Author's personal copy
Induction of Mx protein in Atlantic cod with poly
I:C: Immuno-cross reactive studies of antibodies
to Atlantic salmon Mx with Atlantic cod
B.K. Dasb, K. Urquharta, A.E. Ellisa, B. Colleta,*
aFRS Marine Laboratory, Molecular Genetics, 375 Victoria Road, PO Box 101, Aberdeen AB11 9DB, Scotland, UK
bCentral Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India
Received 11 February 2008; revised 16 April 2008; accepted 27 April 2008
Available online 2 May 2008
salmon antiviral Mx1 protein was used to detect the putative Atlantic cod Mx protein using
Western and dot blotting. A doublet band at about 75 kDa and 65 kDa was detected by Western
blotting in kidney and spleen extracts of cod 3 and 4 days after i.p. injection with poly I:C but
not in control fish injected with PBS. In blood leucocyte lysates, similar immunostaining could
also be detected in Atlantic cod weakly after injection with PBS and more intensely after
injection with poly I:C, suggesting some constitutive expression of Mx protein by leucocytes.
Dot blot analysis showed that the Mx protein level was significantly higher in spleen, kidney,
liver and gill of cod at least up to 4 days after injection with poly I:C when compared with the
Crown Copyright ª 2008 Published by Elsevier Ltd. All rights reserved.
A polyclonal rabbit antiserum directed against the conserved region of the Atlantic
In vertebrates, one of the most important parts of the
innate immune response against viral pathogens is the
system induced by type I interferon (IFN) which includes
an important antiviral protein called Mx. IFN and Mx genes
have been identified in many different fish species  but
not yet in any member of the Gadiformes, including Atlan-
tic cod (Gadus morhua). This species is becoming an impor-
tant farmed species in Norway and Scotland and we have
recently demonstrated that juvenile cod can become
asymptomatic carriers of infectious pancreatic necrosis
virus (IPNV) , an important and widespread disease of
freshwater salmonids and Atlantic salmon post-smolts in
seawater . This raises the possibility of disease interac-
tions between the mariculture of salmon and cod. At
present virtually nothing is known about the interferon
system in cod apart from the recent discovery of interferon
stimulated gene 15 (ISG15), which was highly expressed
following treatment of cod kidney leucocytes with poly
I:C (a well known inducer of IFN) and in vivo following
injection of Vibrio anguillarum bacterin  or IPNV .
* Corresponding author. Tel.: þ44 1224295683; fax: þ44
E-mail address: email@example.com (B. Collet).
1050-4648/$ - see front matter Crown Copyright ª 2008 Published by Elsevier Ltd. All rights reserved.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fsi
Fish & Shellfish Immunology (2008) 25, 321e324
Author's personal copy
It is well established in several fish species that following
injection of poly I:C, Mx transcripts and protein are
produced in several tissues [6e10]. It was also reported
that a rabbit antiserum prepared to a 147 amino acid frag-
ment of the Atlantic halibut Mx protein crossreacted with
chinook salmon Mx proteins . In previous work we
produced a rabbit antiserum to a peptide common to the
three Atlantic salmon Mx protein isoforms  and in the
present work we present evidence that this antiserum
crossreacts with Atlantic cod Mx protein. The antiserum
was used to study the expression of Mx protein in tissue
and blood leucocytes of juvenile cod following injection
of poly I:C.
The Sigma peptide-antiserum service was used to
LNQHYEEKVRPC as previously reported . This peptide
is located in the N terminal region of all three of the Atlan-
tic salmon Mx protein isoforms . Preimmune serum was
collected just before immunization.
Atlantic cod of 60e80 g were kept in 1 m diameter tanks,
ture of10?C. Asample of 30 fishwas taken forstandard viro-
logical and bacteriological culture tests to determine the
disease status of the population and no evidence of patho-
gens was found. Fish were fed a commercial diet (Europa
18%, Skretting, Burgos, Spain) at 2% body weight per day.
Fish were injected intraperitoneally (i.p.) with either
200 ml poly I:C (2 mg ml?1) or 200 ml of PBS (control fish).
Blood samples were withdrawn into heparinized tubes and
kidney, liver, spleen and gills were harvested 2, 3 and 4
days after injection. Four fish were killed (by overdose of
benzocaine) in each group (control and poly I:C) at the
given time points, and the tissue samples taken in PBS
were immediately frozen at ?80?C until further analysis.
Leucocytes were separated from whole blood using the QI
Aamp RNA blood kit (Qiagen) up to step 5 involving leuco-
cyte lysis as per the manufacturer’s instructions. Briefly
1.5 ml of fish blood was mixed with 7.5 ml of buffer EL to
lyse the red cells and incubated on ice for 10e15 min.
The mixture was then centrifuged at 400 ? g for 10 min at
4?C. After centrifugation, the supernatants were discarded
and leucocyte pellets were resuspended with 3 ml of buffer
EL by vortexing. The centrifugation process was repeated
as above and the supernatants were removed completely
after centrifugation. Buffer RLT (600 ml) was added to lyse
the leucocyte pellets and the lysate was stored at ?80?C
The cod tissues (about 50 mg) were extracted by adding
four volumes of 2? SDS sample buffer consisting of 0.1 M
TriseHCl, pH 6.8, 4% SDS, and 20% glycerol. Samples were
sonicated on ice for 4 s, boiled for 5 min and then centri-
fuged at 13,000 rpm for 10 min to remove the tissue debris.
The protein concentration of tissue extracts was measured
using BC protein assay (Bio-Rad) as per the manufacturer’s
For Western blotting, tissue extracts were adjusted to
10 mg protein/20 ml with 1? SDS sample buffer containing
0.2 M dithiothreitol and 0.1% bromophenol blue and 6 ml
leucocyte lysate was mixed with 4 ml of the same SDS
Protein samples (20 ml tissue extract, 10 ml leucocyte
lysate) were run on 10% SDS-PAGE gels along with Dual
colour precision plus protein standards (Bio-Rad) for 1 h
at 100 V. The gel was then blotted onto a NCP membrane
(Bio-Rad) using a Milliblot-Graphite Electroblotter system
at 1.5 mA/cm2for 1 h. Blots were blocked in 1% casein
0.02 M TriseHCl buffer, pH 7.5, 0.5 M NaCl, and 0.3% Tween
20 (TTBS) for 3 h at room temperature and then incubated
overnight with 1:400 dilution of RaMx in blocking buffer at
4?C. The membrane was then washed 3? with TTBS before
incubation with biotinylated horse anti-rabbit Ig as per
Vector Elite ABC kit (Vector Laboratories) instructions for
30 min. The membrane was then washed as previously
tiserum directed against salmon Mx. Lanes 1 and 2, spleen of
poly I:C-injected cod on day 3 and day 4, respectively; lanes
3 and 4, kidney of poly I:C-injected cod on day 3 and day 4, re-
spectively; M, molecular weight marker; C (left), kidney of
control (PBS injected) cod on day 3; C (right), spleen of control
(PBS injected) cod on day 3.
Western blot of spleen and kidney using a rabbit an-
bit antiserum directed against salmon Mx. M, molecular weight
marker; lane 1, day 2 poly I:C-injected cod; lane 2, day 3 poly
I:C-injected cod; lane 3, day 4 poly I:C-injected cod; and lane
4, day 2 PBS-injected cod.
Western blot of blood leucocyte lysates using a rab-
322B.K. Das et al.
Author's personal copy
and incubated with ABC reagents in TTBS for 30 min. The
membrane was subsequently washed 2? 5 min in TTBS
and 1? 5 min in TBS and then incubated with amino-ethyl
carbazole reagent (AEC, Sigma) for 10 min. Finally, the
membrane was washed in two changes of dH2O for 10 min
For dot blot analysis, kidney and spleen (days 2, 3 and
4), liver and gill tissues (day 4) of PBS- and poly I:C-injected
samples were extracted in four volumes of 2? SDS sample
buffer. Samples were sonicated on ice for 4 s, boiled for
5 min and then centrifuged at 13,000 rpm for 10 min to re-
move tissue debris. Protein concentration in each sample
was measured using the BC protein assay (Bio-Rad) follow-
ing the manufacturer’s protocol. Samples were adjusted
to 1 mg protein/ml and 5 ml was blotted onto a nitrocellulose
sheet (Bio-Rad) and air dried for 3 h. Blots were blocked in
1% casein in TTBS for 3 h at room temperature and then
stained following the Vector kit instructions as described
above for Western blots. Negative controls were performed
as above substituting preimmune serum for the RaMx. The
dot blots were analysed in SynGene tools’ Bioimaging sys-
tem Version 3.04.04 (SynGene Laboratory, UK). Comparison
between stimulated and control groups was carried out by
Western blots of kidney and spleen of control cod
injected with PBS showed little staining with RaMx antise-
rum. However, a doublet band at about 75 kDa and another
band at about 65 kDa stained strongly in these tissues at 3
and 4 days following i.p. injection of cod with poly I:C
(Fig. 1). No staining was obtained when the rabbit preim-
mune serum was substituted for the RaMx. This pattern of
staining is virtually identical to that reported for Atlantic
salmon Mx [10,11]. The MW of fish Mx proteins has been re-
ported to be about 70 kDa in rainbow trout , 71 kDa in At-
lantic halibut , and 76 kDa in Atlantic salmon . All of
these authors also reported several lower MW immuno-re-
active bands which they suggested to be degradation prod-
ucts of Mx proteins .
Leucocytes in humans  and Atlantic halibut  are
known to strongly express Mx protein after stimulation
with IFN or poly I:C. In the present work, Western blots of
leucocyte lysates from cod injected with poly I:C showed
similar staining patterns to kidney and spleen, however,
leucocytes from control cod also showed the same staining
pattern (Fig. 2). This might suggest a low level constitutive
expression of Mx, as reported for blood monocytes in hu-
In an attempt to compare the relative staining intensi-
ties of tissues from control cod and cod injected with poly
I:C, dot blots were performed and staining intensity
analysed by image analysis. As shown in Fig. 3 the intensity
of immunostaining of spleen, kidney, gill and liver was
markedly increased up to day 4 post-injection in the poly
I:C-injected fish compared with the controls.
In conclusion, the data indicate that the rabbit antise-
rum produced to Atlantic salmon Mx protein crossreacts
with a poly I:C-inducible protein with MW consistent with
Mx protein of other vertebrates, especially fish, and that
this antibody can be used to study interferon-induced Mx
responses in Atlantic cod nondestructively.
B.K. Das gratefully acknowledges the Department of Bio-
technology, Government of India for the long term DBT
associateship. The Mx antibody was developed under the EU
Wealth Project 501984.
 Robertsen B. The interferon system of teleost fish. Fish Shell-
fish Immunol 2006;20:172e91.
 Garcia J, Urquhart K, Ellis AE. IPNV establishes an asymptom-
atic carrier state in kidney leucocytes of juvenile cod, Gadus
morhua. J Fish Dis 2006;29:409e13.
poly I:C using a rabbit antiserum directed against salmon Mx. Data are mean þ SE (nZ 4). Control fish were injected with PBS. Level
of statistical significance is indicated above the data as follows: *p < 0.05; **p < 0.01 and ***p < 0.001.
Immunoblot analysis of Mx protein in kidney, spleen, gill and liver of Atlantic cod on days 2, 3 and 4 after injection of
Induction of Mx protein in Atlantic cod with poly I:C 323
Author's personal copy Download full-text
 Roberts RJ, Pearson MD. Infectious pancreatic necrosis in
Atlantic salmon, Salmo salar L. J Fish Dis 2005;28:383e90.
 Seppola M, Stenvik J, Steiro K, Solstad T, Robertsen B,
Jensen I. Sequence and expression analysis of an interferon
stimulated gene (ISG15) from Atlantic cod (Gadus morhua
L.). Dev Comp Immunol 2007;31:156e71.
 Das BK, Collet B, Snow M, Ellis AE. Expression kinetics of ISG15
and viral major protein (VP2) in Atlantic cod (Gadus morhua
L.) fry following infection with infectious pancreatic necrosis
virus (IPNV). Fish Shellfish Immunol 2007;23:825e30.
 Lockhart K, Bowden TJ, Ellis AE. Poly I:C induced Mx responses
in Atlantic salmon (Salmo salar L.) parr, post-smolts and
growers. Fish Shellfish Immunol 2004;17:245e54.
 Trobridge GD, Chiou PP, Kim CH, Leong JC. Induction of the Mx
protein of rainbow trout Oncorhynchus mykiss in vitro and in
vivo with poly I:C dsRNA and infectious hematopoietic necrosis
virus. Dis Aquat Org 1997;30:91e8.
 Jensen I, Albuquerque A, Sommer A-I, Robertsen B. Effect of
poly I:C on the expression of Mx proteins and resistance
against infection by infectious salmon anaemia virus in
Atlantic salmon. Fish Shellfish Immunol 2002;13:311e26.
 Bergan V, Robertsen B. Characterisation of Atlantic halibut
(Hippoglossus hippoglossus) Mx protein expression. Dev
Comp Immunol 2004;28:1037e47.
 Jensen V, Robertsen B. Cloning of a Mx cDNA from Atlantic
halibut (Hippoglossus hippoglossus) and characterisation of
Mx mRNA expression in response to double-stranded RNA
and infectious pancreatic necrosis virus. J Interferon Cytokine
 Das BK, Nayak KK, Collet B, Snow M, Ellis AE. Expression of
Mx protein in tissues of Atlantic salmon post-smolts e an
immunohistochemical study. Fish Shellfish Immunol 2007;
 Robertsen B, Trobridge G, Leong J-A. Molecular cloning of
double-stranded RNA inducible Mx genes from Atlantic
salmon (Salmo salar L.). Dev Comp Immunol 1997;21:
 NygaardR, HusgardS,
Robertsen B. Induction of Mx protein by interferon and
 Towbin H, Scmitz A, Jakschies
Horisberger MA. A whole blood immunoassay for the inter-
feron-inducible human Mx protein. J Interferon Res 1992;12:
 Ronni T, Melen K, Malygin A, Julkunen I. Control of IFN-
inducible MxA gene expression in human cells. J Immunol
D, von WussowP,
324B.K. Das et al.