Viral, bacterial and
fungal infections, vol. 3. CABI Publishing; 1999. p. 45577.
[4] Ellis AE. Immunity to bacteria in sh. Fish & Shellsh Immunology
1999;9:291308.
[5] Raida MK, Buchmann K. Temperature-dependent expression of immunerelevant genes in rainbow trout following Yersinia ruckeri vaccination.
Diseases of Aquatic Organisms 2007;77:4152.
[6] Chaves-Pozo E, Munoz P, Lopez-Munoz A, Pelegrin P, Ayala AG, Mulero V, et al.
Early innate immune response and redistribution of inammatory cells in the
bony sh gilthead seabream experimentally infected with Vibrio anguillarum.
Cell and Tissue Research 2005;320:618.
[7] Espenes A, Press CM, Dannevig BH, Landsverk T. Immune complex trapping in
the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss). Cell and Tissue
Research 1995;282:418.
[8] Wiens GD, Glenney GW, Lapatra SE, Welch TJ. Identication of novel rainbow
trout (Oncorynchus mykiss) chemokines, CXCd1 and CXCd2: mRNA expression
after Yersinia ruckeri vaccination and challenge. Immunogenetics
2006;58:30823.

M.K. Raida, K. Buchmann / Fish & Shellsh Immunology 25 (2008) 533541

[9] Welch TJ, Wiens GD. Construction of a virulent, green uorescent proteintagged Yersinia ruckeri and detection in trout tissues after intraperitoneal and
immersion challenge. Diseases of Aquatic Organisms 2005;67:26772.
[10] Murphy K, Travers P, Walport M. Janeways immuno biology. 7th ed. New York
and London: Garland Science Publishing; 2008.
[11] Janeway CA. Presidential address to the American Association of Immunologists the road less traveled by: the role of innate immunity in the adaptive
immune response. Journal of Immunology 1998;161:53944.
[12] Fouz B, Zarza C, Amaro C. First description of non-motile Yersinia ruckeri
serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss
(Walbaum), cultured in Spain. Journal of Fish Diseases 2006;29:33946.
[13] Ellis AE. Fish vaccination. 1st ed. London: Academic Press Limited; 1988.
[14] Gibello A, Blanco MM, Moreno MA, et al. Development of a PCR assay for
detection of Yersinia ruckeri in tissues of inoculated and naturally infected
trout. Applied and Environmental Microbiology 1999;65:34650.
[15] Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I. Evaluation of potential
reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Molecular Biology, http://www.biomedcentral.com/content/pdf/1471-21996-21.pdf,
2005;6.
[16] Ingerslev HC, Pettersen EF, Jakobsen RA, Petersen CB, Wergeland HI. Expression proling and validation of reference gene candidates in immune relevant
tissues and cells from Atlantic salmon (Salmo salar L.). Molecular Immunology
2006;43:1194201.
[17] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta CT) method. Methods
2001;25:4028.
[18] Press CM, Evensen O. The morphology of the immune system in teleost shes.
Fish & Shellsh Immunology 1999;9:30918.
[19] Overturf K, LaPatra S. Quantitative expression of immunological factors in
rainbow trout, Oncorhynchus mykiss (Walbaum), after infection with either
Flavobacterium psychrophilum, Aeromonas salmonicida, or infectious haematopoietic necrosis virus. Journal of Fish Diseases 2006;29:21524.
[20] Hong SH, Peddie S, Campos-Perez JJ, Zou J, Secombes CJ. The effect of intraperitoneally administered recombinant IL-1 beta on immune parameters and
resistance to Aeromonas salmonicida in the rainbow trout (Oncorhynchus
mykiss). Developmental and Comparative Immunology 2003;27:80112.
[21] Martin S, Zou J, Houlihan D, Secombes C. Directional responses following
recombinant cytokine stimulation of rainbow trout (Oncorhynchus mykiss)
RTS-11 macrophage cells as revealed by transcriptome proling. BMC Genomics 2007;8:150.
[22] Secombes CJ, Bird S, Cunningham C, Zou J. Interleukin-1 in sh. Fish &
Shellsh Immunology 1999;9:33543.
[23] Secombes CJ, Zou J, Laing K, Daniels GD, Cunningham C. Cytokine genes in sh.
Aquaculture 1999;172:93102.
[24] Gonzalez SF, Huising MO, Stakauskas R, Forlenza M, Verburg-van
Kemenade BML, Buchmann K, et al. Real-time gene expression analysis in carp
(Cyprinus carpio L.) skin: inammatory responses to injury mimicking infection with ectoparasites. Developmental and Comparative Immunology
2007;31:24454.
[25] Lindenstrom T, Buchmann K, Secombes CJ. Gyrodactylus derjavini infection
elicits IL-1 beta expression in rainbow trout skin. Fish & Shellsh Immunology
2003;15:10715.
[26] Sigh J, Lindenstrom T, Buchmann K. Expression of pro-inammatory cytokines
in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multiliis. Fish & Shellsh Immunology 2004;17:7586.
[27] Tafalla C, Coll J, Secombes CJ. Expression of genes related to the early immune
response in rainbow trout (Oncorhynchus mykiss) after viral haemorrhagic
septicemia virus (VHSV) infection. Developmental and Comparative Immunology 2005;29:61526.
[28] Scapigliati G, Costantini S, Colonna G, Facchiano A, Buonocore F, Bossu P, et al.
Modelling of sh interleukin-1 and its receptor. Developmental and
Comparative Immunology 2004;28:42941.
[29] Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095147.
[30] Bandara G, Mueller GM, Galealauri J, Tindal MH, Georgescu HI, Suchanek MK,
et al. Intraarticular expression of biologically active interleukin-1 receptor
antagonist protein by ex vivo gene transfer. Proceedings of the National
Academy of Sciences of the United States of America 1993;90:107648.
[31] Cominelli F, Bortolami M, Pizarro TT, Monsacchi L, Ferretti M, Brewer MT, et al.
Rabbit interleukin-1 receptor antagonist. Cloning, expression, functional
characterization, and regulation during intestinal inammation. The Journal of
Biological Chemistry 1994;269:696271.
[32] Hung GL, Galealauri J, Mueller GM, Georgescu HI, Larkin LA, Suchanek MK,
et al. Suppression of intraarticular responses to interleukin-1 by transfer of the
interleukin-1 receptor antagonist gene to synovium. Gene Therapy
1994;1:649.
[33] Yang H, Tuzun E, Alagappan D, Yu X, Scott BG, Ischenko A, et al. IL-1 receptor
antagonist-mediated therapeutic effect in murine myasthenia gravis is
associated with suppressed serum proinammatory cytokines, C3, and antiacetylcholine receptor IgG1. Journal of Immunology 2005;175:201825.

541

[34] Engelsma MY, Huising MO, van Muiswinkel WB, Flik G, Kwang J, Savelkoul HFJ,
et al. Neuroendocrineimmune interactions in sh: a role for interleukin-1.
Veterinary Immunology and Immunopathology 2002;87:46779.
[35] Dinarello CA, Abraham E. Does blocking cytokines in sepsis work? American
Journal of Respiratory and Critical Care Medicine 2002;166:11567.
[36] Sangrador-Vegas A, Martin SAM, ODea PG, Smith TJ. Cloning and characterization of the rainbow trout (Oncorhynchus mykiss) type II interleukin-1
receptor cDNA. European Journal of Biochemistry 2000;267:70317.
[37] Iliev DB, Castellana B, MacKenzie S, Planas JV, Goetz FW. Cloning and
expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus
mykiss). Molecular Immunology 2007;44:18037.
[38] Ammit AJ, Lazaar AL, Irani C, ONeill GM, Gordon ND, Amrani Y, et al. Tumor
necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from
human airway smooth muscle cells modulation by glucocorticoids and betaagonists. American Journal of Respiratory Cell and Molecular Biology
2002;26:46574.
[39] Bergamaschi A, Corsi M, Garnier MJ. Synergistic effects of cAMP-dependent
signalling pathways and IL-1 on IL-6 production by H19-7/IGF-IR neuronal
cells. Cellular Signalling 2006;18:167984.
[40] Takezako N, Hayakawa M, Hayakawa H, Aoki S, Yanagisawa K, Endo H, et al.
ST2 suppresses IL-6 production via the inhibition of I kappa B degradation
induced by the LPS signal in THP-1 cells. Biochemical and Biophysical Research
Communications 2006;341:42532.
[41] Lovoll M, Fischer U, Mathisen GS, Bogwald J, Ototake M, Dalmo RA. The C3
subtypes are differentially regulated after immunostimulation in rainbow
trout, but head kidney macrophages do not contribute to C3 transcription.
Veterinary Immunology and Immunopathology 2007;117:28495.
[42] Raida MK, Buchmann K. Bath vaccination of rainbow trout (Oncorhynchus
mykiss Walbaum) against Yersinia ruckeri: effects of temperature on protection
and gene expression. Vaccine 2008;26:105062.
[43] Zhang H, Thorgaard GH, Ristow SS. Molecular cloning and genomic structure of an interleukin-8 receptor-like gene from homozygous clones of
rainbow trout (Oncorhynchus mykiss). Fish & Shellsh Immunology
2002;13:2518.
[44] Jimenez N, Coll J, Salguero FJ, Tafalla C. Co-injection of interleukin 8 with the
glycoprotein gene from viral haemorrhagic septicemia virus (VHSV) modulates the cytokine response in rainbow trout (Oncorhynchus mykiss). Vaccine
2006;24:561526.
[45] Inoue Y, Kamota S, Itoa K, Yoshiura Y, Ototake M, Moritomo T, et al. Molecular
cloning and expression analysis of rainbow trout (Oncorhynchus mykiss)
interleukin-10 cDNAs. Fish & Shellsh Immunology 2005;18:33544.
[46] Zou J, Clark MS, Secombes CJ. Characterisation, expression and promoter
analysis of an interleukin 10 homologue in the puffer sh, Fugu rubripes.
Immunogenetics 2003;55:32535.
[47] Savan R, Igawa D, Sakai M. Cloning, characterization and expression analysis of
interleukin-10 from the common carp, Cyprinus carpio L. European Journal of
Biochemistry 2003;270:464754.
[48] Zhang DC, Shao YQ, Huang YQ, Jiang SG. Cloning, characterization and
expression analysis of interleukin-10 from the zebrarish (Danio rerion). Journal
of Biochemistry and Molecular Biology 2005;38:5716.
[49] DAmico G, Frascaroli G, Bianchi G, Transidico P, Doni A, Vecchi A, et al.
Uncoupling of inammatory chemokine receptors by IL-10: generation of
functional decoys. Nature Immunology 2000;1:38791.
[50] Dumoutier L, Louahed J, Renauld JC. Cloning and characterization of
IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. Journal of Immunology
2000;164:18149.
[51] Wang T, Holland JW, Bols N, Secombes CJ. Cloning and expression of the rst
nonmammalian interleukin-11 gene in rainbow trout (Oncorhynchus mykiss).
FEBS Journal 2005;272:113647.
[52] Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins
1 beta and 6 but not transforming growth factor-beta are essential for the
differentiation of interleukin 17-producing human T helper cells. Nature
Immunology 2007;8:9429.
[53] Bromage ES, Kaattari IM, Zwollo P, Kaattari SL. Plasmablast and plasma cell
production and distribution in trout immune tissues. Journal of Immunology
2004;173:731723.
[54] Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology: the
immune system in health and disease. 6th ed. New York and London: Garland
Science Publishing; 2005.
[55] Hansen JD, Strassburger P. Description of an ectothermic TCR coreceptor, CD8
alpha, in rainbow trout. Journal of Immunology 2000;164:31329.
[56] Fischer U, Utke K, Ototake M, Dijkstra JM, Kollner B. Adaptive cell-mediated
cytotoxicity against allogeneic targets by CD8-positive lymphocytes of
rainbow trout (Oncorhynchus mykiss). Developmental and Comparative
Immunology 2003;27:32337.
[57] Utke K, Bergmann S, Lorenzen N, Kollner B, Ototake M, Fischer U. Cellmediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral
haemorrhagic septicaemia virus. Fish & Shellsh Immunology 2007;22:18296.