Open Access

Comparative analysis of the acute response of the trout, O. mykiss, head kidney to in vivo challenge with virulent and attenuated infectious hematopoietic necrosis virus and LPS-induced inflammation

  • Simon MacKenzie1Email author,
  • Joan C Balasch1,
  • Beatriz Novoa2,
  • Laia Ribas3,
  • Nerea Roher4,
  • Aleksei Krasnov5 and
  • Antonio Figueras2
Contributed equally
BMC Genomics20089:141

DOI: 10.1186/1471-2164-9-141

Received: 01 November 2007

Accepted: 26 March 2008

Published: 26 March 2008

Abstract

Background

The response of the trout, O. mykiss, head kidney to bacterial lipopolysaccharide (LPS) or active and attenuated infectious hematopoietic necrosis virus (IHNV and attINHV respectively) intraperitoneal challenge, 24 and 72 hours post-injection, was investigated using a salmonid-specific cDNA microarray.

Results

The head kidney response to i.p. LPS-induced inflammation in the first instance displays an initial stress reaction involving suppression of major cellular processes, including immune function, followed by a proliferative hematopoietic-type/biogenesis response 3 days after administration. The viral response at the early stage of infection highlights a suppression of hematopoietic and protein biosynthetic function and a stimulation of immune response. In fish infected with IHNV a loss of cellular function including signal transduction, cell cycle and transcriptional activity 72 hours after infection reflects the tissue-specific pathology of IHNV infection. attIHNV treatment on the other hand shows a similar pattern to native IHNV infection at 24 hours however at 72 hours a divergence from the viral response is seen and replace with a recovery response more similar to that observed for LPS is observed.

Conclusion

In conclusion we have been able to identify and characterise by transcriptomic analysis two different types of responses to two distinct immune agents, a virus, IHNV and a bacterial cell wall component, LPS and a 'mixed' response to an attenuated IHNV. This type of analysis will lead to a greater understanding of the physiological response and the development of effective immune responses in salmonid fish to different pathogenic and pro-inflammatory agents.

Background

The orchestration of a successful immune response to infection requires an integrated tissue response coordinated by specific cytokine and chemokine release. Pathogen-specific immune responses are coordinated and dependent upon the activation of specific pathogen recognition receptors (PRRs), molecular moieties present upon sub-sets of leukocytes such as macrophages or dendritic cells. PRRs respond to pathogens or their PAMPs (Pathogen Associated Molecular Patterns) by the initiation of distinct transcriptomic programmes which will dictate the cellular/tissue response [13]. In mammals different transcriptional programmes have been identified by microarray analysis for specific PAMPs (viral, bacteria and yeast) by both macrophages and dendritic cells which initiate the immune response by secreting molecules such as pro-inflammatory cytokines [4, 5].

The availability of salmonid-specific gene chips [68] has provided the means to begin to characterise the salmonid immune response at a global gene level both in vitro and in vivo. This technology will provide a deeper understanding of overall cellular and tissue processes during immune activation. A number of recent reports concerning PAMP recognition [9], activated macrophage transcriptomics [8] and immunomics [1016] and genome-wide surveys [17] show that fish and fish macrophages clearly respond differentially to different pathogens. This therefore should lead to different physiological/immunological responses in vivo upon which the survival of the organism is based.

The head kidney or anterior kidney located posterior to the cranium, is the central hematopoietic organ in salmonids and other fish species. In addition, it contains adrenalin-producing chromaffin cells and also plays a major endocrine role in secretion of cortisol, the major glucocorticoid and mineralocorticoid in fish [1821]. The head kidney can thus integrate the neuro-immuno-endocrine milieu in normal and pathological states. However, few global gene regulation studies concerning the molecular regulation of head kidney function during infection or PAMP stimulation in salmonids [15] have been reported although many studies have used this tissue as a primary source of macrophage-like cells for studies on the activation of the immune system [22, 23].

The Novirhabdovirus infectious hematopoietic necrosis virus (IHNV) is probably one of the most important fish viral pathogens, responsible for great mortalities in farmed salmonids [24, 25]. As for all the Rhabdoviridae, the genome of IHNV consists of a single-stranded negative-sense RNA which has been entirely sequenced [26, 27]. Their genome codes for five structural proteins: a nucleoprotein (N), a polymerase-associated protein (P), a matrix protein (M), an RNA-dependent RNA polymerase (L) and a surface glycoprotein (G) responsible for immunogenicity [2830]. An additional gene, only present in some fish rhabdoviruses, located between the G and L genes, encodes a non-structural protein NV, whose putative role in virus replication remains to be fully evaluated [31] but appears to be linked to viral growth and pathogenicity [30]. The strong early immune response elicited by IHNV and other related RNA viruses has favored the development of several vaccines using a reverse genetics approach [32, 33]; however, recently a DNA vaccine against IHN has been registered in Canada (Novartis Animal Health Canada, Inc).

LPS, the major constituent of the external layer of the outer layer of Gram-negative bacteria, is a widely used PAMP-preparation which induces potent immune responses in which the lipid A portion of the molecule is primarily responsible for the endotoxic properties observed in experimental animals [34, 35]. Fish present a remarkable tolerance to LPS challenge in comparison to mammals which has been postulated to be due to differences in receptor-mediated recognition of LPS [36]. In vivo challenge to high concentrations of LPS in fish does not result in endotoxin-mediated mortality [23].

We have carried out in vivo challenges using either live or attenuated IHNV (infectious hematopoietic necrosis virus) or bacterial lipopolysaccharide (E. coli LPS) in trout (Oncorhynchus mykiss). Total RNAs from head kidney tissue were sampled, 1 and 3 days post intra-peritoneal injection (i.p.), and analysed by gene chip analysis. We have identified a generalised immune/stress/hematopoietic gene response to all treatments and a large set of viral-specific genes responding to IHNV infection. Gene ontology analysis presents two distinct physiological responses to either LPS or IHNV in which IHNV pathogenesis can be clearly identified. The response to LPS indicates a general inflammatory response followed by a significant hematopoietic response. Here we present a comparison of the differential gene expression patterns induced in vivo by a generic PAMP, E. coli LPS, and a viral pathogen, IHNV, and an attenuated form of the viral pathogen, attIHNV in the head kidney of the rainbow trout.

Results and Discussion

Fish survival and pathogenesis

The epizootiology of IHN in young fish has been thoroughly described [27, 3742] and includes widespread hemorrhages in kidney, liver and musculature leading to anemia, and extensive necrosis of major hematopioetic tissues (head kidney and spleen). No mortalities, external signs or histological lesions were observed in fish injected with IHNV or attIHNV at sampling time points. The remaining or non-sampled fish displayed the referred pathological features of IHN, arriving to a 100% mortality 7–9 days and 15–17 days after challenge with virulent or attenuated IHNV, respectively. In fish challenged with LPS no mortalities were recorded in experimental groups. This is a typical response in which fish do not show an appreciable 'septic shock' type response. A molecular mechanism has been proposed addressing LPS-tolerance in fish which may be due to differential signalling from Toll-like receptors in which the classical TLR4 paradigm differs from that observed in mammals. In fact, rainbow trout macrophages have been shown to be about 1000 times less sensitive to LPS than mammalian macrophages [9].

Overview of viral and LPS-induced differential gene expression in the head kidney

In order to examine the transcriptional profile of head kidneys (HK) dissected from trout treated with either intra-peritoneal LPS or infection with IHNV or attIHNV we used a salmonid-specific cDNA microarray platform previously validated for studies involving stress, toxicity and immune response in trout [68, 43].

Total numbers of genes significantly expressed in each of the treatments is shown in fig. 1 and a list of 20 selected genes, ranked by the expression levels was included in tables 1, 2, 3. Applying a selection criteria based in a classical cut-off value of >2 fold change over the differentially expressed genes (p < 0.01) emphasizes the stronger global induction of gene expression following the inoculation of both active and attenuated forms of IHNV (fig. 1b). Within differentially expressed gene groups (>1 fold change, p < 0.01; fig. 1a), bacterial LPS induces a higher response in comparison to viral groups. Although the number of ranked selected genes (p-value < 0.01) is considerably lower than in viral treatments, about 56.5% (n = 58) and 60% (n = 45) of regulated genes at 24 and 72 hours respectively achieve more than 2 fold change in its expression levels, in contrast with attenuated (15%; n = 413 at 24 h and 25%; n = 591 at 72 h) or active (26%, n = 570 at 24 h and 35.5%; n = 428 at 72 h) viral inoculation of IHNV.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig1_HTML.jpg
Figure 1

Gene response to lipolysaccharide and viral (active and attenuated IHNV) in the head kidney of trout ( O. mykiss ). (a) Total gene number (>1 fold change; p < 0.01) expressed on the microarray in each treatment and in each time point (b) Differentially expressed genes (p < 0.01; FDR < 0.05) with a 2-fold increase in expression levels are shown for each treatment and time point.

Table 1

List of top 20 up and down regulated genes in the head kidney of trout, 24 and 72 hours after i.p. injection of LPS. Genes were selected for significant differential expression at p < 0.01 (FDR < 0.05). Values are expressed as FC, fold change.

24 hours UP

   

72 hours UP

   

Clone ID

Clone Name

FC

p

Clone ID

Clone Name

FC

p

est03d11

Unknown-42

8.42

0.000

est03d11

Unknown-42

3.81

0.000

est04b01

Similar to rRNA (Vangl2)

4.54

0.004

est03e02

Hypothetical-fish 15

3.39

0.001

EXOB1_H05

Transposase-6

3.16

0.000

est04e04

MHC class 1b antigen

3.17

0.006

EXOB2_D06

NADH dehydrogenase subunit 5-1

2.76

0.000

EXOB2_H06

High affinity immunoglobulin epsilon receptor alpha

2.73

0.003

est02g07

Ig mu heavy chain disease protein

2.73

0.001

CA387837

Nucleolar protein NAP57

2.27

0.000

est02a11

Ig kappa chain V-IV region B17-2

2.71

0.002

EXOB4_C11

High affinity immunoglobulin gamma Fc receptor I precursor

2.15

0.008

EST1-3A_G09

Unknown-75

2.32

0.001

ENH2_F09

Beta-2-microglobulin-1

1.99

0.004

CA383564

Coatomer epsilon subunit 1

2.25

0.001

EXOB2_A09

60S ribosomal protein L5-2

1.80

0.005

est04e04

MHC class 1b antigen

2.24

0.007

HST0001_C05

Cell division control protein 42 homolog

1.72

0.009

EST1-3A_F12

Transposase-59

2.12

0.001

EXOB3_F09

MHC class I heavy chain-1

1.71

0.001

HK0002_D09

Prothymosin alpha

2.03

0.003

est01g04

5-aminolevulinate synthase

1.58

0.010

est02e09

Ig heavy chain V-III region HIL

2.00

0.005

    

EXOB4_D12

Hpa repeat-1

1.99

0.001

    

HK0003_E06

DnaJ homolog subfamily C member 9

1.96

0.005

    

HK0003_E08

Unknown-194

1.90

0.007

    

est04g12

Envelope protein

1.90

0.004

    

HK0002_H08

TATA-binding protein associated factor 2N

1.79

0.003

    

CA387665

Cytochrome P450 2J2

1.79

0.000

    

est01h01

Unknown-19

1.76

0.009

    

est03b11

Acidic leucine-rich nuclear phosphoprotein 32 A-1

1.68

0.008

    

24 hours DOWN

   

72 hours DOWN

   

EXOB2_D07

Ependymin related protein-1

-2.28

0.002

est04c10

Unknown-56

-2.04

0.000

HST0001_C04

Hemoglobin alpha chain

-2.42

0.004

CA367764

Calmodulin-1

-2.13

0.001

EST1-3A_D08

Glyceraldehyde-3-phosphate dehydrogenase-6

-2.49

0.009

est03c04

Matrix metalloproteinase-9

-2.21

0.000

EXOB1_H06

CC chemokine SCYA110-2

-2.55

0.005

HKT0001_E07

Actin, alpha skeletal 1

-2.28

0.002

EST1-3A_H06

Transcription factor jun-B-1

-2.89

0.003

est01f03

Deltex protein 1

-2.37

0.001

est03f08

Transaldolase

-2.91

0.002

est01e02

Thymosin beta-4-1

-2.73

0.002

EXOB2_H01

Unknown-100

-2.91

0.002

EXOB3_G05

Actin, cytoplasmic 2

-2.78

0.000

est02f08

Serine protease-like protein-1

-2.97

0.000

EST1-3A_D08

Glyceraldehyde-3-phosphate dehydrogenase-6

-3.14

0.000

EST1-3A_A09

Serine protease-like protein-2

-3.96

0.003

EST1-3A_B03

Hypothetical-fish 44

-3.16

0.007

CA371363

Glucose-6-phosphate isomerase-1

-4.35

0.000

CA348284

CCAAT/enhancer binding protein beta

-3.22

0.003

HST0001_D08

Beta-globin

-4.46

0.000

CA374193

Chemokine receptor CXCR4

-3.71

0.000

est04e05

Glutathione peroxidase-gastrointestinal

-4.54

0.000

utu04f08

Actin, alpha skeletal 5

-3.81

0.001

est01c04

Unknown-11

-6.17

0.001

KVkm2_F01

Unknown-224

-4.21

0.000

EXOB2_D05

Matrix metalloproteinase 9-2

-6.63

0.000

est01f01

DNA-binding protein inhibitor ID-1

-4.64

0.000

est01e10

Tolloid-like protein (nephrosin)-1

-6.69

0.001

est02f08

Serine protease-like protein-1

-4.64

0.001

CA348284

CCAAT/enhancer binding protein beta

-7.36

0.006

EST1-3A_A09

Serine protease-like protein-2

-5.52

0.000

est03c04

Matrix metalloproteinase-9

-7.44

0.000

EXOB3_H01

Matrix metalloproteinase-13

-5.88

0.007

EXOB2_G12

Tolloid-like protein (nephrosin)-2

-9.26

0.000

EXOB2_H01

Unknown-100

-12.08

0.000

EST1-3A_B03

Hypothetical-fish 44

-14.01

0.000

est01e10

Tolloid-like protein (nephrosin)-1

-12.97

0.000

EXOB3_H01

Matrix metalloproteinase-13

-17.18

0.000

EXOB2_G12

Tolloid-like protein (nephrosin)-2

-13.47

0.000

Table 2

List of top 20 up and down regulated genes in the head kidney of trout, 24 and 72 hours after i.p. injection of attIHNV. Genes were selected for significant differential expression at p < 0.01 (FDR < 0.05). Values are expressed as FC, fold change.

24 hours UP

   

72 hours UP

   

Clone ID

Clone Name

FC

p

Clone ID

Clone Name

FC

p

EXOB2_B10

Hemoglobin beta chain

19.46

0.000

est03e08

Hypothetical-fish 36

3.06

0.000

HST0001_C04

Hemoglobin alpha chain

11.19

0.000

CA343473

C3a anaphylatoxin chemotactic receptor

2.98

0.000

KVkm2_A01

Unknown-219

10.93

0.000

EXOB2_G11

Stanniocalcin-1

2.93

0.000

HST0001_D04

Alpha-globin I-2

10.93

0.000

EXOB1_B02

Hypothetical-fish 34

2.84

0.000

EXOB4_A03

Carbonic anhydrase

10.57

0.000

KVkm2_H07

Unknown-226

2.77

0.000

CA366564

Huntingtin

8.52

0.000

EXOB3_A01

Unknown-103

2.73

0.000

EXOB2_B12

RING finger protein 103

8.34

0.000

CA363723

Cyclin C

2.64

0.000

HST0001_C07

Unknown-213

7.60

0.000

EXOB4_H01

Unknown-132

2.63

0.000

ENH2_E07

Unknown-3

7.04

0.000

est04c02

Unknown-53

2.61

0.000

EXOB4_G09

Histone H14

6.94

0.000

HK0002_G04

Fructose-bisphosphate aldolase A

2.45

0.000

EXOB4_H06

Alpha-globin 1-3

6.63

0.000

EST1-3A_H03

Unknown-76

2.42

0.000

utu01e09

Embryonic alpha-type globin2+collagen alpha 2(1)

4.94

0.000

CA358107

Ectonucleoside triphosphate diphosphohydrolase 1

2.42

0.000

HK0002_G02

Creatine kinase, sarcomeric mitochondrial precursor

4.49

0.000

CA378361

Ubiquitin ligase SIAH1

2.40

0.000

HK0001_C08

Galectin-9 (VHSV-induced protein)-3

4.14

0.000

HK0001_H12

Unknown-162

2.35

0.000

CA361101

Regulator of G-protein signaling 1-2

4.03

0.000

CA342204

TNF receptor associated factor 3

2.33

0.000

est02a11

Ig kappa chain V-IV region B17-2

3.19

0.000

EST1-3A_H05

Adenosine deaminase 3

2.31

0.000

CA343473

C3a anaphylatoxin chemotactic receptor

3.00

0.000

est04f10

Hypothetical-fish 1

2.30

0.000

est04b01

Similar to rRNA (Vangl2)

2.69

0.000

KVkm2_H11

Unknown-228

2.30

0.000

HST0001_D02

ATP-binding cassette, sub-family F, member 2

2.58

0.000

HKT0001_A11

Hypothetical-fish 11

2.28

0.000

CA384134

G1/S-specific cyclin D2

2.56

0.000

CA355893

DnaJ homolog subfami.B member 2

2.26

0.000

24 hours DOWN

   

72 hours DOWN

   

est01e10

Tolloid-like protein (nephrosin)-1

-2.60

0.000

EXOB2_G02

Profilin-1

-2.13

0.000

est03c04

Matrix metalloproteinase-9

-2.70

0.000

EXOB2_F12

60S ribosomal protein L7a-1

-2.13

0.000

HST0001_C03

Plasminogen precursor-2

-2.72

0.000

est02g11

Cytochrome c oxidase subunit I-1

-2.14

0.000

KVkm2_H10

Unknown-227

-2.76

0.000

HK0001_H03

60S ribosomal protein L26

-2.14

0.000

CA34828

CCAAT/enhancer binding protein beta

-3.05

0.000

HK0003_A12

Heterogeneous nuclear ribonucleoprotein A1-1

-2.16

0.000

EXOB2_D05

Matrix metalloproteinase 9-2

-3.07

0.000

EXOB1_H12

NADH dehydrogenase subunit 4

-2.23

0.000

est02f08

Serine protease-like protein-1

-3.21

0.000

HK0002_D07

Unknown-172

-2.24

0.000

EST1-3A_A09

Serine protease-like protein-2

-3.25

0.000

EXOB3_G03

60S acidic ribosomal protein P2

-2.31

0.000

HK0003_C11

Tropomyosin alpha 3 chain-1

-3.34

0.000

EXOB2_G09

Cytochrome c oxidase subunit I-2

-2.34

0.000

utu03e06

Parvalbumin alpha-3

-3.50

0.000

HST0001_D04

Alpha-globin I-2

-2.37

0.000

CA370329

Lysozyme C precursor

-3.90

0.000

ENH2_H06

Unknown-5

-2.46

0.000

HK0003_C08

Parvalbumin alpha-2

-3.99

0.000

est04c05

Ferritin heavy chain-1

-2.54

0.000

utu04h1

Myosin light chain 2-2

-4.13

0.000

P_46

ATPase 6

-2.56

0.000

utu02c02

Myosin heavy chain, skeletal, adult 1-1

-4.30

0.000

utu02b07

Cytochrome c oxidase subunit II

-2.64

0.000

Hete0002_A07

Metallothionein-IL

-4.49

0.000

KVkm2_H10

Unknown-227

-2.67

0.000

HK0003_E07

Myosin light chain 2-1

-4.49

0.000

EST1-3A_H07

Cytochrome b-1

-2.86

0.000

HK0002_D07

Unknown-172

-5.04

0.000

est02h09

Nonhistone chromosomal protein HMG-17

-3.07

0.000

EXOB1_A03

Metallothionein A

-5.05

0.000

CA370329

Lysozyme C precursor

-3.08

0.000

est01c04

Unknown-11

-6.17

0.000

EXOB1_C02

Unknown-83

-3.44

0.000

HK0002_F05

Myosin heavy chain, skeletal, fetal

-7.31

0.000

EXOB2_G01

Leukocyte cell-derived chemotaxin 2

-3.56

0.000

Table 3

List of top 20 up and down regulated genes in the head kidney of trout, 24 and 72 hours after i.p. injection of active IHNV. Genes were selected for significant differential expression at p < 0.01 (FDR < 0.05). Values are expressed as FC, fold change.

24 hours UP

   

72 hours UP

   

Clone ID

Clone Name

FC

p

Clone ID

Clone Name

FC

p

CA343473

C3a anaphylatoxin chemotactic receptor

5.35

0.000

HST0001_D04

Alpha-globin I-2

15.48

0.000

est04c02

Unknown-53

4.26

0.000

est02a11

Ig kappa chain V-IV region B17-2

10.06

0.000

est02b02

PEST-containing nuclear protein

4.00

0.000

EXOB4_D02

Beta actin-2

9.60

0.000

EXOB3_A01

Unknown-103

3.91

0.000

EXOB2_B10

Hemoglobin beta chain

9.58

0.000

CA358107

Ectonucleoside triphosphate diphosphohydrolase 1

3.69

0.000

HK0003_A03

Thymosin beta-4-2

7.56

0.000

EXOB4_H01

Unknown-132

3.63

0.000

est02f08

Serine protease-like protein-1

6.41

0.000

HK0002_G04

Fructose-bisphosphate aldolase A

3.59

0.000

est02h09

Nonhistone chromosomal protein HMG-17

6.00

0.000

EXOB1_B03

Unknown-81

3.59

0.000

est03c04

Matrix metalloproteinase-9

5.91

0.000

EXOB4_E09

Hypoxanthine-guanine phosphoribosyltransferase

3.55

0.000

est01e06

Coronin-1B

5.51

0.000

CA378361

Ubiquitin ligase SIAH1

3.43

0.000

est02g07

Ig mu heavy chain disease protein

5.44

0.000

HK0003_G05

Unknown-201

3.43

0.000

EXOB4_H05

Unknown-133

5.24

0.000

est04f10

Hypothetical-fish 1

3.41

0.000

est01e02

Thymosin beta-4-1

5.17

0.000

EST1-3A_G03

Unknown-74

3.40

0.000

EXOB3_D02

Eukaryotic translation elongation factor 1 alpha 1

4.60

0.000

CA378435

Protein phosphatase 2C delta isoform

3.35

0.000

EXOB2_A01

MHC class II invariant chain-like protein 1

4.44

0.000

HK0002_H10

Unknown-182

3.33

0.000

EXOB3_H01

Matrix metalloproteinase-13

4.34

0.000

EXOB1_F02

Transcription regulator protein BACH1

3.28

0.000

EXOB1_A05

Ribosomal protein L6-1

4.33

0.000

est04b01

Similar to rRNA (Vangl2)

3.26

0.000

EST1-3A_F05

Heat shock 70 kDa protein 1

4.27

0.000

EST1-3A_H05

Adenosine deaminase 3

3.21

0.000

EXOB2_G0

Profilin-1

4.00

0.000

HK0001_H12

Unknown-162

3.05

0.000

HKT0001_H03

Microtubule-associated protein RP/EB

3.95

0.000

CA368716

Membrane-bound transcription factor site 2 protease

3.00

0.000

CA370329

Lysozyme C precursor

3.91

0.000

24 hours DOWN

   

72 hours DOWN

   

utu01a03

40S ribosomal protein S3-1

-3.22

0.000

EXOB1_B02

Hypothetical-fish 34

-4.48

0.000

HKT0001_H03

Microtubule-associated protein RP/EB

-3.24

0.000

HK0002_G04

Fructose-bisphosphate aldolase A

-4.80

0.000

utu04g05

40S ribosomal protein S9-3

-3.27

0.000

CA384134

G1/S-specific cyclin D2

-4.93

0.000

EST1-3A_D03

60S ribosomal protein L23

-3.32

0.000

EST1-3A_H03

Unknown-76

-5.18

0.000

EXOB1_H08

ADP, ATP carrier protein 3

-3.43

0.000

est03f04

F-box/WD-repeat protein 11

-5.28

0.000

EST1-3A_F05

Heat shock 70 kDa protein 1

-3.54

0.000

CA368716

Membrane-bound transcription factor site 2 protease

-5.31

0.000

EXOB1_A03

Metallothionein A

-3.55

0.000

CA384029

Chromobox protein homolog 4

-5.47

0.000

EXOB2_G09

Cytochrome c oxidase subunit I-2

-3.55

0.000

EXOB3_A01

Unknown-103

-5.54

0.000

HST0001_C03

Plasminogen precursor-2

-3.56

0.000

EXOB4_H01

Unknown-132

-5.99

0.000

EXOB3_E01

Na/K ATPase alpha subunit-2

-3.76

0.000

EST1-3A_H05

Adenosine deaminase 3

-6.23

0.000

HK0001_D01

Ubiquitin

-3.83

0.000

EXOB1_B03

Unknown-81

-6.29

0.000

HK0002_B07

Heat shock 70kDa protein 8

-4.24

0.000

HK0001_H12

Unknown-162

-6.44

0.000

Hete0002_A07

Metallothionein-IL

-4.50

0.000

HK0002_H10

Unknown-182

-6.59

0.000

utu02a08

Ubiquitin and ribosomal protein S27a-2

-4.43

0.000

est04c02

Unknown-53

-6.62

0.000

est02a11

Ig kappa chain V-IV region B17-2

-4.84

0.000

EXOB4_E09

Hypoxanthine-guanine phosphoribosyltransferase

-6.63

0.000

utu02c02

Myosin heavy chain, skeletal, adult 1-1

-4.97

0.000

est02b02

PEST-containing nuclear protein

-6.85

0.000

utu04f11

40S ribosomal protein S3-2

-6.00

0.000

est04f10

Hypothetical-fish 1

-7.01

0.000

est03c10

Alanine-glyoxylate aminotransferase 2

-7.41

0.000

EST1-3A_G03

Unknown-74

-7.55

0.000

est01c04

Unknown-11

-8.11

0.000

CA378361

Ubiquitin ligase SIAH1

-7.82

0.000

HK0002_D07

Unknown-172

-8.74

0.000

EXOB2_G11

Stanniocalcin-1

-9.69

0.000

However, using the aforementioned selection criteria (>2 fold change; p < 0.01) the magnitude of the transcriptomic response, measured as the number of differentially expressed genes, shows a clear difference between active viral treatment and LPS, the former eliciting an extensive immune, apoptotic and transcriptional response (see the analysis of functional classes below; figs. 2, 3, 4, 5, 6, 7). As shown by the gene representation of the two viral treatments mirroring strength of induction at 1–1.5 fold change levels, transcriptomic responses also include an extensive repertoire of genes expressed below 1.5 fold with p < 0.01 that clearly outnumber the 2-fold expressed genes (Fig. 1a) and can be ascribed to low-level transcriptional, metabolic and homeostasis maintenance programs (see tables 4, 5 and figs. 2, 3, 4, 5, 6, 7). Whilst it may be expected that an attenuated form of the virus does not induce a similar magnitude of response, the species-specific onset of the immune response elicited by the highly antigenic viral glycoprotein (G) of attIHNV may undoubtedly contribute to the observed gene expression pattern, as described in several DNA vaccination assays for fish pathogenic rhabdoviruses [14, 16, 33].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig2_HTML.jpg
Figure 2

Over represented GO functional classes in LPS array experiments. Functional categories with Yates corrected Chi squared p < 0.05 were selected. Number of regulated genes for each category is shown in parenthesis.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig3_HTML.jpg
Figure 3

Over represented GO functional classes in attIHNV array experiments. Functional categories with Yates corrected Chi squared p < 0.05 were selected. Number of regulated genes for each category is shown in parenthesis

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig4_HTML.jpg
Figure 4

Over represented GO functional classes in IHNV array experiments. Functional categories with Yates corrected Chi squared p < 0.05 were selected. Number of regulated genes for each category is shown in parenthesis.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig5_HTML.jpg
Figure 5

Gene Ontology analysis for LPS treatment. Results show categories with significant difference, (pair comparison independent test, p < 0.05), chosen from categories which had a p-value < 0.01 from individual treatment groups. Data is shown as mean fold change ± std.error.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig6_HTML.jpg
Figure 6

Gene Ontology analysis for attIHNV treatment. Results show categories with significant difference, (pair comparison independent test, p < 0.05), chosen from categories which had a p-value < 0.01 from individual treatment groups. Data is shown as mean fold change ± std.error.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-9-141/MediaObjects/12864_2007_Article_1335_Fig7_HTML.jpg
Figure 7

Gene Ontology analysis for IHNV treatment. Results show categories with significant difference, (pair comparison independent test, p < 0.05), chosen from categories which had a p-value < 0.01 from individual treatment groups. Data is shown as mean fold change ± std.error.

Table 4

Genes common to all treatments. Genes selected were expressed in a minimum of 5 of the 6 experiments and compared against all experiments in the database (n = 155). Only genes specific to these experiments were chosen (Fischer's exact Test, p < 0.01). Values are expressed as FC, fold change.

Clone ID

Clone Name

LPS (FC)

AttIHNV (FC)

IHNV (FC)

  

24 hr

72 hr

24 hr

72 hr

24 hr

72 hr

CA384637

60 kDa heat shock protein-1

-

1.47

1.18

1.25

-1.25

1.29

est03b11

Acidic leucine-rich nuclear phosphoprotein 32 A-1

1.68

1.41

1.17

1.32

1.27

1.34

utu01f04

Actin, alpha skeletal 2

-

-1.31

-1.48

1.18

1.52

1.29

utu01g11

Actin, alpha skeletal 3

1.67

-1.64

-1.47

-1.25

1.34

1.42

utu04d04

Actin, alpha skeletal 4

2.00

-1.30

-1.18

-

1.61

1.37

EST1-3A_H05

Adenosine deaminase 3

1.57

-

1.14

2.31

3.21

-6.23

HST0001_D04

Alpha-globin I-2

-1.30

-

10.93

-2.37

-3.11

15.48

CA364941

Annexin A1-1

-2.08

-1.78

-1.36

-1.46

-1.26

1.68

F_122

ARP2/3 complex 21 kDa subunit

-

-.192

-1.41

-1.23

-1.57

1.33

P_10

Aryl hydrocarbon receptor

1.35

-

1.32

1.80

1.47

-2.32

CA367764

Calmodulin-1

-

-2.13

-1.26

-1.29

-1.73

1.49

CA348284

CCAAT/enhancer binding protein beta

-7.36

-3.22

-3.05

-

-2.16

1.76

CA363762

Cell death activator CIDE-B

1.32

-

1.35

1.74

2.08

-2.53

CA383564

Coatomer epsilon subunit 1

2.25

-

1.20

1.43

1.24

1.53

HK0003_G01

Creatine kinase, M-2

-

1.61

-2.02

1.32

-1.54

-1.40

HK0002_G02

Creatine kinase, sarcomeric mitochondrial precursor

-1.68

-

4.49

-1.54

-1.82

1.20

CA358998

Cysteine-rich protein 1

-1.75

-2.71

-1.37

-1.51

-1.62

2.75

utu01g04

Cytochrome oxidase subunit III-2

-

-1.36

-2.55

-2.04

-2.08

2.33

EXOB1_C10

Cytochrome P450 2K4-2

1.60

1.34

1.20

-1.44

-

-1.14

utu01d06

Deoxyribonuclease gamma precursor

1.50

-

-1.19

-1.26

1.46

-1.31

utu01e09

Embryonic alpha-type globin2+collagen alpha 2(1)

-1.62

1.42

4.94

-1.41

-1.96

-

HK0001_D12

Estrogen-responsive B box protein

1.29

-

-1.22

-1.14

2.11

-1.38

HK0002_G04

Fructose-bisphosphate aldolase A

1.65

-

1.25

2.45

3.59

-4.80

HK0001_A11

Fumarate hydratase, mitochondrial precursor

-

-1.58

-1.12

-1.21

1.43

-1.25

CA371363

Glucose-6-phosphate isomerase-1

-4.35

-1.68

-1.40

-1.15

-1.37

2.81

EXOB3_H05

Glucose-6-phosphate isomerase-2

-2.07

-

-1.51

-2.08

-1.77

1.81

CA366403

Heat shock 27 kDa protein-1

1.44

-

1.21

1.61

2.49

-2.43

HST0001_C04

Hemoglobin alpha chain

-2.42

-

11.19

-1.82

-2.93

2.87

EXOB2_B10

Hemoglobin beta chain

-1.86

-

19.46

-2.07

-2.45

9.58

EXOB4_C11

High affinity immunoglobulin gamma Fc receptor I precursor

-

2.15

-1.70

-1.73

-1.88

1.71

est02a11

Ig kappa chain V-IV region B17-2

2.71

-

3.19

-2.07

-4.84

10.06

est02g07

Ig mu heavy chain disease protein

2.73

-

2.38

1.56

-2.14

5.44

EXOB2_D05

Matrix metalloproteinase 9-2

-6.63

-2.62

-3.07

1.71

-1.93

3.20

EXOB3_H01

Matrix metalloproteinase-13

-17.18

-5.88

-1.32

2.06

1.44

4.34

est03c04

Matrix metalloproteinase-9

-7.44

-2.21

-2.70

1.68

-2.92

5.91

EXOB3_F09

MHC class I heavy chain-1

-

1.71

-1.43

-1.20

-2.87

-3.02

P_4

Myeloperoxidase

1.55

-

1.23

1.88

2.32

-3.19

EXOB3_E09

N-myc downstream regulated protein-2

-1.48

1.67

1.16

1.21

-

-1.32

EXOB2_G02

Profilin-1

-

-2.04

-1.79

-2.13

-2.12

4.00

est02f08

Serine protease-like protein-1

-2.97

-4.64

-3.21

1.32

-1.84

6.41

EST1-3A_A09

Serine protease-like protein-2

-3.96

-5.52

-3.25

1.32

-2.91

3.75

HK0002_H08

TATA-binding protein associated factor 2N

1.79

-

-1.32

-1.22

1.71

-1.32

est01e02

Thymosin beta-4-1

-

-2.73

-2.03

-1.32

-1.47

5.17

est01e10

Tolloid-like protein (nephrosin)-1

-6.69

-12.97

-2.60

-1.07

-3.11

2.98

EXOB2_G12

Tolloid-like protein (nephrosin)-2

-9.26

-13.47

-1.67

1.44

-1.75

3.70

Hete0002_E09

Transposase-56

2.06

1.96

1.42

1.30

2.04

1.67

EST1-3A_F12

Transposase-59

2.12

1.62

1.28

1.23

-

1.20

EXOB3_F11

Ubiquitin-conjugating enzyme E2-18 kDa

-

1.47

1.48

1.68

1.61

-1.84

Table 5

Genes specific to viral treatments. Genes selected were expressed in a minimum of 3 of the 4 experiments and compared against all experiments in the database (n = 155). Only genes specific to these experiments were chosen (Fischer's exact Test, p < 0.001). Values are expressed as FC, fold change. 13 Unknowns were removed.

Clone ID

Clone Name

AttIHNV (FC)

IHNV (FC)

  

24 hr

72 hr

24 hr

72 hr

CA387866

Arachidonate 5-lipoxygenase-2

1.16

1.34

1.27

1.03

CA384555

B-cell-specific coactivator OBF-1

1.48

1.76

1.37

-1.09

CA343473

C3a anaphylatoxin chemotactic receptor

3.00

2.98

5.35

-1.50

ENH2_F03

Cathepsin K-1

-2.30

1.22

-3.01

1.42

EXOB4_F03

Cathepsin S

-1.46

-1.28

-1.65

2.80

CA376117

CD231

1.31

1.55

2.04

-1.66

CA363762

Cell death activator CIDE-B

1.35

1.74

2.08

-2.53

EXOB4_E08

Cytochrome c oxidase subunit VIIa-related

1.08

-1.14

1.18

-2.47

CA381440

Double-stranded RNA-specific adenosine deaminase

1.45

1.53

1.69

-1.67

CA351392

Guanine nucleotide exchange factor DBS

1.19

1.93

2.97

-3.13

EXOB4_C11

High affinity immunoglobulin gamma Fc receptor I precursor

-1.70

-1.73

-1.88

1.71

HKT0001_A11

Hypothetical-fish 11

1.20

2.28

1.49

-1.85

est03c07

Ig kappa chain V-I region WEA

1.60

2.07

1.41

1.21

est02g07

Ig mu heavy chain disease protein

2.38

1.56

-2.14

5.44

CA363978

Inhibitor of kappaB kinase gamma

1.16

2.13

2.21

-2.36

EXOB3_F09

MHC class I heavy chain-1

-1.43

-1.20

-2.87

-3.02

CA341859

Nuclear factor NF-kappa-B

1.40

1.12

1.63

1.28

CA378435

Protein phosphatase 2C delta isofor

1.44

2.05

3.35

-3.87

EXOB1_B07

PRPF39 protein

1.19

1.22

1.39

 

est03e10

Putative inorganic polyphosphate/ATP-NAD kinase

-1.11

-1.05

1.33

1.29

est03b12

Secretory granule proteoglycan core protein

-1.83

-1.83

-2.97

2.77

ENH2_B08

Splicing factor, arginine/serine-rich 8

1.10

1.24

1.25

-1.49

CA380121

Telomerase reverse transcriptase

1.33

1.36

1.34

-1.33

EXOB1_G12

Thioredoxin

-1.24

-1.61

-1.56

1.46

CA342204

TNF receptor associated factor

1.23

2.33

2.64

-2.89

CA378736

Tyrosine-protein kinase BTK

1.28

1.61

1.81

-1.80

EXOB3_G04

Tyrosine-protein kinase HCK

-1.07

-1.38

-1.39

1.40

P_54

VEGF4

1.23

1.34

1.37

 

Not surprisingly, both viral treatments display a 10% increase in the number of genes regulated >2 fold at 72 hours post-treatment. Previous studies on the pathogenicity of IHNV in salmonids typically describe a 24 h delay in detectable viral titres and internal measures of immunological disturbance, followed by a rapid increase in the differential expression of viral-related and acute phase response genes in major haematopoietic organs and liver, respectively [42, 44]. In addition, a tissue-specific effect determines the dynamics of the transient cellular populations during inflammation and, subsequently, the tissue-dependent gene expression profile. It has been suggested [45] that the transient double stranded RNA intermediates produced by the accelerated replication of IHNV 24 hours post infection seem to regulate the expression of trout Toll-like Receptor 3 (rtTLR3) in a unpredictable manner, strongly dependent on viral growth and host lymphocyte recirculation cycles. Interestingly, the pattern of rtTLR3 expression in response to Yersinia ruckeri, a gram-negative bacterial trout pathogen shows a remarkably lower magnitude in terms of fold change compared with the viral challenge [45], mimicking the magnitude of the response between LPS and IHNV/attIHNV treatments described above (fig 1b). Although both treatments display increases in the number of regulated genes the patterns of gene expression are also significantly different. The number of genes regulated show that LPS induces in the majority a down-regulation of gene expression at both time points. Similar results were observed in LPS-stimulated macrophages derived from trout, O. mykiss, analysed with the same platform [8]. Highest fold changes in individual genes were observed in down-regulated genes in the LPS groups (table 1). On the other hand, viral treatments induce a higher induction of transcriptomic and cell cycle/apoptotic activity where induction and suppression processes display a similar weighting (tables 2 and 3).

The viral treatments show the highest differential gene expression counts obtained when compared across all available experiments in our gene (KuopioChip) database. This observation, together with the extensive transcriptional onset observed in the analysis of functional ontology categories in viral treatments (see below), follows that of Abbas et al. (2006) suggesting that a specific immune response mobilizes more transcriptional remodelling than the majority of physiological responses.

Specific vs. common gene responses

From the selected differentially expressed genes (p < 0.01) in LPS and viral treatments, the differential response in LPS and viral treatments (significant differential expression in at least 5 of the 6 experiments) was evaluated across 155 experiments in our gene database (KuopioChip) in order to ascertain those genes co-expressed (Fischer's exact P < 0.01) in each or both experimental conditions (minimum requirement was significant differential expression in at least 5 of the 6 experiments). A total of 49 genes were identified from which many genes showed opposite responses representative of directional responses to viral and LPS-induced stimuli (table 4 and 5).

Interestingly genes regulated by the attIHNV treatment show a mixture of effects both similar and different to LPS and IHNV suggesting that different mechanisms, non-specific and specific response, of activation are induced by the attenuated virus in the head kidney. Alpha globin for example shows down regulation by LPS whereas attIHNV initially induces expression, 11 fold, followed by suppression, -2.37 fold. On the other hand IHNV causes suppression at 24 hours followed by strong induction, 15 fold. LPS and attIHNV show similar profiles for: Glucose-6-phosphate isomerase, annexin 1, the nephrosin proteins, calmodulin-1, CEBP-beta, cysteine-rich protein-1, cytochrome oxidase and cytochrome p-450. The majority of the genes are related to immunity and the inflammatory response. Suppression of HK proteases, nephrosin and MMPs, seems to be one of the most characteristic effects of LPS (Nephrosins FC; 24 hours; -6.69 and -9.26 and 72 hours -12.97 and -13.47) whereas viral treatment causes a similar initial effect followed by induction of expression (FC at 72 hours; attIHNV 1.44 and IHNV 2.98 and 3.7). Serine protease-like proteins 1 and 2(spl1 and spl2) are also highly suppressed by LPS, attIHNV and IHNV at 24 hours. This suppression continues in the LPS treatment, however in viral samples we observe a significant increase in expression in the 72 hour samples (attIHNV; 1.32 both spl1 and 2, IHNV; 6.4 and 3.75). Several cytoskeleton related genes including ARP2/3, the actin and profilin-1 are down-regulated by both LPS and attIHNV whereas IHNV induces up-regulation of all genes in this group at 72 hours post-infection.

Viral treatments induce a typical adaptive immune response (table 5). The antigen processing/presenting loading pathways include the differential expression of MHC Class I and lymphoid and myeloid cell lineages, as shown by the regulation of B-cell specific coactivator OBF-1, essential for the response of B-cells to antigens and required for the formation of germinal centers, the conservation of BTK B-cell, HCK neutrophil and TNFalpha receptor signaling pathways, the cathepsin mediated antigen processing and the interferon-inducible RNA-specific adenosine deaminase ADAR1 (table 5).

Not surprisingly, NF-kappa-B (NFkB) signal transduction suffered from moderate to severe regulation in the head kidney of infected trout (tables 3 and 4). NFkB is considered a pleiotropic transcription factor expressed in several cell types undergoing amongst others inflammatory assaults. Both active IHNV and attIHNV treated fish showed differential regulation of Inhibitor of kappaB kinase gamma, member of a family of proteins which inactivate NFkB by trapping it in the cell cytoplasm [46] and are actively expressed in virus infected cells [47].

The anaphylactic arm of the complement activation acts in a double fashion through the component C3a/C3aReceptor: as a chemotactic mediator involved in endotoxic responses and, simultaneously, as a regulator of homing and mobilizations of hematopoietic stem cells (see below). Both complement related serine proteases (spl) and C3 proteolytic components were differentially expressed in LPS-induced or viral treatments. However, the highest levels of complement expression seem to be strongly dependent of viral invasions in fish head kidney (tables 3 and 5)

Stromal protease activity and extracellular matrix remodelling in trout head kidney

Although the precise function of nephrosin, an astacin metalloproteinase [48] remains unresolved, it has been shown to be involved in the late stages of granulocyte differentiation and cell migration and/or tissue infiltration processes in challenged carp and zebrafish [49, 50]. In trout treated with LPS, the two nephrosin proteins genes are severely down regulated (between -6 and -10 fold) at both time points (fig. 1). The rest of metalloproteinases (MMP 9-2, 13) also showed a marked decrease of expression in LPS treated trout (less than -6 fold change) at both time points and at 24 h in IHNV or attIHNV infected fish (-1.3 to -3 fold; see table 2 and additional file 1), therefore suggesting a decrease in extracellular matrix remodeling and leukocyte movement.

In mammals, MMP 9 has been implicated in the signal processing and maturation of dendritic cells, IL-8 mediated activation of neutrophils and undifferentiated hematopoietic stem cells [51, 52]. Whilst the existence of functionally differentiated dendritic cells in fish is still controversial [53, 54], the mammalian leukocyte response outlined for IL-8 and MMP 9 seems to be conserved in teleosts, although the exact pattern of organ distribution, the intensity of gene regulation and the voluble species-specific expression of IL-8 in fish HK remains obscure [55, 56]. The chemotatic properties of IL-8 on leukocytes are amplified by the recruitment of neutrophils mediated by CXC chemokines. Our data show a moderate (-3.71 fold change) down-regulation of trout CXCR4. Mammalian CXCR4 forms with TNFalpha, several HSP proteins and GDF5 an activation cluster involved in monocyte LPS signal transduction [57] and also acts synergistically with the colony-stimulating factor mediated mobilization of hematopoietic stem cells [57, 58].

The MMP gene family appears to be induced by LPS in trout macrophages [8], and a widescreen transcriptomic analysis of carp metalloproteinases detected a large amount of MMP9 mRNA mainly in hematopoietic organs, HK and spleen [59, 60]. Fish metalloproteinases thus probably act in a mammalian fashion, showing and up-regulation by the synergistic stimulation of Tumor Necrosis Factor alpha (TNFalpha) and LPS. In agreement with the low level expression of MMPs observed 24 h post injection, no significant amounts of TNFalpha mRNA were detected in LPS treated trout, in contrast with IHNV infected fish. Both myeloperoxidase, a lysosomal hemoprotein characteristic of mononuclear phagocytes, and TNF were up regulated 24 h post injection of attenuated IHNV in trout HK (2.3 and 2.6 fold, respectively). In the active viral group, however, TNFalpha was found to be inactive (see below). The moderate up-regulation of TNFalpha 3 days post attIHNV stimulation (table 2) may suggest, according to the well described model of macrophage stimulation by LPS [8], the onset of a proliferative myeloid response in the principal hematopoietic organ.

Intracellular thymosin beta-4 (TBX4) is considered the main G-actin sequestering peptide in mammals [61]. The putative functions of extracellular TB4X include induction of hemostasis, wound and tissue healing, chemotaxis, induction of metalloproteinases, granulocyte-mediated inhibition of inflammatory processes and regulation of hematopoietic stem cell proliferation [62]. Not surprisingly, the TBX4 gene is >2 fold down regulated in trout HK 72 h post injection of LPS (table 1), but strongly upregulated (>5–7 fold) in IHNV infected trout undergoing an incipient hemorrhagic symptomology. This suggests that, in fish as in mammals, the pleiotropic effects of TBX4 are mediated by the remodeling of intracellular actin cytoskeleton and/or components of the extracellular matrix [63].

The moderate/severe down-regulation of CXCR4 1 day post LPS challenge (-3.7 fold change), metalloproteinases and TB4X observed at 24 h and 72 h (less than -6 and -2 fold change levels, respectively; table 1) thus favors a scenario of delayed reactive state to LPS stimulation in the recirculation and traffic of trout head kidney hematopoietic cells following i.p. administration of bacterial LPS. Moreover, the head kidney itself shows a low level stromal extracellular matrix remodeling in trout treated with LPS, as suggested by the down regulated expression of actin, MMP, nephrosins and Thymosin beta 4 (table 1). However, at 3 days post IHNV infection the transcriptomic footprints in the head kidney reveal an immunological shift orientated toward a somewhat impaired adaptive arm activation coupled with a strong hemostatic and extracellular matrix sculpting response: the systemic spreading of IHNV clearly inhibits TNFalpha, MHC class I and several macrophage and cell cycle/differentiation markers (tables 3, 4; see below) favoring a MHC class II, immunoglobulin and MMP/TBX4 enhanced immune response.

Complement response to LPS-induced and viral challenges in trout head kidney

Contrary to the liver, the head kidney cannot be considered an acute phase reactive organ. Following a xenobiotic assault, complement related serine proteases, modulators and C3 proteolytic peptides are synthesized primarily in the liver. However, minor but biologically significant extra-hepatic (mainly in active immune cells, gills, skin, heart, gonad and renal tissues) synthesis of complement components has recently been demonstrated in fish as in other vertebrates [64, 65]. Therefore, complement inflammatory, chemotactic, opsonic and lytic activities extend the effect of the innate arm of immune responses to the core of major hematopoietic organs.

In trout injected with LPS, complement related serine proteases (spl1 and spl2) homologous to the MASP proteins involved in the activation of the classical complement pathway [66, 67] remained down regulated throughout the treatment (-3 and -4 fold change at 24 h and 72 h respectively; table 1) and no expression of C3 genes were detectable in head kidney (table 1), in concordance with the biased adaptive response observed in this group (see below). A recent microarray analysis of acute phase reactivity in the liver of catfish (Ictalurus punctatus) showed an enhanced (greater than >2 fold) complement response in fish infected with Edwardsiella ictaluri, a gram negative pathogen responsible for enteric septicemia in catfish [13] In trout, Lovoll et al. (2007b) found a similar expression pattern in hepatocytes treated with LPS, but, in sharp contrast with the highest expression levels of complement genes in liver, a minor up regulation of C3 genes in head kidney and spleen was observed. Additionally, gene expression showed a strong tissue and isoform dependence: C3-4 was found to be down regulated in HK following stimulation with LPS, and more interestingly, not all trout isoforms maintained similar levels of gene expression [68].

The tolerance of fish to the standard LPS doses used in rodent immune challenges is well known, and has been linked to the peculiarities of PAMP receptors in fish [36] that may preclude a strong endotoxic shock response. Thus, the strongest cellular and tissue responses to i.p. injection LPS in fish are thought to be restricted to activated monocyte/macrophages and lymphocytes, portals of entry (gills, intestine, skin) and acute phase organs (liver). However, the dynamics of the teleostean immune response is of primary interest in these organs, as in the head kidney, that functions either as a cradle for immunological priming of leukocyte populations or as a major node in the complex and the still poorly understood network of neuro-immune-endocrine interactions in fish. In that respect, our results uncover a striking difference between lipopolysaccharide and viral treatments concerning head kidney transcriptomic dynamics: in attenuated and active IHNV groups, the complement related serine proteases and anaphylatoxin receptors (C3aR) maintain up regulated expression levels at both time points, with the maximal expression (>5–6 fold change) in active viral groups.

The C3aR shows an unequivocal upregulated expression at 24 h and 72 h in animals infected with attIHNV, and at 24 h in animals infected with active IHNV (our results also show a tenuous down regulation of C3aR (-1.5 fold change) in IHNV treated fish at 72 h; see additional file 1). At 72 h, the response to IHNV consist of a mixture of adaptive (MHC, IG) and innate (C3aR, complement related serine proteases, lysozyme C) immune and stress (HSP70, hemostasis) responses against a background of metalloproteinase-mediated matrix remodeling (table 3). Therefore, as suggested in this and previous studies with mammals [6971], the expression of C3a/C3aR may probably contribute to the homing/mobilizations and differentiation of hematopoietic stem cells in response to the generalized immune and stress response elicited by an aggressive and extremely pathogenic virus in fish. Moreover, in mammals several C3 cleavage fragments, including C3a has been demonstrated to be linked to the CXCR4-mediated responsiveness of hematopoietic stem cells [71]. The moderate down regulation of CXCR4 in LPS treated fish (table 1) and upregulation (>1.5 fold) in attIHNV infected fish (see additional file 1), together with the strong upregulation of C3aR in IHNV treated fish suggests a conservation of complement mediated functional responses in the hematopoietic head kidney. The dynamics and trafficking of hematopoietic and differentiated cells in fish are, nonetheless, far from being fully understood.

Specific immune responses to LPS-induced and viral challenges in trout head kidney

Genes involved in the immunoglobulin system increased in all treatments displaying different kinetics. Ig gamma Fc receptor (CD64) gene expression increased late in the LPS treatment whereas IgM heavy chain (B-cells) and Ig kappa chain V-IV region B17-2 (involved in antigen presentation) increased acutely. Under attIHNV conditions a similar regulation is observed for the latter genes in which IgM expression is sustained at 72 hours and Ig Kappa suppressed. CD64 expression is suppressed by attenuated virus, whereas IHNV suppresses all three genes at the early stage and induces expression (1.7, 10 and 5.4 fold respectively) at the later stage of infection. This may reflect migration of leukocytes to the primary sites of infection/inflammation and/or differential recruitment of leukocyte sub-populations to the infected head kidney in both IHNV/attIHNV treatments.

The coatomer protein (COP) epsilon known to play a role in the formation and maturation of phagosomes [72] is induced in all treatments, and the Class I major histocompatibility complex (MHC) antigen, well known to be involved in antigen presentation in dendritic cells is activated late by LPS and suppressed by viral treatments (-3 fold; tables 2, 3), whereas Class II MHC showed a moderate up regulation (4.4 fold) in fish infected with IHNV at 72 h.

In fish, as in mammals, the interaction of MHC molecules with T cell receptors (TCR) seems to activate subsets of cytotoxic T lymphocytes (CTL) and T helper cells (Th) in a similar fashion [73]. Homologues of mammalian MHC, several proteins associated to antigen presentation, beta2 microglobulin and CD8+ and CD4+ (markers for CTL and Th, respectively) have been recently characterized in trout and other fish species [74, 75], thus reinforcing the conservation of antigen processing pathways in immune cells. However, the trafficking, recirculation and cell-to-cell communication against a quiescent/activated immune background have not yet been properly described in fish. Moreover, the translation of classical bacterial or viral inflammatory murine models encompasses several technical (full characterization of immune processes, absence/presence of mediators, conservation of activation/inhibition pathways) and species-specific difficulties, the latter related to the high variability of the interspecific thresholds of immune activation in fish and, last but not least, the relative virulence and co-evolutionary trade-offs of pathogens.

Our and several recent studies have attempted to resolve these issues weighting the organ related immune response to an established model of LPS-induced inflammation or viral infection. In this regard, the expression of CD8 and CD4 (coreceptors of MHC Class I and II binding, respectively) in trout seems to be restricted mainly to thymus and to a certain extent, to spleen, even though non-infected fish maintain a widespread low level expression in several hematopoietic or lymphocyte infiltrated organs [74, 75]. Overturf and LaPatra (2006) were unable to find elevated levels of CD8 expression in the HK of trout infected with bacteria or IHNV at 24 h or 5 days post infection, although in liver and spleen a positive dose-response correlation followed the infection (24 h) expression of CD8 and C3 [76]. In a similar experiment, 72 hours after IHNV challenge Hansen and LaPatra (2002) observed a surprising tissue-specific shutdown of MHC Class IIB mRNA in head kidney and spleen of infected trout, thus suggesting an enhanced CD8 response coupled with activation of MHC Class I antigen presentation following IHNV infection [77], as observed in a cohabitant model of fish viral infection described recently [78]. Similarly, an infection by Vibrio anguilarum seemed to depress the short term (up to 4 days) expression of MHC class II genes in head kidney, liver and spleen of turbot, Scophthalmus maximus [79]. However, an elevated expression of MHC Class II genes has been described in trout following i.m. DNA vaccination with recombinant IHNV [80] and xenobiotic inflammation [81]. In trout challenged with VSHV, a member of Rhabdoviridae, the dynamics of T cell expansion, and thus the onset of MHC mediated adaptive immune response, were found to be correlated with the expected waves of viral replication, with peak a week after viral challenge [82], mimicking the delayed (up to 10 days) expression of MHC mRNAs that were also observed in japanese flounder (Paralichthys olivaceus) leukocytes infected with Neoheterobothrium hirame, a monogenean parasite [83].

As described in other species [84, 85], our results suggest a predilection for CD4/Th lymphocyte response in the head kidney of trout challenged with IHNV at 72 post infection, coupled with a strong spl-induced complement cleavage activation and MMP/TBX4 extracellular matrix sculpting, together with a decreased TNFalpha mediated activation of monocyte/macrophage populations, a shutdown of MHC Class I and also a low level regulation of apoptosis, as shown by the inhibition of Ubiquitin ligase S1AH1 and Galectin-9 (see table 3 and additional file 1).

The i.p. administration of LPS activates the MHC Class I pathway of antigen processing in concordance with previous studies with LPS-activated trout macrophages [8]. However, the transcription factor CCAAT/enhancer binding protein-alpha (C/EBP-alpha), best known for its role in driving myeloid cells towards the granulocytic line [86] but also known to be induced during macrophage differentiation, and the MMP/TBX4 response was inhibited in head kidney 72 h post injection. Deltex protein 1 (DTX) also appears to be down regulated 72 h post infection with LPS (table 1). Although still poorly understood, the cross-talk between Deltex protein 1 (DTX1) and the evolutionary conserved Notch and NFkappaB signaling pathways [87, 88] allows the normal development and maturation of differentiated lymphocyte populations in hematopoietic and lymphopoietic organs in mammals. This suggests an impaired trafficking of lymphoid/myeloid HK cells in the early response to LPS in trout.

Myeloperoxidase (MPO) was up-regulated by all treatments at 24 hours returning to baseline in LPS samples, increasing in attIHNV and actively inhibited in IHNV samples (tables 1, 2, 3, 4). Myeloperoxidase is expressed in neutrophils and monocytes and plays a role in the oxygen dependent mechanism of phagocytosis. The macrophage scavenger receptor MARCO [89] was also inhibited in IHNV samples. Therefore, the microbiocidal function was diminished in the head kidney of IHNV infected trout.

Taken together, these results may suggest a minor function of the head kidney in the short term (24–72 hrs) activation of the immune response to virulent IHNV, or, alternatively, an inducible and maybe antagonistic early differential expression of MHC Class I or II mediated antigen processing in the head kidney, heavily influenced either by the type and infective dynamics of pathogen (LPS vs. viral) or the portals of entry (organ/tissue). From the experimental infections described above, the liver and spleen also seem to act as a major acute phase reactive organs at the initial stages of viral invasion, and the head kidney can be more properly defined as an inductor of a delayed adaptive response as much as a major regulator of erythropoietic and myeloid differentiation. Nevertheless the dual expression of the MHC antigen processing/presenting machinery appears to be strongly influenced by the species-specific immunological sensibility [90, 91], the virulence of pathogens and the timing of cellular differentiation in immune organs. This pathogen/host species-specific branching of MHC mediated immune response in fish requires, however, further analysis.

Haemoglobin metabolism in LPS-induced and viral infected trout head kidney

Hemoglobin genes (α, β) are suppressed by both LPS and IHNV at 24 hours, 2–3 fold suppression, however attIHNV strongly induces expression of both proteins(11 and 19 fold respectively) followed by suppression at 72 hours. IHNV induces expression (3 and 10 fold α and β respectively) at 72 hours whereas LPS samples return to baseline levels.

Interestingly, 5-aminolevulinate synthase, the key enzyme involved in heme synthesis was up-regulated in both attIHNV samples and in a LPS-specific manner at 72 hours. A similar response has been observed in head kidney tissue from Atlantic salmon, S. salar, infected with Piscirickettsia [15].

The coordinated expression of both hemoglobin genes and 5-aminolevulinate synthase suggest erythropoietic activity in the head kidney. Furthermore, a significant increase in cellular proliferation in PU-1-ve cells, 72 hours post-LPS administration, in the head kidney of LPS-treated rainbow trout has been reported (Ribas et al, 2007 in press) thus activation of hematopoietic, potentially erythropoietic, mechanisms during the early stages of infectious processes may be a standard for the non-specific immune response in fish.

HSP induced stress response to LPS-induced and viral challenges in trout head kidney

Active IHNV inhibits the expression of heat shock protein (HSP) 27 (-2.4 fold) and induces the expression of 70 and 90 (2.8 and 2.3 fold respectively) 3 days post infection from a previous inactive state at 24 h (-4.24 fold). The attIHNV showed a down regulation of HSP70 (-2 fold) at the same time point.

HSPs have been implicated in the generalized stress response associated to xenobiotics and/or inflammatory reactions in fish [44, 85, 92]. However, the reliability of HSP as an indicator of stress or pathologic/immune disturbances has recently thoroughly criticized [93] because of the great variability of measured HSP expression: despite the correlated expression of HSP with altered states in stressed or injured fish, the sensitivity and intensity of HSP response can vary in a species-specific manner, and among tissues, HSP families, season, developmental stages and stressor. Not being an acute phase response organ, it is, thus, difficult to speculate about the fate of HSP repaired enzymatic and/or cytoskeleton proteins in infected head kidney.

Conclusion

For the functional analysis of biological roles of regulated genes, our two-step approach in the first instance establishes a list of differentially expressed genes whose ascribed biological roles are evaluated and secondly by identifying overrepresented GO functional categories using the KuopioChip analysis software (see materials and methods). This methodological approach is not exempt of limitations. The selected cut-offs for minimal gene expression, the co-expressed patterns of gene expression, the non lineal genome-proteome crosstalk and the limited transcript enrichment of the array can either limit the amount and quality of transcriptomic responses assessed or exclude transient but biologically relevant genetic responses correlated with the abruptness and organ-dependent systemic damage in infected fish.

As a guideline for elucidating the biological response of viral/LPS-induced challenge, the comparison of differentially expressed genes by the GO categories showed a marked induction of metalloproteinases and other collagen and extracellular matrix sculptors in LPS treated trout, coupled with a decrease in genes controlling the basal metabolism and an increase in the activity of immune related mediators of MHC antigen presenting and immunoglublin-mediated opsonisation (figures 2 and 5) 72 h post infection.

Several genes involved in signal transduction and protein biosynthesis were active in attIHNV groups 72 h post infection, following a decrease in cytoskeleton remodelling. As in active IHNV infected trout, the functional GO categories are strongly enriched in genes active during inflammatory, immune and defence responses. In both attenuated and active IHNV treated fish, the immune response at 72 h clearly outweighs the metalloproteinase orientated LPS response, but, as described above, the response of the trout head kidney transcriptome to IHNV infection was more robust and diversified in number and immune related activation pathways.

Methods

Animal protocol an experimental infections

Rainbow trout, Oncorhynchus mykiss, were obtained from two commercial fish farms. Fish were maintained in flow through tanks under ambient conditions of light (photoperiod 10L/14D) and temperature (15 ± 2°C). Fish were fed with commercial trout pellets ad libitum and acclimated for at least two weeks prior to use in experiments. The health status of the animals and water quality were checked on a daily basis. Challenge experiments were carried out separately for each treatment and were performed in recirculating tanks with aeration at 14°C. Trout (n = 10/tank) weighing between 70–100 g were separated into 6 different tanks, 3 control and 3 experimental tanks. For experimental stimulations the fish were mildly anaesthetized (MS-222, 40 ppm, stage II of anaesthesia according to Iwama et al. 1989) and intraperitoneal (i.p.) injections carried out. Fish were intraperitoneally injected with saline (control) and LPS (6 mg/Kg; serotype 026:B6, Sigma, #L-8274), or with 100 μl of a 106 pfu/ml dilution of IHNV or attIHNV or culture medium (negative control). At defined time points, 24 and 72 hours post i.p. injection, animals (total n = 6), control and experimental, were selected from each tank (n = 2) and sacrificed by over anesthetization (MS-222, 100 ppm, stage III of anaesthesia [94]). Head kidneys (HK) were immediately dissected out, pooled and processed for total RNA purification using Tri Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's protocol [95, 96].

Viruses and cell line

IHNV (French isolate 32/87) was used and propagated in the fish epithelial cell-line EPC derived from common carp (Cyprinus carpio) [97]. EPC cells were cultured in Eagle's minimum essential medium (MEM, Gibco) supplemented with foetal bovine serum (FBS), penicillin (100 IU ml-1), streptomycin (100 μg ml-1), buffered with 7,5% sodium bicarbonate and incubated at 20°C. The virus was inoculated on EPC grown in MEM with antibiotics and 2% FCS at 14°C. When the cytopathic effect was complete, the supernatant was harvested and centrifuged to eliminate cell debris. The virus stock was titrated according to Reed & Muench [98] in EPC 96 well plates. Attenuated IHNV was generated by reverse genetic engineering of virulent IHNV by M. Bremont as described elsewhere [31].

RNA isolation

RNA from head kidneys of experimental and control/sham-injected fish were extracted with the Trizol reagent (Life Technologies) or Tri Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. DNase treatment was performed to remove contaminating DNA from preparations. RNA was precipitated using ethanol 100% and ammonium acetate (pH 5.2). The RNA was stored at -80°C in ethanol 70% until use.

Microarray design and analyses

The design of the microarray is described in detail elsewhere [6, 7] and a full description of the platform and data presented in this manuscript are accessible through the public GEO depositories (accession number GPL6155 and GSE10272). In brief, the platform included 1380 genes printed in six replicates each. Random clones from common and subtracted cDNA libraries (976) were compared with the known vertebrate proteins using blastx and 686 genes were identified; the functional annotations were transferred from the putative homologs. These clones were supplemented with 297 genes selected by the categories of Gene Ontology. Overall, each microarray was enriched in a number of functional classes, such as stress and defense response (145 and 105 genes, respectively), cell cycle (62 genes), signal transduction (114 genes), chaperone activity (41 genes), and apoptosis (79 genes).

Total RNA obtained from head kidney tissue was verified for quantity and integrity by denaturing electrophoresis and labeling with Cy3- and Cy5-dCTP (Amersham Pharmacia) was completed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer, and cDNA was purified with Microcon YM30 (Millipore). We used a dye swap experimental design [99] and each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignment was reversed. All head kidney samples were analyses using the dye swap protocol. The slides were pre-treated with 1% BSA, fraction V, 5 × SSC, 0.1% SDS (30 min at 50°C) and washed with 2 × SSC (3 min) and 0.2 × SSC (3 min) and hybridized overnight in cocktail containing 1.3 × Denhardt's, 3 × SSC 0.3% SDS, 2.1 μg/μl polyadenylate and 1 μg/μl yeast tRNA. All chemicals were from Sigma-Aldrich. Scanning was performed with ScanArray 5000 and images were processed with QuantArray (GSI Luminomics). The measurements in spots were filtered by criteria I/B ≥ 3 and (I-B)/(S I + S B ) ≥ 0.6, where I and B are the mean signal and background intensities and S I , S B are the standard deviations. After subtraction of mean background, locally weighted non-linear regression (Lowess) normalization [100] was performed separately for each slide. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Student's t-test (p < 0.01). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes [101]. For the analysis of the functional profiling of samples, all genes that showed significant differential expression (p < 0.01) in at least one sample were used. Due to the large number of genes, the statistical significance of over represented functional categories in each experiment (figs. 2, 3, 4) was assessed using the Yates correction to Chi square test (corrected p < 0.05). The log (p-level) ranked up or down-regulated genes were analyzed interrogating the functional classes of Gene Ontology (GO) [102] and compared by the sums of ranked genes (Student's t-test, p < 0.05). A list of LPS and virus specific responsive genes (figs 5, 6, 7) was obtained by GO data mining of a database (KuopioChip) of 155 previous immune and stress related experiments [7, 43].

Quantitative RT PCR

Primers were designed to amplify 194–305 bp fragments. RNA was processed with Rnase-free Dnase (Promega). Synthesis of cDNA with Superscript III reverse transcriptase (Invitrogen) was primed with oligo(dT). Analyses were carried out using Dynamo SYBR Green kit (Finnzymes) and ABI Prism 7700 (Amersham-Pharmacia)

In order to quantify mRNA expression, real time PCR (Q-PCR) was carried out. cDNA was diluted 1:50 for target mRNA and 1:100 for 18S and used as a template with the primers Q-PCR Fw and Q-PCR Rv (see additional file 1, table 7). Wells (20 μl final volume) contained 10 μl of iQ™ SYBR Green Supermix (Bio-Rad), 500 nM concentration of forward and reverse primers and 5 μl of cDNA. Controls lacking cDNA and controls containing RNA were included. Reactions were run in a MyiQ thermocycler (BioRad) under the following protocol: 5 min initial denaturation at 95°C, followed by 40 cycles of 10 sec denaturation at 95°C and 30 sec at 60°C, and a final melting curve of 81 cycles (from 55°C to 95°C). All samples were run in triplicate and fluorescence was measured at the end of every extension step. CT (threshold cycle) values for each sample were expressed as "fold differences", calculated relative to untreated controls and normalized for each gene against those obtained for 18S (see additional file 1, Table 8).

Notes

Declarations

Acknowledgements

This work was supported by Plan Nacional AGL2005-12134 from the Ministerio de Educación y Ciencia, España and the EU-funded project IMAQUANIM, EC contract number FOOD-CT-2005-007103.

Authors’ Affiliations

(1)
Unitat de Fisiologia Animal, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Universitat Autonoma de Barcelona (UAB)
(2)
Instituto de Investigaciones Marinas, CSIC
(3)
Dpt of Infectious Disease Epidemiology, Imperial College Faculty of Medicine
(4)
Departament de Fisiologia, Facultat de Biología, Universitat de Barcelona
(5)
Akvaforsk, Institutt for akvakulturforskning

References

  1. Bianchi ME: DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of leukocyte biology. 2007, 81 (1): 1-5. 10.1189/jlb.0306164.PubMedGoogle Scholar
  2. Sepulcre MP, Lopez-Castejon G, Meseguer J, Mulero V: The activation of gilthead seabream professional phagocytes by different PAMPs underlines the behavioural diversity of the main innate immune cells of bony fish. Molecular immunology. 2007, 44 (8): 2009-2016. 10.1016/j.molimm.2006.09.022.PubMedGoogle Scholar
  3. Zhong J, Kyriakis JM: Dissection of a signaling pathway by which pathogen-associated molecular patterns (PAMPs) recruit the JNK and p38 MAPKs and trigger cytokine release. J Biol Chem. 2007, 282 (33): 24246-54.PubMedGoogle Scholar
  4. Giri MS, Nebozhyn M, Showe L, Montaner LJ: Microarray data on gene modulation by HIV-1 in immune cells: 2000-2006. Journal of leukocyte biology. 2006, 80 (5): 1031-1043. 10.1189/jlb.0306157.PubMedGoogle Scholar
  5. Li J, Pritchard DK, Wang X, Park DR, Bumgarner RE, Schwartz SM, Liles WC: cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages. Journal of leukocyte biology. 2007, 81 (1): 328-335. 10.1189/jlb.0206124.PubMedGoogle Scholar
  6. Koskinen H, Pehkonen P, Vehniainen E, Krasnov A, Rexroad C, Afanasyev S, Molsa H, Oikari A: Response of rainbow trout transcriptome to model chemical contaminants. Biochemical and biophysical research communications. 2004, 320 (3): 745-753. 10.1016/j.bbrc.2004.06.024.PubMedGoogle Scholar
  7. Krasnov A, Koskinen H, Pehkonen P, Rexroad CE, Afanasyev S, Molsa H: Gene expression in the brain and kidney of rainbow trout in response to handling stress. BMC genomics. 2005, 6 (1): 3-10.1186/1471-2164-6-3.PubMedPubMed CentralGoogle Scholar
  8. MacKenzie S, Iliev D, Liarte C, Koskinen H, Planas JV, Goetz FW, Molsa H, Krasnov A, Tort L: Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol. Molecular immunology. 2006, 43 (9): 1340-1348. 10.1016/j.molimm.2005.09.005.PubMedGoogle Scholar
  9. Iliev DB, Liarte CQ, MacKenzie S, Goetz FW: Activation of rainbow trout (Oncorhynchus mykiss) mononuclear phagocytes by different pathogen associated molecular pattern (PAMP) bearing agents. Molecular immunology. 2005, 42 (10): 1215-1223. 10.1016/j.molimm.2004.11.023.PubMedGoogle Scholar
  10. Byon JY, Ohira T, Hirono I, Aoki T: Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination. Fish Shellfish Immunol. 2005, 18 (2): 135-147. 10.1016/j.fsi.2004.06.008.PubMedGoogle Scholar
  11. Ewart KV, Belanger JC, Williams J, Karakach T, Penny S, Tsoi SC, Richards RC, Douglas SE: Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology. Dev Comp Immunol. 2005, 29 (4): 333-347. 10.1016/j.dci.2004.08.004.PubMedGoogle Scholar
  12. Morrison RN, Cooper GA, Koop BF, Rise ML, Bridle AR, Adams MB, Nowak BF: Transcriptome profiling the gills of amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.): a role for tumor suppressor p53 in AGD pathogenesis?. Physiological genomics. 2006, 26 (1): 15-34. 10.1152/physiolgenomics.00320.2005.PubMedGoogle Scholar
  13. Peatman E, Baoprasertkul P, Terhune J, Xu P, Nandi S, Kucuktas H, Li P, Wang S, Somridhivej B, Dunham R, Liu Z: Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) after infection with a Gram-negative bacterium. Dev Comp Immunol. 2007Google Scholar
  14. Purcell MK, Nichols KM, Winton JR, Kurath G, Thorgaard GH, Wheeler P, Hansen JD, Herwig RP, Park LK: Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus. Molecular immunology. 2006, 43 (13): 2089-2106. 10.1016/j.molimm.2005.12.005.PubMedGoogle Scholar
  15. Rise ML, Jones SR, Brown GD, von Schalburg KR, Davidson WS, Koop BF: Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection. Physiological genomics. 2004, 20 (1): 21-35. 10.1152/physiolgenomics.00036.2004.PubMedGoogle Scholar
  16. Yasuike M, Kondo H, Hirono I, Aoki T: Difference in Japanese flounder, Paralichthys olivaceus gene expression profile following hirame rhabdovirus (HIRRV) G and N protein DNA vaccination. Fish Shellfish Immunol. 2006Google Scholar
  17. Kasahara M, Naruse K, Sasaki S, Nakatani Y, Qu W, Ahsan B, Yamada T, Nagayasu Y, Doi K, Kasai Y, Jindo T, Kobayashi D, Shimada A, Toyoda A, Kuroki Y, Fujiyama A, Sasaki T, Shimizu A, Asakawa S, Shimizu N, Hashimoto S, Yang J, Lee Y, Matsushima K, Sugano S, Sakaizumi M, Narita T, Ohishi K, Haga S, Ohta F, Nomoto H, Nogata K, Morishita T, Endo T, Shin IT, Takeda H, Morishita S, Kohara Y: The medaka draft genome and insights into vertebrate genome evolution. Nature. 2007, 447 (7145): 714-719. 10.1038/nature05846.PubMedGoogle Scholar
  18. Engelsma MY, Huising MO, van Muiswinkel WB, Flik G, Kwang J, Savelkoul HF, Verburg-van Kemenade BM: Neuroendocrine-immune interactions in fish: a role for interleukin-1. Veterinary immunology and immunopathology. 2002, 87 (3–4): 467-479. 10.1016/S0165-2427(02)00077-6.PubMedGoogle Scholar
  19. Kobayashi I, Sekiya M, Moritomo T, Ototake M, Nakanishi T: Demonstration of hematopoietic stem cells in ginbuna carp (Carassius auratus langsdorfii) kidney. Dev Comp Immunol. 2006, 30 (11): 1034-1046. 10.1016/j.dci.2006.01.005.PubMedGoogle Scholar
  20. To TT, Hahner S, Nica G, Rohr KB, Hammerschmidt M, Winkler C, Allolio B: Pituitary-interrenal interaction in zebrafish interrenal organ development. Molecular endocrinology (Baltimore, Md. 2007, 21 (2): 472-485. 10.1210/me.2006-0216.Google Scholar
  21. Willett CE, Cortes A, Zuasti A, Zapata AG: Early hematopoiesis and developing lymphoid organs in the zebrafish. Dev Dyn. 1999, 214 (4): 323-336. 10.1002/(SICI)1097-0177(199904)214:4<323::AID-AJA5>3.0.CO;2-3.PubMedGoogle Scholar
  22. Joerink M, Ribeiro CM, Stet RJ, Hermsen T, Savelkoul HF, Wiegertjes GF: Head kidney-derived macrophages of common carp (Cyprinus carpio L.) show plasticity and functional polarization upon differential stimulation. J Immunol. 2006, 177 (1): 61-69.PubMedGoogle Scholar
  23. Mackenzie S, Liarte C, Iliev D, Planas JV, Tort L, Goetz FW: Characterization of a highly inducible novel CC chemokine from differentiated rainbow trout (Oncorhynchus mykiss) macrophages. Immunogenetics. 2004, 56 (8): 611-615. 10.1007/s00251-004-0698-6.PubMedGoogle Scholar
  24. Bootland LM, Leong JC: Infectious hematopoietic necrosis virus. Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections, Woo PTK & Bruno DW, eds CAB International, Oxon, UK. 1999, 57-121.Google Scholar
  25. Hoffmann B, Beer M, Schutze H, Mettenleiter TC: Fish rhabdoviruses: molecular epidemiology and evolution. Current topics in microbiology and immunology. 2005, 292: 81-117.PubMedGoogle Scholar
  26. Schutze H, Enzmann PJ, Kuchling R, Mundt E, Niemann H, Mettenleiter TC: Complete genomic sequence of the fish rhabdovirus infectious haematopoietic necrosis virus. The Journal of general virology. 1995, 76 ( Pt 10): 2519-2527.Google Scholar
  27. Schutze H, Mundt E, Mettenleiter TC: Complete genomic sequence of viral hemorrhagic septicemia virus, a fish rhabdovirus. Virus genes. 1999, 19 (1): 59-65. 10.1023/A:1008140707132.PubMedGoogle Scholar
  28. Lee JY, Cho WJ, Do JW, Kim HJ, Park JW, Park MA, Sohn SG, Jeong G, Hah YC: Monoclonal antibodies raised against infectious haematopoietic necrosis virus (IHNV) G protein and a cellular 90 kDa protein neutralize IHNV infection in vitro. The Journal of general virology. 1996, 77 ( Pt 8): 1731-1737.Google Scholar
  29. McAllister PE, Wagner RR: Structural proteins of two salmonid rhabdoviruses. Journal of virology. 1975, 15 (4): 733-738.PubMedPubMed CentralGoogle Scholar
  30. Thoulouze MI, Bouguyon E, Carpentier C, Bremont M: Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout. Journal of virology. 2004, 78 (8): 4098-4107. 10.1128/JVI.78.8.4098-4107.2004.PubMedPubMed CentralGoogle Scholar
  31. Biacchesi S, Thoulouze MI, Bearzotti M, Yu YX, Bremont M: Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. Journal of virology. 2000, 74 (23): 11247-11253. 10.1128/JVI.74.23.11247-11253.2000.PubMedPubMed CentralGoogle Scholar
  32. Biering E, Villoing S, Sommerset I, Christie KE: Update on viral vaccines for fish. Developments in biologicals. 2005, 121: 97-113.PubMedGoogle Scholar
  33. Lorenzen N, Lorenzen E, Einer-Jensen K, LaPatra SE: DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout. Fish Shellfish Immunol. 2002, 12 (5): 439-453. 10.1006/fsim.2002.0422.PubMedGoogle Scholar
  34. Bishop RE: Fundamentals of endotoxin structure and function. Contributions to microbiology. 2005, 12: 1-27.PubMedGoogle Scholar
  35. Raetz CR, Whitfield C: Lipopolysaccharide endotoxins. Annual review of biochemistry. 2002, 71: 635-700. 10.1146/annurev.biochem.71.110601.135414.PubMedPubMed CentralGoogle Scholar
  36. Iliev DB, Roach JC, Mackenzie S, Planas JV, Goetz FW: Endotoxin recognition: in fish or not in fish?. FEBS letters. 2005, 579 (29): 6519-6528. 10.1016/j.febslet.2005.10.061.PubMedPubMed CentralGoogle Scholar
  37. Amend DF, Yasutake WT, Mead RW: A haematopoietic virus disease of rainbow trout and sockeye salmon. Trans Am Fish Soc. 1969, 796–804-Google Scholar
  38. Drolet BS, Rohovec JS, Leong JA: The route of entry and progression of infectious hematopoietic necrosis virus in Oncorhynchus mykiss (Walbaum): a sequential immunohistochemic study. J Fish Dis. 1994, 337–47-Google Scholar
  39. Romero A, Figueras A, Tafalla C, Thoulouze MI, Bremont M, Novoa B: Histological, serological and virulence studies on rainbow trout experimentally infected with recombinant infectious hematopoietic necrosis viruses. Diseases of aquatic organisms. 2005, 68 (1): 17-28. 10.3354/dao068017.PubMedGoogle Scholar
  40. Wolf K: Infectious hematopoietic necrosis. In: Wolf K (ed) Fish viruses and fish viral diseases. Cornell University Press, Ithaca, NY,. 1988, 83-114.Google Scholar
  41. Yamamoto T: Multiplication of Infectious Hematopoietic Necrosis Virus in rainbow trout following immersion infection: Whole-body assay and immunohistochemistry. J Aquat Anim Health. 1990, 271-280. 10.1577/1548-8667(1990)002<0271:MOIHNV>2.3.CO;2.Google Scholar
  42. Yamamoto T, Batts WN, Arakawa CK, Winton JR: Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry. J Aq Animal Health. 1990, 2 (4): 271-280. 10.1577/1548-8667(1990)002<0271:MOIHNV>2.3.CO;2.Google Scholar
  43. Krasnov A, Afanasyev S, Oikari A: Hepatic responses of gene expression in juvenile brown trout (Salmo trutta lacustris) exposed to three model contaminants applied singly and in combination. Environmental toxicology and chemistry / SETAC. 2007, 26 (1): 100-109. 10.1897/06-056R.1.PubMedGoogle Scholar
  44. Purcell MK, Kurath G, Garver KA, Herwig RP, Winton JR: Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination. Fish Shellfish Immunol. 2004, 17 (5): 447-462. 10.1016/j.fsi.2004.04.017.PubMedGoogle Scholar
  45. Rodriguez MF, Wiens GD, Purcell MK, Palti Y: Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics. 2005, 57 (7): 510-519. 10.1007/s00251-005-0013-1.PubMedGoogle Scholar
  46. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M: A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature. 1997, 388 (6642): 548-554. 10.1038/41493.PubMedGoogle Scholar
  47. O'Donnell SM, Holm GH, Pierce JM, Tian B, Watson MJ, Chari RS, Ballard DW, Brasier AR, Dermody TS: Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus. Journal of virology. 2006, 80 (3): 1077-1086. 10.1128/JVI.80.3.1077-1086.2006.PubMedPubMed CentralGoogle Scholar
  48. Hung CH, Huang HR, Huang CJ, Huang FL, Chang GD: Purification and cloning of carp nephrosin, a secreted zinc endopeptidase of the astacin family. J Biol Chem. 1997, 272 (21): 13772-13778. 10.1074/jbc.272.21.13772.PubMedGoogle Scholar
  49. Boutet I, Long Ky CL, Bonhomme F: A transcriptomic approach of salinity response in the euryhaline teleost, Dicentrarchus labrax. Gene. 2006, 379: 40-50. 10.1016/j.gene.2006.04.011.PubMedGoogle Scholar
  50. Song HD, Sun XJ, Deng M, Zhang GW, Zhou Y, Wu XY, Sheng Y, Chen Y, Ruan Z, Jiang CL, Fan HY, Zon LI, Kanki JP, Liu TX, Look AT, Chen Z: Hematopoietic gene expression profile in zebrafish kidney marrow. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101 (46): 16240-16245. 10.1073/pnas.0407241101.PubMedPubMed CentralGoogle Scholar
  51. Laterveer L, Lindley IJ, Heemskerk DP, Camps JA, Pauwels EK, Willemze R, Fibbe WE: Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8. Blood. 1996, 87 (2): 781-788.PubMedGoogle Scholar
  52. Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G: Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact. Blood. 2000, 96 (8): 2673-2681.PubMedGoogle Scholar
  53. Donate C, Roher N, Balasch JC, Ribas L, Goetz FW, Planas JV, Tort L, Mackenzie S: CD83 expression in sea bream macrophages is a marker for the LPS-induced inflammatory response. Fish Shellfish Immunol. 2007, 23 (4): 877-85.PubMedGoogle Scholar
  54. Ohta Y, Landis E, Boulay T, Phillips RB, Collet B, Secombes CJ, Flajnik MF, Hansen JD: Homologs of CD83 from elasmobranch and teleost fish. J Immunol. 2004, 173 (7): 4553-4560.PubMedGoogle Scholar
  55. 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 (27-28): 5615-5626. 10.1016/j.vaccine.2006.04.061.PubMedGoogle Scholar
  56. 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. Dev Comp Immunol. 2005, 29 (7): 615-626. 10.1016/j.dci.2004.12.001.PubMedGoogle Scholar
  57. Triantafilou K, Triantafilou M, Dedrick RL: A CD14-independent LPS receptor cluster. Nature immunology. 2001, 2 (4): 338-345. 10.1038/86342.PubMedGoogle Scholar
  58. Levesque JP, Hendy J, Takamatsu Y, Simmons PJ, Bendall LJ: Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. The Journal of clinical investigation. 2003, 111 (2): 187-196.PubMedPubMed CentralGoogle Scholar
  59. Johnson MC, Sangrador-Vegas A, Smith TJ, Cairns MT: Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) matrix metalloproteinase-9. Fish Shellfish Immunol. 2004, 17 (5): 499-503. 10.1016/j.fsi.2004.05.005.PubMedGoogle Scholar
  60. Takeuchi K, Kubota S, Kinoshita M, Toyohara H, Sakaguchi M: Cloning and characterization of cDNA for carp matrix metalloproteinase 9. Fisheries Sci. 2002, 610–617-Google Scholar
  61. Muller CS, Huff T, Hannappel E: Reduction of thymosin beta4 and actin in HL60 cells during apoptosis is preceded by a decrease of their mRNAs. Molecular and cellular biochemistry. 2003, 250 (1-2): 179-188.PubMedGoogle Scholar
  62. Huff T, Muller CS, Otto AM, Netzker R, Hannappel E: beta-Thymosins, small acidic peptides with multiple functions. The international journal of biochemistry & cell biology. 2001, 33 (3): 205-220. 10.1016/S1357-2725(00)00087-X.Google Scholar
  63. Sun HQ, Yin HL: The {beta}-Thymosin Enigma. Ann N Y Acad Sci. 2007, 1112: 45-55.PubMedGoogle Scholar
  64. Lovoll M, Dalmo RA, Bogwald J: Extrahepatic synthesis of complement components in the rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2007, 23 (4): 721-31.PubMedGoogle Scholar
  65. Laufer J, Katz Y, Passwell JH: Extrahepatic synthesis of complement proteins in inflammation. Molecular immunology. 2001, 38 (2-3): 221-229. 10.1016/S0161-5890(01)00044-X.PubMedGoogle Scholar
  66. Nakao M, Kajiya T, Sato Y, Somamoto T, Kato-Unoki Y, Matsushita M, Nakata M, Fujita T, Yano T: Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio). J Immunol. 2006, 177 (8): 5471-5479.PubMedGoogle Scholar
  67. Nakao M, Osaka K, Kato Y, Fujiki K, Yano T: Molecular cloning of the complement (C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio). Immunogenetics. 2001, 52 (3-4): 255-263. 10.1007/s002510000273.PubMedGoogle Scholar
  68. 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 (3-4): 284-295. 10.1016/j.vetimm.2007.03.005.PubMedGoogle Scholar
  69. Ratajczak J, Reca R, Kucia M, Majka M, Allendorf DJ, Baran JT, Janowska-Wieczorek A, Wetsel RA, Ross GD, Ratajczak MZ: Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow. Blood. 2004, 103 (6): 2071-2078. 10.1182/blood-2003-06-2099.PubMedGoogle Scholar
  70. Ratajczak MZ, Reca R, Wysoczynski M, Kucia M, Baran JT, Allendorf DJ, Ratajczak J, Ross GD: Transplantation studies in C3-deficient animals reveal a novel role of the third complement component (C3) in engraftment of bone marrow cells. Leukemia. 2004, 18 (9): 1482-1490. 10.1038/sj.leu.2403446.PubMedGoogle Scholar
  71. Reca R, Mastellos D, Majka M, Marquez L, Ratajczak J, Franchini S, Glodek A, Honczarenko M, Spruce LA, Janowska-Wieczorek A, Lambris JD, Ratajczak MZ: Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1. Blood. 2003, 101 (10): 3784-3793. 10.1182/blood-2002-10-3233.PubMedGoogle Scholar
  72. Botelho RJ, Hackam DJ, Schreiber AD, Grinstein S: Role of COPI in Phagosome Maturation. J Biol Chem. 2000, 275 (21): 15717-15727. 10.1074/jbc.M910068199.PubMedGoogle Scholar
  73. Nakanishi T, Fischer U, Dijkstra JM, Hasegawa S, Somamoto T, Okamoto N, Ototake M: Cytotoxic T cell function in fish. Dev Comp Immunol. 2002, 26 (2): 131-139. 10.1016/S0145-305X(01)00055-6.PubMedGoogle Scholar
  74. Dijkstra JM, Somamoto T, Moore L, Hordvik I, Ototake M, Fischer U: Identification and characterization of a second CD4-like gene in teleost fish. Molecular immunology. 2006, 43 (5): 410-419. 10.1016/j.molimm.2005.03.005.PubMedGoogle Scholar
  75. Moore LJ, Somamoto T, Lie KK, Dijkstra JM, Hordvik I: Characterisation of salmon and trout CD8alpha and CD8beta. Molecular immunology. 2005, 42 (10): 1225-1234. 10.1016/j.molimm.2004.11.017.PubMedGoogle Scholar
  76. Overturf K, LaPatra S: Quantitative expression (Walbaum) of immunological factors in rainbow trout, Oncorhynchus mykiss (Walbaum), after infection with either Flavobacterium psychrophilum, Aeromonas salmonicida, or infectious haematopoietic necrosis virus. J Fish Dis. 2006, 29 (4): 215-224. 10.1111/j.1365-2761.2006.00707.x.PubMedGoogle Scholar
  77. Hansen JD, La Patra S: Induction of the rainbow trout MHC class I pathway during acute IHNV infection. Immunogenetics. 2002, 54 (9): 654-661. 10.1007/s00251-002-0509-x.PubMedGoogle Scholar
  78. Jorgensen SM, Hetland DL, Press CM, Grimholt U, Gjoen T: Effect of early infectious salmon anaemia virus (ISAV) infection on expression of MHC pathway genes and type I and II interferon in Atlantic salmon (Salmo salar L.) tissues. Fish Shellfish Immunol. 2007, 23 (3):
  79. Zhang YX, Chen SL: Molecular identification, polymorphism, and expression analysis of major histocompatibility complex class IIA and B genes of turbot (Scophthalmus maximus). Marine biotechnology (New York, NY. 2006, 8 (6): 611-623.Google Scholar
  80. Boudinot P, Blanco M, de Kinkelin P, Benmansour A: Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout. Virology. 1998, 249 (2): 297-306. 10.1006/viro.1998.9322.PubMedGoogle Scholar
  81. Hoeger B, Kollner B, Dietrich DR, Hitzfeld B: Water-borne diclofenac affects kidney and gill integrity and selected immune parameters in brown trout (Salmo trutta f. fario). Aquatic toxicology (Amsterdam, Netherlands). 2005, 75 (1): 53-64.Google Scholar
  82. Boudinot P, Boubekeur S, Benmansour A: Rhabdovirus infection induces public and private T cell responses in teleost fish. J Immunol. 2001, 167 (11): 6202-6209.PubMedGoogle Scholar
  83. Matsuyama T, Fujiwara A, Nakayasu C, Kamaishi T, Oseko N, Tsutsumi N, Hirono I, Aoki T: Microarray analyses of gene expression in Japanese flounder Paralichthys olivaceus leucocytes during monogenean parasite Neoheterobothrium hirame infection. Diseases of aquatic organisms. 2007, 75 (1): 79-83. 10.3354/dao075079.PubMedGoogle Scholar
  84. Tassakka AC, Savan R, Watanuki H, Sakai M: The in vitro effects of CpG oligodeoxynucleotides on the expression of cytokine genes in the common carp (Cyprinus carpio L.) head kidney cells. Veterinary immunology and immunopathology. 2006, 110 (1–2): 79-85. 10.1016/j.vetimm.2005.09.005.PubMedGoogle Scholar
  85. Sakai M, Kono T, Savan R: Identification of expressed genes in carp (Cyprinus carpio) head kidney cells after in vitro treatment with immunostimulants. Developments in biologicals. 2005, 121: 45-51.PubMedGoogle Scholar
  86. Rangatia J, Vangala RK, Treiber N, Zhang P, Radomska H, Tenen DG, Hiddemann W, Behre G: Downregulation of c-Jun expression by transcription factor C/EBPalpha is critical for granulocytic lineage commitment. Molecular and cellular biology. 2002, 22 (24): 8681-8694. 10.1128/MCB.22.24.8681-8694.2002.PubMedPubMed CentralGoogle Scholar
  87. Moran ST, Cariappa A, Liu H, Muir B, Sgroi D, Boboila C, Pillai S: Synergism between NF-{kappa}B1/p50 and Notch2 during the Development of Marginal Zone B Lymphocytes. J Immunol. 2007, 179 (1): 195-200.PubMedGoogle Scholar
  88. Liu WH, Lai MZ: Deltex regulates T-cell activation by targeted degradation of active MEKK1. Molecular and cellular biology. 2005, 25 (4): 1367-1378. 10.1128/MCB.25.4.1367-1378.2005.PubMedPubMed CentralGoogle Scholar
  89. Kraal G, van der Laan LJ, Elomaa O, Tryggvason K: The macrophage receptor MARCO. Microbes and infection / Institut Pasteur. 2000, 2 (3): 313-316.PubMedGoogle Scholar
  90. Bergmann SM, Fichtner D, Skall HF, Schlotfeldt HJ, Olesen NJ: Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence. Diseases of aquatic organisms. 2003, 55 (3): 205-210. 10.3354/dao055205.PubMedGoogle Scholar
  91. Quillet E, Dorson M, Le Guillou S, Benmansour A, Boudinot P: Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout. Fish Shellfish Immunol. 2007, 22 (5): 510-519. 10.1016/j.fsi.2006.07.002.PubMedGoogle Scholar
  92. Krasnov A, Koskinen H, Rexroad C, Afanasyev S, Molsa H, Oikari A: Transcriptome responses to carbon tetrachloride and pyrene in the kidney and liver of juvenile rainbow trout (Oncorhynchus mykiss). Aquatic toxicology (Amsterdam, Netherlands). 2005, 74 (1): 70-81.Google Scholar
  93. Iwama GK, Afonso LO, Todgham A, Ackerman P, Nakano K: Are hsps suitable for indicating stressed states in fish?. The Journal of experimental biology. 2004, 207 (Pt 1): 15-19. 10.1242/jeb.00707.PubMedGoogle Scholar
  94. Iwama GK, McGeer JC, Pawluck MP: The effect of five fish anaesthetics on acid-base balance, hematocrit, cortisol an adrenaline in rainbow trout. Can J Zool. 1989, 2065-2073.Google Scholar
  95. Chomcynski P: A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques. 1993, 532-537.Google Scholar
  96. Chomcynski P, Sacchi N: Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162 (1): 156-169.Google Scholar
  97. Tomasec J, Fijan N: Virusne bolestiriba (viral dseases of fish). Final report on research under a part of project 6n/1966, Zagreb. 1971Google Scholar
  98. Reed LJ, Muench H: A simple method of estimating fifty percent endpoints. The American Journal of Hygiene. 1938, 27: 493-497.Google Scholar
  99. Yang YH, Speed T: Design issues for cDNA microarray experiments. Nature reviews. 2002, 3 (8): 579-588.PubMedGoogle Scholar
  100. Cleveland WS: Local regression models. Statistical Models. Edited by: J.M. Chambers TJHE. 1999, S, Pacific Grove, Wadsworth and Brooks/Cole, 309–376-Google Scholar
  101. Storey JD, Tibshirani R: Statistical significance for genomewide studies. Proceedings of the National Academy of Sciences of the United States of America. 2003, 100 (16): 9440-9445. 10.1073/pnas.1530509100.PubMedPubMed CentralGoogle Scholar
  102. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000, 25 (1): 25-29. 10.1038/75556.PubMedPubMed CentralGoogle Scholar

Copyright

© MacKenzie et al; licensee BioMed Central Ltd. 2008

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.