Developmental and Comparative Immunology 36 (2012) 93103

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Developmental and Comparative Immunology

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

Molecular characterization of nucleotide binding and oligomerization domain

(NOD)-2, analysis of its inductive expression and down-stream signaling
following ligands exposure and bacterial infection in rohu (Labeo rohita)
B. Swain a, M. Basu a, B.R. Sahoo a, N.K. Maiti a, P. Routray b, A.E. Eknath a, M. Samanta a,
a
Fish Health Management Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, Orissa 751002, India
b
Aquaculture Production and Environment Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, Orissa 751002, India

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

Article history: Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor
Received 13 May 2011 (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses
Revised 16 June 2011 or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and
Accepted 17 June 2011
characterized from rohu (Labeo rohita) which is highly commercially important sh species in the Indian
Available online 13 July 2011
subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open read-
ing frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular
Keywords:
mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 491 aa and 111200 aa),
Indian major carp
Labeo rohita
one NACHT domain (at 271441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenet-
NOD-2 ically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited signicant similarity
RICK (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive
IFN-c expression across the developmental stages, and highlighted the embryonic innate defense system in
Aeromonas hydrophila sh. Tissue specic analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distri-
Edwardsiella tarda bution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation,
Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2
and its associated downstream molecules RICK and IFN-c were signicantly enhanced in the treated sh
compared to control. These ndings suggested the key role of NOD-2 in augmenting innate immunity in
sh in response to bacterial and viral infection. This study may be helpful for the development of preven-
tive measures against infectious diseases in sh.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Innate immune system is the rst line of defense system against
pathogens and pathogenic products. While higher vertebrates like
birds and mammals show presence of higher level of immune de-
fense mechanism like adaptive immunity, which is a slower pro-
cess, insects and other lower vertebrates like sh and amphibian
depend upon innate immune system, a more faster one but less
specic than adaptive immunity (Akira et al., 2006; Magnadottir,
2006). Innate immune system recognizes pathogens or conserved
pathogen derived structures like agellin, lipopolysaccharides
(LPS), lipoproteins, peptidoglycan (PGN) and nucleic acid by
germ-line-encoded pattern recognition receptors (PRRs) that are
distributed in extracellular, membrane and cytoplasmic compart-
ments. Three major classes of PRRs have been identied: (a) the
Toll-like receptors (TLRs) that recognize ligand on either the extra-
cellular surface or within the endosome, (b) the NOD-like receptors
Corresponding author. Tel.: +91 674 2465421; fax: +91 674 2465407.
E-mail address: msamanta1969@yahoo.com (M. Samanta).
(NLRs) that function as cytoplasmic sensors, and (c) RIG I-like
receptors (RLRs) class of cytoplasmic PRR that recognize viruses
(Akira et al., 2006; Franchi et al., 2009; Chen et al., 2009).
NLR is more recently identied member in the intracellular PRR
family. This family of receptors is characterized with presence of
three domains. At C-terminal, a leucin rich repeat (LRR) domain,
at the center a nucleotide oligomerization (NACHT) domain, and
a proteinprotein interaction domain at the N-terminal end (Chen
et al., 2009; Benko et al., 2008). The C-terminal LRR domain recog-
nizes specic ligand to which the NLR is to be associated, the
NACHT domain mediates self-regulation and oligomerization and
the N-terminal domain generates downstream signals (Jin and
Flavell, 2010). Based on various N-terminal domains, like pyrin do-
main (PYD), caspase recruitment domain (CARD) or baculovirus
inhibitor of apoptosis repeat domain (BIR), NLR is subdivided into
three classes viz., NALP (NACHT, LRR and PYD containing proteins),
NOD (NACHT, LRR and CARD containing proteins) and NAIP (for
neuronal apoptosis inhibitor protein) (Akira et al., 2006; Wilman-
ski et al., 2008; Opitz et al., 2009; Proell et al., 2008; Fritz et al.,
2006). NOD subfamily consists of ve members NOD1, NOD2,

0145-305X/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2011.06.018
94 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103
NOD3, NOD4 and NOD5. NOD-2 detects muramyl dipeptide (MDP)
found in both Gram positive and negative bacterial peptidoglycan,
and has two amino terminal caspase recruitment domains (CARD1
and CARD2) that trigger down-stream signaling (Inohara et al.,
1998, 2003; Kim et al., 2008; Huang et al., 2008; Laing et al., 2008).
Prior to the activation by ligand, NLR protein remains in an inac-
tive form due to folding of LRR region and its binding to NACHT,
and on the arrival of ligand the LRR region unfolds and mediate signal
transduction (Bourhis et al., 2007; Monie et al., 2009; Jozaki et al.,
2009). Following recognition of bacterial PGN by NOD-2, oligomeri-
zation of the NACHT domains initiates the recruitment of interacting
proteins, the RIP2 or RICK (receptor interacting serinethreonine pro-
tein kinase-2) via CARDCARD-interaction. Recruitment of RICK
causes activation of Ijj complex and stimulates NF-jB activation
by ubiquitinylation of Ijj-c subunit of Ijj complex. NOD2 also acti-
vates MAPK (mitogen-activated protein kinase) pathway that results
in activation of specic transcription factors like AP-1, and induces
the expression of pro-inammatory cytokines and chemokines viz.,
IL-1b, IL-6, IL-8, TNF and IFN-c (Monie et al., 2009; Chen et al.,
2010a,b; Hasegawa et al., 2008; Rosenzweig et al., 2009; Inohara
et al., 2000).
NOD-2 has been extensively studied in mammals, including hu-
man (Hisamatsu et al., 2003; Voss et al., 2006), mouse (Iwanaga
et al., 2003) and porcine (Tohno et al., 2008), and recently the exis-
tence of some members of NLR family has been described in lower
vertebrates and invertebrates. In zebrash, three distinct classes of
NLR subfamily (NLR-A, B and C) were reported. NLR-A resembled
mammalian NODs, NLR-B to mammalian NALPs, and NLR-C was
unique to the teleost sh (Laing et al., 2008). In channel catsh (Ict-
alurus punctatus), ve NLRs (NOD1, NOD2, NLRC3, NLRC5 and
NLRX1) were reported, and were induced in Edwardsiella ictaluri
infection (Sha et al., 2009). Recently NOD1 and NOD2 genes were
reported in grass carp (Chen et al., 2010a,b) and two splice variants
of NOD-2 (NOD-2 a and b) was identied in rainbow trout (Chang
et al., 2011).
India ranks third among the world freshwater sh producers
(FAO, 2003), and among the freshwater cultured sh species, rohu
(Labeo rohita) is the most commercially important and highly fa-
vored sh in the Indian subcontinent. Diseases are one of the lim-
iting factors in the success and failure of the aquaculture industry.
Fishes are immune-primitive animal, and to protect themselves
against diseases, they mostly depend on their innate immune re-
sponses elicited by various PRRs. NOD-2 is one of the important
PRR and is expected to play a signicant role in protecting sh
against diseases.
In rohu, information on various PRRs is meager, and there is no
information on NOD-2 gene, its sequence, basal and inductive
expression prole and its signaling mechanism following infection.
Therefore, the present study was undertaken to clone and charac-
terize NOD-2 gene, to study its expression pattern in ontogeny, ba-
sal expression in various organs, and to investigate its role in PGN
and LTA stimulation, during Aeromonas hydrophila and Edwardsi-
ella tarda infections and in response to poly I:C exposure. Here
we report the complete cDNA sequence of rohu NOD-2 gene, its
inductive expression, phylogenetic relationship among mammals
and other sh species and analysis of its downstream signaling cas-
cades following ligand stimulation and infection.
2. Materials and methods
2.1. Fish
Rohu (L. rohita) weighing 50 g, was obtained from the Central
Institute of Freshwater Aquaculture (CIFA), and was stocked in
500 L aerated tanks, with each tank containing 50 sh. Before the
start of the experiment, acclimatization was carried out for
3-weeks and the sh were fed twice a day with commercial carp
diet with daily two-third water exchange. The water temperature
varied from 25 to 28 C and the pH of the water varied from 7.4
to 7.6 during the experiment.
2.2. Bacteria
A. hydrophila (ATCC-35654) and E. tarda (ATCC-15947) were cul-
tured in LB broth (USB, USA) at 37 C for 16 h with constant shak-
ing. Viable count was determined as colony forming unit (CFU)
following 10 fold serial dilutions and plating on nutrient agar.
2.3. Cloning of rohu NOD-2 gene
To amplify and clone rohu NOD-2 gene, three sets of PCR prim-
ers (Supplementary Table S1A) were designed based on the nucle-
otide sequences of NOD-2 gene in Ctenopharyngodon idella
(Accession No. GQ141736.1) and zebrash (Accession No.
XM_692832.3). From rohu gill, total RNA was extracted, cDNA
was prepared and PCR was carried out using 1 ll cDNA in a 50 ll
reaction volume under the conditions of one cycle of initial dena-
turation at 94 C for 5 min followed by 40 cycles of 94 C for 30 s,
60 C for 30 s, 72 C for 1 min and a nal extension at 72 C for
5 min. One-fth of the PCR product was analyzed in 2% agarose
gel, the single specic band was puried with agarose gel DNA
extraction kit (Roche, Germany), cloned in pGEM-T Easy vector
(Promega, Madison, USA) and both strand sequencing was carried
out with T7 and SP6 primer (ABI prism 3000). Obtained sequence
was conrmed by BLAST.
To obtain full-length rohu NOD-2 cDNA sequence, 50 and 30 -rapid
amplication of cDNA end (RACE) were performed using cDNA
prepared with strand switching mechanism at 50 end of RNA tran-
script by SMARTer RACE cDNA amplication kit (Clontech, USA).
To amplify 30 and 50 -end region, NOD-2 gene specic forward (GSP
FW-30 ) and reverse (GSP RV-50 ) primers (Supplementary Table
S1A) were designed from partial rohu NOD-2 sequence obtained
previously. Touchdown-PCR was carried out for 30 and 50 RACE using
GSP-FW-30 /UPM and GSP-RV-50 /UPM primer sets (Supplementary
Table S1A), respectively under the conditions of one cycle of initial
denaturation at 94 C for 2 min; followed by ve cycles of 94 C/
30 s, 72 C/3 min; next ve cycles of 94 C/30s, 70 C/30s, 72 C/
3 min; next 25 cycles 94 C/30 s, 60 C/30 s, 72 C/3 min; and one
cycle at 72 C/5 min. Nested PCR for 50 and 30 RACE was performed
using NOD-2 specic GSP FW-30 /NUP for 30 and GSP RV-50 /NUP for
50 nested primer sets (Supplementary Table S1A). For nested PCR,
1 ll of primary RACE-PCR product was used as a template with
the following conditions: initial one cycle of 94 C/5 min, 35 cycle
of 94 C/30 s, 60 C/30 s and 72 C/90 s followed by one cycle of
72 C/5 min. The PCR products were cloned in pGEM-T Easy vector,
sequenced and validated through BLAST search (Altschul et al.,
1990). The full-length cDNA sequence of rohu NOD-2 (rNOD-2)
was submitted to the GenBank.
2.4. Sequence analysis
Amino acids in rNOD-2 were predicted from full-length cDNA
sequence by Lasergene DNA star software, and were aligned with
other species NOD-2 by CLUSTALW (Higgins, 1994). The alignment
le generated by CLUSTALW was submitted in ESPript2.2 program
(http://www.espript.ibcp.fr/ESPript) to build the blocks. The
molecular weight of the protein was calculated using protein
molecular weight calculator (http://www.sciencegateway.org/
tools/proteinmw.htm). Phylogenetic tree of NOD-2 among rohu
and other species was obtained by neighbor joining method of
MEGA4 program (Tamura et al., 2007). The branches were vali-
dated by bootstrap analysis from 1000 replications using default
 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103
speed of that program. Secondary structure of NOD-2 was pre-
dicted by SMART algorithm (http://smart.embl-heidelberg.de),
and the percentages of similarity and identity of amino acids were
calculated by the MatGAT program (Campanella et al., 2003).
2.5. In vivo expression of NOD-2 gene
To study NOD-2 gene expression during various developmental
stages of rohu, mature rohu were bred by induced breeding tech-
niques. Fertilized eggs were collected from CIFA hatchery, and
were maintained at ambient temperature (2829 C). Develop-
mental stages from fertilized eggs to hatchlings were periodically
observed under the inverted microscope. Samples were collected
separately at different hours of developments viz., 0 h (fertilized
egg), 4, 5, 8, 9, 14, 16 h and hatchlings at 48 and 72 h. Total RNA
was extracted using TRIzol reagent, cDNA was prepared using oli-
go-dT primer, and NOD-2 expression was analyzed by quantitative
real-time PCR (qRT-PCR) assay keeping b-actin as an internal con-
trol (primers: Supplementary Table S1B).
To study the NOD-2 basal expression, three rohu ngerlings
(50 g) were sampled and gill, liver, kidney, intestine, blood, skin,
heart, brain and muscle were collected separately. In each sample,
total RNA was extracted, cDNA was prepared and expression of
NOD-2 transcripts was analyzed by quantitative real-time PCR
(qRT-PCR) assay keeping b-actin as internal control.
For in vivo treatment, healthy rohu ngerlings (50 g) were di-
vided into control and treated groups keeping three sh in each
group. Puried poly I:C (Sigma, USA) was diluted in DEPC treated
water at 10 mg/ml. DEPC treated water (100 ll) containing
300 lg of diluted poly I:C was intra-peritoneally (i.p.) injected to
treated group, and control sh received only 100 ll of DEPC-trea-
ted water. Lipoteichoic acid (LTA) from Streptococcus pyogenes
(cat: L3140; Sigma, USA) and peptidoglycans (PGN) from Staphylo-
coccus aureus (cat: 77140; Sigma, USA) were diluted in PBS and
were injected at 20 lg/sh through caudal vein (i.v.). Control sh
received only 100 ll of PBS through i.v. route.
For live bacteria challenge, sh were intra-peritoneally (i.p.) in-
jected with 100 ll of PBS containing A. hydrophila or E. tarda sus-
pension (1  106 CFU/sh) separately. The control group was
injected with 100 ll of PBS only, and kept separately in the aerated
tank.
After the designated time course, control and treated group of
sh were sacriced and tissues were collected separately. Total
RNA was extracted from each sample using TRIzol reagent and
cDNA was prepared with oligo-dT primer and high delity reverse
transcriptase kit (Roche, Germany). qRT-PCR was carried out to
compare NOD-2 gene expression between treated and control
group after normalizing it with b-actin expressions. Data was ex-
pressed as mean standard error (s.e.) and the signicant differ-
ence between the control and treated group at each time point
was determined by the Student t-test using Microsoft Excel 2010
with p < 0.05 as signicance level.
2.6. Cell culture
Primary culture of rohu kidney and heart cell was carried out fol-
lowing the previously described protocol (Hwang et al., 2010) with
minor modications. Briey, the kidney or heart from healthy rohu
ngerling (50 g) was surgically isolated in Hanks balanced salt
solution (HBSS), washed twice and then transferred into RPMI-
1640 medium. It was dissected into small pieces by scissor and
was passed through 21-gauge needle, treated with 100 U of collage-
nase (Sigma, Germany) and was placed on magnetic stirrer for
30 min. Medium containing cell suspension was centrifuged at
3000 rpm at 4 C for 5 min. Pellet was washed twice, and was
dissolved in 12 ml of RPMI-1640 medium containing 10% FBS
95
(GIBCOBRL, USA), 100 IU/ml of penicillin and 100 lg/ml of strepto-
mycin (Sigma). Cells were distributed at 1 ml/well in a 24 well cell
culture plate (TPP, Switzerland) and were cultured at 28C in a 5%
CO2 atmosphere. On 4th day, half of the medium was replaced with
fresh growth medium, at 7th day the cells were washed twice with
RPMI-1640 medium without antibiotics and serum, and were used
for ligand stimulation. For each ligand exposure triplicate wells
were used. PBS (20 ll) containing 20 lg of LTA or PGN was added
separately in each well and were designated as treated group and
the control wells received only PBS. After 2 and 4 h of ligand expo-
sure, total RNA was extracted with TRIzol reagent, cDNA was
prepared and qRT-PCR was carried out to analyze NOD-2, RICK
and IFN-c gene expression between treated and control group after
normalizing with b-actin expressions.
2.7. RNA isolation and 1st strand cDNA synthesis
Total cellular RNA from different organs/tissues (gill, liver, kid-
ney, intestine, blood, skin, heart, brain and muscle) of sh or from
cell culture wells were extracted with TRIzol reagent (Invitrogen,
USA). RNA concentration was measured by UV-spectrophotometer
and the integrity was assessed by observing the band intensity of
28S and 18S ribosomal RNA on 1% agarose gel. For the rst strand
cDNA synthesis, 1 lg of total RNA was treated with 1 unit of DNase
I (MBI, Fermentas) and reverse transcription was carried out using
high delity reverse transcriptase (RT) kit (Roche, Germany).
2.8. Real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) analysis of the target
genes; NOD2, RICK, IFN-c and the reference gene b-actin (primers;
Supplementary Table S1B) were performed in LightCycler 480 II
real-time PCR detection system (Roche, Germany). Amplications
were carried out in 10 ll reaction volume, containing 1.0 ll of
cDNA, 0.25 ll of FW and RV primer (2.5 lM each), 5 ll of 2 light-
Cycler 480 SYBR Green I master mix (Roche, Germany) and 3.5 ll
of PCR grade H2O. PCR amplications were performed in triplicate
wells under the following conditions: initial denaturation at 95 C
for 10 min followed by 45 cycles of 94 C for 10 s, 60 C (for NOD2
and b-actin) or 58 C (for RICK) or 53 C (for IFN-c) for 10 s and
72 C/10 s. The reaction carried out without cDNA was used as neg-
ative control. The PCR efciencies were determined by analysis of
serial dilutions of cDNA, and efciencies were close to 100% allow-
ing use of the 2DDCT method for calculation of relative gene
expression of the target gene NOD2, RICK and IFN-c with that of
reference gene b-actin. The specicity of the amplication product
was veried in ethidium bromide-stained agarose gel (2%).
3. Results
3.1. Rohu NOD-2 (rNOD-2) gene sequence analysis
The full-length cDNA of rohu NOD-2 (rNOD-2) consisted of
3176 bp with a 165 bp 50 un-translated region (UTR), a 2949 bp
open reading frame (ORF) having translation initiation codon
(AUG) at nucleotide (nt) 166168, encoding 982 amino acid (aa)
and a 62 bp 30 -UTR. Secondary structure prediction by SMART re-
vealed that rNOD-2 consisted of two N-terminal CARD domains,
CARD1 (491 aa) and CARD2 (111200 aa), one middle NACHT do-
main (271441aa) and seven C-terminal LRR domains (at 731758,
759786, 812839, 840867, 868895, 896923 and 924951 aa)
(Fig. 1A and B; GenBank Accession No. JF923468). Analysis with the
SignalP program revealed a 24 aa putative consensus secretion sig-
nal peptide in rNOD-2, and upon cleavage between Ala24 and
Glu25 it was expected to generate a mature protein of 958 aa
96 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103
residues. The estimated molecular mass of complete rNOD-2 was
109.65 kDa, and the mature protein (without signal peptide) was
107.2 kDa.
3.2. Structural and phylogenetic analysis of rohu NOD-2
The deduced rNOD-2 amino acids sequence was compared with
other species full-length NOD-2 and its various domains (CARDs,
NACHT and LRR) to identify the similarity and identity of amino
acids by MatGAT program. As observed in Table 1, rNOD-2 exhib-
ited highest amino acid similarity and identity with freshwater
shes viz.; grass carp (94.2% and 88.6%) and zebrash (91.2% and
84.1%) than that of marine water sh, and as expected dog, chim-
panzee, and human were at the bottom of the table. Except the
NACHT domain, other domains (CARDs and LRR) in rNOD-2 showed
highest similarity and identity with grass carp. To identify the con-
served amino acid sequence motifs, the predicted amino acid
sequence of rNOD-2 was compared with other species NOD-2 by
CLUSTALW, and several strictly conserved regions (identical amino
acids in black box) and well conserved regions (>50% consensus)
were identied in CARD, NACHT and LRR domains across the spe-
cies (Supplementary Fig. S1A and B). Most strikingly, we observed
a stretch of 25aa in LRR region of rohu, raibow trout, fugu, and also
in rabbit but not in other species (Supplementary Fig. S1B).
To analyze the evolutionary relationship, a molecular phyloge-
netic tree was constructed based on pair-wise alignments of
rNOD-2 amino acid sequence with other species. The inferred phy-
logeny of the NOD-2 gene family showed two distinct clusters; one
for mammals and the other for shes. In the sh cluster, evolution-
al diversion of NOD-2 gene among freshwater sh (rohu, grass carp
and zebrash) and marine sh (rainbow trout and fugu) was
clearly observed (Fig. 2). In the phylogenetic tree, rNOD-2 showed
the closest relationship with grass carp whereas, higher verte-
brates viz., rat, mouse, rabbit, cattle, pig, dog, chimpanzee and hu-
man NOD-2 formed a separate cluster and were distantly related to
rNOD-2. The evolutionary closeness between grass carp and rohu
NOD-2 possibly implied a similar biological function.
3.3. NOD-2 gene expression during developmental stages
In rohu, developmental prole of NOD-2 gene expression was
analyzed by quantitative real-time PCR (qRT-PCR), and it revealed
constitutive expression of NOD-2 gene throughout the embryonic
developmental stages, hatched embryos and larvae (Fig. 3). These
data may suggest the protective role of NOD-2 during the early
stages of development in pathogenically hostile environments.
3.4. Tissue distribution of rNOD-2
To examine the basal expression of NOD-2 in healthy rohu n-
gerlings, quantitative real-time PCR (qRT-PCR) analysis was carried
out in nine different organs and tissues. To eliminate the individual
variation of NOD-2 expression, three sh were sampled and ana-
lyzed separately by qRT-PCR and their mean value was considered.
It was evident that NOD-2 was constitutively expressed in all the
organs examined viz.; gill, liver, kidney, intestine, blood, skin,
heart, brain, and muscle. Among the tissues, comparatively highest
level of NOD-2 expression was detected in liver followed by blood,
and an equivalent expression was in gill, skin and heart and the
lowest expression was observed in intestine and brain (Fig. 4).
3.5. Modulation of NOD-2 expression by PGN and LTA
To analyze the modulation of rNOD-2 gene in response to their
ligand stimulation, NOD-2 gene expression in rohu kidney and
heart cell culture was analyzed by qRT-PCR following PGN and
LTA stimulation. In kidney cell culture, LTA triggered the signicant
up-regulation (p > 0.05) of rNOD-2 gene (12 fold) at 2 h post
stimulation and was receded to almost half (6 fold) at 4 h as com-
pared to control. Similar course of NOD-2 activation at 2 h (7
fold), and 4 h (3.5 fold) were noticed in PGN treatment. In heart
cell culture a different kinetics was observed. LTA mediated
NOD-2 expression was around 2 fold (p > 0.05) both at 2 and 4 h
as compared to control. Whereas, PGN mediated induction was
slightest at 2 h and a signicant (p > 0.05) induction (7.5 fold)
was observed at 4 h which was almost comparable to expression
in kidney cell at 2 h post stimulation (Fig. 5A).
Next we examined the in vivo expression of rNOD-2 gene modula-
tion in the blood of rohu ngerlings following intra venous (i.v.) injec-
tion of LTA and PGN, and analyzed rNOD-2 gene transcripts at 2 and
4 h post treatment by qRT-PCR. As observed in Fig. 5B, LTA and PGN
mediated rNOD-2 gene expression in blood remained unchanged at
2 h, but a signicant (p > 0.05) induction (4 fold) was noticeable at
4 h post treatment in the treated sh compared to control sh.
3.6. Differential expression of NOD-2 in bacterial infection
To investigate whether NOD-2 expression could be modulated
in bacterial infection, articial infection with E. tarda was carried
out for different time course (6, 12 and 24 h), and in vivo rNOD-2
gene expression was analyzed in gill, liver, kidney, intestine and
blood by qRT-PCR. The most striking observation was that at
12 h post infection, rNOD-2 expression was signicantly
(p > 0.05) induced in liver (5 fold) and blood (6 fold) but not
in gill, kidney and intestine of the infected sh compared to con-
trol. At 24 h post infection, enhanced expression of rNOD-2 main-
tained a stable state (p > 0.05) in blood but declined to basal level
in liver (Fig. 6A).
We next examined rNOD-2 expression in A. hydrophila infected
rohu ngerlings at 24 and 48 h by qRT-PCR assay. As indicated in
Fig. 6B, the expression of rNOD-2 remained unchanged in gill and
kidney but signicant (p > 0.05) induction was observed in blood
at 24 and 48 h (5 fold) in the infected group of sh compared
to control. In liver, at 24 h there was down-regulation (p > 0.05)
but at 48 h a signicant (p > 0.05) up-regulation (2 fold) was ob-
served. In the intestine rNOD-2 was down regulated at 24 h and it
continued up to 48 h (p > 0.05) in the infected sh compared to
control. The pattern of rNOD-2 expression in various organs/tissues
following two types of bacterial infection (A. hydrophila and E. tar-
da) revealed almost similar pattern, blood is the highest responder
of rNOD-2 followed by liver.
3.7. Modulation of NOD-2 expression by poly I:C treatment
To assess whether poly I:C, a double stranded RNA mimicking
structure, could affect NOD-2 gene expression, rohu ngerlings
were i.p. injected with poly I:C, and three organs (gill, liver and kid-
ney) were analyzed for rNOD-2 gene transcripts at 24 and 36 h
post treatment by qRT-PCR assay. Kinetics of rNOD-2 expression
signicantly varied among the tissues (Fig. 7). In the treated sh,
at 24 h substantial increase (p > 0.05) in rNOD-2 transcripts were
observed in gill (1.8 fold) and kidney (2.4 fold) and at 36 h it de-
creased to normal level. However, in liver a steady state of increase
in rNOD-2 was observed at 24 h (1.3 fold) and 48 h (2.3 fold).
These results might suggest that during viral infection, the NOD-
2 response in various organs is diverse and liver was equally
important to other immunological organs.
3.8. Analysis of NOD-2 down-stream signaling pathway
The down-stream of NOD signaling pathway is RICK which is in-
volved in signal transduction and activation of nuclear factor kappa
 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103 97

Fig. 1. Rohu NOD-2 full-length cDNA showing ORF, UTR and various domains. Un-translated regions (UTR) at 50 and 30 were shown in upper case, ORF in lower case, start and
stop codon were underlined. CARD1 and CARD2 were marked with lightened and darken gray color. The central NACHT domain was shown with the gray background and
italic bold letter. Alternative LRR domains were shown with light gray and dark gray boxes (A). Schematic representation of domain organization of NOD-2 proteins (B).
GenBank Accession No: JF923468.
98 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103

Table 1
Similarity and identity of rohu NOD-2 and its domains with other species.

NOD-2 CARD-1 CARD-2 NACHT LRR

Similarity Identity Similarity Identity Similarity Identity Similarity Identity Similarity Identity
Zebrash 91.2 84.1 84.0 72.3 84.4 75.6 98.2 95.3 83.3 75.0
Rainbow trout2a 81.1 67.4 76.1 60.2 68.5 55.8 90.7 79.7 89.3 77.4
Rainbow trout2b 79.1 64.9 40.9 31.8 68.5 55.8 90.7 79.7 89.3 77.4
Grass carp 94.2 88.6 96.6 85.2 92.2 86.7 96.5 91.2 87.3 81.7
Fugu 77.2 61.2 70.3 57.1 64.4 45.3 86.5 76.6 89.0 70.5
Dog 48.8 35.5 58.2 33.7 50.0 28.6 74.9 60.2 65.1 49.8
Rat 61.7 43.4 56.0 30.4 48.9 26.7 74.3 60.8 54.0 41.7
Pig 64.1 45.5 54.9 33.7 46.7 28.9 75.4 59.1 70.2 46.8
Chimpanzee 61.9 45.0 57.1 37.0 52.2 28.9 74.9 59.6 67.8 47.1
Mouse 62.2 44.0 56.0 31.5 51.1 28.9 74.3 60.2 67.1 45.7
Human 62.1 45.0 57.1 37.0 51.1 28.9 74.9 59.6 67.8 47.5
Rabbit 62.2 45.7 58.9 36.3 53.3 30.0 73.7 59.1 70.4 55.7
Cattle 64.0 44.3 54.9 31.5 48.9 28.6 74.9 57.9 69.0 48.9

100 Human 4.5

82 Chimpanzee
4
93 Rabbit

fold changes from the calibrator (0h)

3.5
Dog

100 Pig 3
93
100 Cattle 2.5
Mouse
2
100 Rat
Grass-carp 1.5
96
100 Rohu 1
Zebrafish
0.5
Fugu
0
80 Rainbow-trout-NOD2a 0h 4h 5h 8h 9h 14h 16h 48h 72h
100 Rainbow-trout-NOD2b
Fig. 3. NOD-2 gene expression at different developmental stages. Total RNA was
extracted from different stages of development (denoted by hours) and quantitative
0.05
real-time PCR (qRT-PCR) was carried out to analyze the expression of NOD-2 gene
in different developmental stages. Expressions of NOD-2 mRNA transcripts were
Fig. 2. Phylogenetic relationship of rohu NOD-2 with mammalian and other sh
represented as a ratio relative to b-actin (internal control) levels in the same
species. NOD-2 amino acid sequences were aligned by using CLUSTALW program
samples. NOD-2 expression at 0 h was chosen as calibrator (1) and the relative
within DNASTAR and the un-rooted phylogenetic tree was generated by the
expression of NOD-2 in other developmental stages was represented as fold
neighbor-joining method within the MEGA 4 program. The tree was boosted 1000
changes from the calibrator. The results were expressed as mean standard error
times and the percentages were shown. The full-length NOD-2 amino acids
(bars in the graph) from three samples (n = 3).
sequences used for tree construction, were retrieved from following database:
human NP_071445.1; mouse NP_665856.2; dog XP_544412.2; pig NP_001098765.1;
cattle NP_001002889.1; chimpanzee NP_001098710.1; rabbit XP_002721391.1; rat
NP_001099642.1; zebrash XP_697924.3; takifugu AAY97878.1; grass carp 6
ACX71753.1; rainbow trout NOD2a, ADV31549.1; NOD2b, ADV31550.1.
Fold changes from the calibrator (brain)

B (NF-jB) or mitogen-activated protein kinase (MAPK). We inves-

4
tigated in vitro activation of RICK by analyzing its gene transcripts
in LTA and PGN treated kidney and heart cell culture and in vivo by
3
analysis in blood of rohu ngerlings by qRT-PCR assay. At 4 h post
treatment, signicant (p > 0.05) increase in RICK expression was
observed in PGN treated kidney (4.5 fold) and heart (5 fold) cell 2

culture and in blood (7 fold) of the treated sh compared to con-

trol. In response to LTA treatment, substantial increase of RICK 1
expression was observed in heart cell culture at 2 and 4 h, but
highly signicant (p > 0.05) increase (7 fold) was observed in 0
blood at 4 h post treatment in the treated group compared to Gill Liver Kidney Intestine Blood Skin Heart Brain Muscle
control (Fig. 8A and B).
Fig. 4. Basal expression of NOD-2 gene in various tissues. Total RNA was extracted
from gill, liver, kidney, intestine, blood, skin, heart, brain, and muscle. Quantitative
3.9. IFN-c induction following LTA, PGN and poly I:C treatment real-time PCR (qRT-PCR) was carried out to analyze the expression of NOD-2 gene
among the tissues. Expressions of NOD-2 mRNA transcript were represented as a
In NOD-signaling pathway, IFN-c is one of the downstream effec- ratio relative to b-actin (internal control) levels in the same samples. Brain
expressed lowest NOD-2 and was chosen as calibrator (1), and the relative
tor molecules. To examine whether IFN-c could be induced by PGN, expression of NOD2 in other tissues was represented as fold changes from the
LTA and poly I:C we analyzed its expression both in vitro and in vivo calibrator. The results were expressed as mean standard error (bars in the graph)
by qRT-PCR assay. PGN induced IFN-c expression both in vitro from three sh (n = 3).
 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103 99

A 14 A 10

9 *
12 8
*
fold changes from the control

fold changes from control

10 6 *
5

8
4
3
6
2

4 1

0

6h
Cont

12h
24h

Cont

12h
24h

6h

6h

6h
Cont

12h
24h

Cont

12h
24h

Cont

12h
24h
6h
2
treat treat treat treat treat
Gill Liver Kidney Intestine Blood
0
B
Cont

Cont
Cont

Cont
2h
4h
2h
4h

2h
4h
2h
4h

___________________ ___________________ ___________________ ___________________ 6

*
LTA PGN LTA PGN *
___________________ ___________________ 5
Kidney Heart

fold changes from control

4
B 6
fold changes from the control

3
5

4 2 *

3 1
*
* *
2
0
Cont
24h
48h

Cont
24h
48h

Cont
24h
48h

Cont
24h
48h

Cont
24h
48h
1 treat treat treat treat treat
Gill Liver Kidney Intestine Blood
0
Cont

Cont
2h
4h

2h
4h

___________________
LTA PGN
___________________
Blood
Fig. 5. NOD-2 gene expression in response to LTA and PGN. Primary culture of rohu
kidney and heart cell and rohu ngerling was stimulated by LTA and PGN for 2 and
4 h. Total RNA was extracted and qRT-PCR was carried out to analyze the expression
of NOD-2. All data were represented as a ratio relative to b-actin (internal control)
in the same samples and data were expressed as fold induction relative to the
control. The results were expressed as mean standard error (bar in the graph)
from triplicate cell culture wells or from three sh (n = 3). (A) NOD-2 expression in
kidney and heart cell culture and (B) in blood. Signicant difference (p < 0.05)
between the control and treated group was indicated with asterisks ().
(kidney and heart cell culture) and in vivo (blood). In the PGN-trea-
ted kidney cell culture, at 2 h post treatment there was 3.5 fold in-
crease in IFN-c that touched up to 7 fold (p < 0.05) at 4 h, and in
heart cell culture it was 3 fold (p < 0.05) at 2 h, and 10 fold in-
crease (p < 0.05) at 4 h in the treated cells compared to control. In
the PGN-treated blood, slightest increase in IFN-c was observed at
2 h post treatment in comparison to un-treated control blood and
a signicant increase (p < 0.05) in IFN-c induction (3.5 fold) was
noticed at 4 h post treatment. LTA mediated IFN-c expression was
much more appreciable (3 fold) in heart cell culture at 2 and 4 h
(Fig. 9A), and in blood a steady state of IFN-c induction was observed
at 2 h (1.5 fold) and at 4 h (2.5 fold) (p < 0.05) in the treated group
compared to control (Fig. 9B).
In response to in vivo poly I:C treatment, we observed signi-
cant increase (p < 0.05) in IFN-c (6 fold) in liver at 24 h and it in-
creased to 7 fold at 36 h of post treatment in the treated group of
___________________ Fig. 6. Modulation of NOD-2 expression following bacterial infections. Live bacteria
(1  106) were injected into rohu ngerlings by i.p. After the designated time
course, total RNA was extracted from gill, liver, kidney, intestine and blood; real-
time quantitative PCR was conducted to analyze the expression of NOD-2. b-Actin
was used as an internal control. The results were expressed as mean standard
error (bars) from three independent experiments (n = 3). (A) Edwardsiella tarda
infection (B) Aeromonas hydrophila infection. Signicant difference (p < 0.05)
between the control and treated group was indicated with asterisks ().
sh compared to control. However, expression of IFN-c gene tran-
scripts were down-regulated (p < 0.05) in gill and kidney at 24 h
post treatment, but at 36 h a substantial increase (2 fold) in
IFN-c expression was observed in kidney of the treated sh com-
pared to control (Fig. 9C). These results indicated the immunolog-
ical signicance of liver in inducing innate immune response.
4. Discussion
The innate immune system is the only defense weapon of inver-
tebrates and a fundamental defense mechanism of sh (Magnadot-
tir, 2006). It plays an important instructive role in acquired
immune response and homeostasis, and is equally important in
higher vertebrates. Innate immune response is rapid, nonspecic
and does not require priming. Several pattern recognition recep-
tors (PRRs) play the vital role in inducing innate immune response.
NOD-2 is one of the members of NLR family which acts as a cyto-
plasmic sensor of detecting pathogen associated molecular pattern
(PAMPs), and upon recognition it activates the intra-cellular signal-
ing cascades to induce a battery of innate immune genes. In mam-
mals, NOD receptors have been studied in great detail, however in
100 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103

4.0 A 7

3.5 6 *
*

fold chnages from the control

* 5
3.0 *
4
2.5
3
fold changes from control

*
* *
2.0
2
1.5 1

0
1.0

Cont
Cont

Cont
Cont
2h
4h
2h
4h

2h
4h
2h
4h
___________________ ___________________ ___________________ ___________________
0.5
LTA
___________________ PGN ___________________
LTA PGN
Kidney Heart
0
Cont

Cont

Cont
24h
36h

24h
36h

24h
36h

fold chnages from the control

B 9
treat treat treat 8
*
7
Gill Liver Kidney
6
Fig. 7. NOD-2 expression following poly I:C treatment. Poly I:C was injected into
5
rohu ngerlings by i.p. Total RNA was extracted from gill, liver and kidney at 24 and
4
36 h of post injection, and quantitative real-time PCR was conducted to analyze the
expression of NOD-2 gene expression. The data was normalized to b-actin and were
*
3
expressed as fold induction relative to control. The results were expressed as
mean standard error (bars) from three independent experiments (n = 3). Signif- 2
icant difference (p < 0.05) between the control and treated group was indicated 1
with asterisks ().
0

Cont
Cont

sh it has partially been studied in zebrash (Laing et al., 2008),
channel catsh (Sha et al., 2009), grass carp (Chen et al., 2010a,b)
and rainbow trout (Chang et al., 2011).
India ranks third among the world freshwater sh producers
(FAO, 2003) with aqua farming of Indian major carps (IMC) viz., L.
rohita, Catla catla and Cirrhinus mrigala out of which more than
80% is of rohu (L. rohita). Rohu is of prime importance not only in
India, but is equally important in Bangladesh, Pakistan, Myanmar,
Vietnam and Nepal. Diseases are one of bottleneck in aqua-farming
and considerable economic losses occur due to the diseases caused
by variety of infectious agents. Among these, the bacterial patho-
gens belonging to the genus Aeromonas, Psedumonas, Edwardsiella,
Flavobacterium, etc. are mainly responsible for severe mortality
and morbidity of Indian major carps (Bootsma et al., 1977; Kumar
et al., 1986; Karunasagar et al., 1989; Shome et al., 1996). Keeping
in view the importance of innate immunity during diseases, we fo-
cused our work on the identication, characterization of NOD-2
gene in rohu (rNOD-2), its modulation during infection, its signaling
cascades and the induction of down-stream effector molecule. Here
we report the identication of NOD-2 gene, its full length cloning
and sequencing, its ontogeny, tissue distribution, expressional up-
regulation following A. hydrophila and E. tarda infection and expo-
sure to PGN, LTA and poly I:C.
Rohu NOD-2 gene (rNOD-2) comprised of 3176 bp in length and
was closely related to grass carp NOD-2 (gcNOD2) having 3130 bp
(Chen et al., 2010a,b). Previously, in gcNOD2, a 24 aa signal peptide
was reported and we predicted the presence of a similar signal
peptide in rohu. These data differ signicantly from other species
NOD-2 including zebrash (Laing et al., 2008), which was devoid
of signal peptide, and justied the cytosolic localization of NOD
proteins. Existence of putative signal peptide in rohu and grass
carp NOD-2 needs further investigation to locate their cellular dis-
tribution. Two splice variants of NOD-2 were reported in rainbow
trout (Chang et al., 2011). Although we have identied a single
2h
4h
2h
4h
___________________ ___________________
LTA PGN
___________________
Blood
Fig. 8. RICK expression in response to LTA and PGN. Rohu kidney and heart cell
primary culture, and rohu ngerling was stimulated by LTA and PGN for 2 and 4 h.
Total RNA was extracted and qRT-PCR was carried out to analyze the expression of
RICK. All data were represented as a ratio relative to b-actin (internal control) in the
same samples and data were expressed as fold induction relative to the control. The
results were expressed as mean standard error (bar in the graph) from triplicate
cell culture wells or from three sh (n = 3). RICK expression in kidney and heart cell
culture (A) and in blood (B). Signicant difference (p < 0.05) between the control
and treated group was indicated with asterisks ().
NOD-2 gene transcript, but the existence of splice variants cannot
be ruled out in rohu. Seven LRR regions were identied in rohu,
rainbow trout and fugu NOD-2, whereas in grass carp and zebrash
it was six. Whether the number of LRR, and their location did play
any signicant role need further study.
Previously it was shown that innate immunity plays exclusive
role to protect sh embryo against microbes 4 weeks post
fertilization in zebrash (Chang et al., 2010; Kanther and Rawls,
2010) and 3 days post fertilization in carp (Huttenhuis et al.,
2006). Very recently, b-2 microglobulin, lysozyme, TLR-22 like gene
and transferrin expression was reported during ontogenesis of rohu
(Nayak et al., 2011). In addition to the previously reported defense
factors, we predicted the critical role of NOD-2 in protecting sh
eggs, hatchlings and larvae from pathogens. Our data showed that
in rohu, NOD-2 was expressed during various embryonic develop-
mental stages, although the pattern of relative expression was not
linear among the stages. This was the rst observation of NOD-2
gene expression during ontogenesis in rohu. This data suggested
that along with other innate immune factors, NOD-2 might play a
key role in sh eggs, hatched embryos and larvae in protecting them
under the pathogenically hostile environments.
 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103 101

A B
16
5.0
14 * *
4.5
fold chnages from control

fold chnages from control

12 4.0
3.5 *
10
3.0
8 * 2.5
6 2.0
* 1.5
4
* 1.0
2 0.5
0 0.0

Cont

Cont
Cont

Cont
Cont

Cont

4h

4h
2h

2h
2h
4h

2h
4h
2h
4h

2h
4h
___________________ ___________________ ___________________ ___________________ ___________________ ___________________
LTA
___________________ PGN LTA
___________________ PGN L T A PGN
___________________
Kidney Heart
Blood
C
10
9 *
8
fold changes from control

7
*
6
5
4
3
2
1
* *
0
Cont
24h
36h

24h
36h

24h
36h
Cont
Cont

treated treated treated

Gill Liver Kidney
Fig. 9. IFN-c expression in response to LTA, PGN and poly I:C. Rohu kidney and heart cell primary culture was stimulated with LTA and PGN for 2 and 4 h and rohu ngerlings
were i.v. injected with LTA, PGN for 2 and 4 h and i.p. injected with poly I:C for 24 and 36 h. Total RNA was extracted and qRT-PCR was carried out to analyze the expression of
IFN-g. All data were represented as a ratio relative to b-actin (internal control) in the same samples and data were expressed as fold changes relative to the control. The results
were expressed as mean standard error (bar in the graph) from triplicate cell culture wells or from three sh (n = 3). LTA and PGN-mediated IFN-c expression in kidney and
heart cell culture (A) and in blood (B). Poly I:C mediated IFN-c expression in various organs (C). Signicant difference (p < 0.05) between the control and treated group was
indicated with asterisks ().

In rohu, NOD-2 was constitutively expressed in all the tested or- by these ligands and the works are in progress to understand their
gans/tissues, however the pattern of expression was markedly dif- role.
ferent from zebrash (Laing et al., 2008), channel catsh (Sha et al., In mammals, NOD-2 is well recognized for its antibacterial
2009) and grass carp (Chen et al., 2010a,b). PGN and LTA present in immune responses (Kawai and Akira, 2006). In rohu, we investi-
both Gram-positive and negative bacterial cell wall contain ligands gated expression of NOD-2 gene transcript in response to A.
for TLR-2 as well as NOD1 and NOD-2 receptors. We investigated hydrophila and E. tarda infection, and we noticed signicant
rNOD-2 expression in response to LTA and PGN and our data up-regulation of rNOD2 transcript in blood (46 fold) and liver
showed that both ligands can induce rNOD-2 expression in vitro (25 fold) of the treated sh as compared to control. This data
as well as in vivo. Once activated by MDP (muramyl dipeptide) a highlighted the critical role of NOD-2 in sh and was in sharp
common component of all types of PGN, NOD-2 activates down- contrast to the channel catsh where NOD1was activated but
stream signaling pathway through RICK (McDonald et al., 2005). not NOD2 (Sha et al., 2009).
We examined RICK expression in vitro and in vivo. The data re- In human, NOD-2 was reported for its innate immune anti-viral
vealed the transcriptional up regulation of RICK by LTA and PGN, responses (Sabbah et al., 2009) and in grass carp activation of
and thus indicated the activation of NOD signaling pathway in NOD1 and NOD2 was reported in reovirus infection (Chen et al.,
rohu. In addition to NOD-2, NOD1 and TLR-2 may also be induced 2010a,b). In rohu, NOD-2 up-regulation by poly I:C was noticed
102 B. Swain et al. / Developmental and Comparative Immunology 36 (2012) 93103
in all the organs, although the kinetics varied. In agreement with
the previous observation our data highlighted the importance of
NOD-2 molecule in sensing viral infection across the species.
Activation of NOD signaling induced both type-I and type-II
IFN (Chang et al., 2011; Takahashi et al., 2006; Rosenzweig
et al., 2009). Our data showed that in rohu, NOD-2 expression
was correlated with the increase in IFN-c gene transcript both
in vitro (kidney and heart cell culture) and in vivo (blood), when
stimulated with PGN, LTA and poly I:C. These results were con-
sistent with the observation in grass carp, where reovirus infec-
tion resulted in enhanced expression of NOD-2 and IFN-c (Chen
et al., 2010a,b). These ndings together established the critical
role of NOD-2 in eliciting innate immune response against bac-
terial and viral infection through the cross talk of IFN-c gene
in sh.
In conclusion, it is reported that NOD-2 gene is identied and
characterized in rohu. Its function as an important pattern recogni-
tion receptor (PRR) has been shown by analyzing expressional up-
regulation of rNOD-2 and its associated down-stream molecule
RICK and IFN-c following PGN, LTA and poly I:C treatment and A.
hydrophila and E. tarda infection. This study suggests the key role
of NOD-2 in inducing innate immunity in sh and warrant further
study to understand the possible interplay between TLR and NODs
in sh.
Acknowledgments
This work was partially supported by the Grant from NAIP-ICAR
(Project Code C4-C30018) and partially from NFBSARA-ICAR (Pro-
ject Code: AS-2001).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.dci.2011.06.018.
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