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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
534
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.
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
536
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).
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.
537
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).
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.
538
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).
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).
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
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).
541
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