BMC Veterinary Research
BioMed Central
Open Access
Research article
First detection, isolation and molecular characterization of
infectious salmon anaemia virus associated with clinical disease in
farmed Atlantic salmon (Salmo salar) in Chile
Marcos G Godoy1, Alejandra Aedo1, Molly JT Kibenge2, David B Groman3,
Carmencita V Yason4, Horts Grothusen1, Angelica Lisperguer4,
Marlene Calbucura1, Fernando Avendaño5, Marcelo Imilán5, Miguel Jarpa5
and Frederick SB Kibenge*2
Address: 1Biovac SA, Puerto Montt, Chile, 2Department of Pathology and Microbiology, OIE Reference Laboratory for ISA, Atlantic Veterinary
College, University of Prince Edward Island, Charlottetown, PE, Canada, 3Aquatic Diagnostic Services, Atlantic Veterinary College, University of
Prince Edward Island, Charlottetown, PE, Canada, 4Regional Diagnostic Virology Laboratory, Atlantic Veterinary College, University of Prince
Edward Island, Charlottetown, PE, Canada and 5Marine Harvest S.A., Puerto Montt, Chile
Email: Marcos G Godoy - mgodoy@biovac.cl; Alejandra Aedo - aaedo@biovac.cl; Molly JT Kibenge - mkibenge@upei.ca;
David B Groman - groman@upei.ca; Carmencita V Yason - yason@upei.ca; Horts Grothusen - horst.grothusen@biovac.cl;
Angelica Lisperguer - Angelica.Lisperguer@marineharvest.com; Marlene Calbucura - Marlene.calbucura@biovac.cl;
Fernando Avendaño - Fernando.Avendano@marineharvest.com; Marcelo Imilán - Marcelo.Imilan@marineharvest.com;
Miguel Jarpa - Miguel.Jarpa@marineharvest.com; Frederick SB Kibenge* - kibenge@upei.ca
* Corresponding author
Published: 4 August 2008
BMC Veterinary Research 2008, 4:28
doi:10.1186/1746-6148-4-28
Received: 19 March 2008
Accepted: 4 August 2008
This article is available from: http://www.biomedcentral.com/1746-6148/4/28
© 2008 Godoy et al; licensee 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.
Abstract
Background: Infectious salmon anaemia (ISA) is a viral disease of marine-farmed Atlantic salmon
(Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae.
The virus is considered to be carried by marine wild fish and for over 25 years has caused major
disease outbreaks in marine-farmed Atlantic salmon in the Northern hemisphere. In the Southern
hemisphere, ISAV was first detected in Chile in 1999 in marine-farmed Coho salmon (Oncorhynchus
kisutch). In contrast to the classical presentation of ISA in Atlantic salmon, the presence of ISAV in
Chile until now has only been associated with a clinical condition called Icterus Syndrome in Coho
salmon and virus isolation has not always been possible. During the winter of 2007, unexplained
mortalities were registered in market-size Atlantic salmon in a grow-out site located in Chiloé in
Region X of Chile. We report here the diagnostic findings of the first significant clinical outbreak
of ISA in marine-farmed Atlantic salmon in Chile and the first characterization of the ISAV isolated
from the affected fish.
Results: In mid-June 2007, an Atlantic salmon marine farm site located in central Chiloé Island in
Region X of Chile registered a sudden increase in mortality following recovery from an outbreak
of Pisciricketsiosis, which rose to a cumulative mortality of 13.6% by harvest time. Based on the
clinical signs and lesions in the affected fish, and laboratory tests performed on the fish tissues, a
confirmatory diagnosis of ISA was made; the first time ISA in its classical presentation and for the
first time affecting farmed Atlantic salmon in Chile. Rapid sequencing of the virus-specific RT-PCR
products amplified from the fish tissues identified the virus to belong to the European genotype
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(Genotype I) of the highly polymorphic region (HPR) group HPR 7b, but with an 11-amino acid
insert in the fusion glycoprotein, and ability to cause cytopathic effects (CPE) in CHSE-214 cell line,
characteristics which make it distinct from common European Genotype ISAV isolates from
Europe and North America.
Conclusion: In conclusion, the present work constitutes the first report of a case of ISA in farmed
Atlantic salmon in Chile. The clinical signs and lesions are consistent with the classical descriptions
of the disease in marine-farmed Atlantic salmon in the Northern hemisphere. The outbreak was
caused by ISAV of European genotype (or Genotype I) of HPR 7b but distinct from common
European Genotype ISAV isolates.
Background
Infectious salmon anaemia (ISA) is a viral disease of
marine-farmed Atlantic salmon (Salmo salar) caused by
ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae [1]. In the Northern hemisphere, the
first registered outbreak of ISA was in 1984 in Atlantic
salmon "parr", on the southwestern coast of Norway [2].
Subsequently the disease was reported in Canada in 1996
[3], in Scotland in 1998 [4], in Faeroe Islands in 1999 [5],
and in Maine, USA, in 2000 [6]. The clinical disease in
farmed Atlantic salmon is characterized by variable mortality ranging from 0 to 50% with ascites, exophthalmia,
petechiation of the visceral adipose tissue, haemorrhagic
liver necrosis, renal interstitial haemorrhage and tubular
nephrosis, filamental sinus congestion of the gills, splenic
congestion with concomitant erythrophagocytosis, and
congestion of the lamina propria of the stomach and
foregut [2,7-10]. ISAV remains an emerging fish pathogen
that continues to cause severe economic losses to the
salmon-farming industry in an increasing number of
countries, although the disease can be successfully eradicated as was managed by Scotland in the late 1990s [11].
In the Southern hemisphere, ISAV was first detected in
Chile in 1999 in marine-farmed Coho salmon (Oncorhynchus kisutch) and was shown to be of the North American
genotype [12]. In contrast to the classical presentation of
ISA in Atlantic salmon, the presence of ISAV in Chile up
until now has only been associated with a clinical condition called Icterus Syndrome in Coho salmon and virus
isolation has not always been possible [13]. Between
2001 and 2003, ISAV of the North American genotype was
found in apparently normal Atlantic salmon in Lake Llanquihue (Oscar Gárate, personal communication), which
is not in the same zone as where the present disease outbreak occurred. It is not known why the North American
variant of ISAV has not caused typical ISA outbreaks as
those known to occur in Eastern Canada and Maine, USA,
and yet the virus has become widespread in the Atlantic
salmon industry in Chile.
ISAV belongs to the family Orthomyxoviridae, together
with influenza viruses [1]. However, the virus is sufficiently different from influenza viruses to be assigned to
its own genus, Isavirus. Members of this genus are enveloped particles of 90–140 nm diameter with surface projections consisting of a combined haemagglutininesterase (HE) glycoprotein, which is the receptor binding
haemagglutinin with receptor destroying enzyme activity
demonstrated to be an acetylesterase [14,15] encoded on
segment 6 [14], and a separate fusion (F) glycoprotein
encoded on segment 5 [16]. The genome consists of eight
segments of linear, single-stranded negative sense RNA
ranging in length from 1.0 to 2.4 kb with a total molecular
size of approximately 14.3 kb [17]. Sequence analysis of
several ISAV isolates on the eight segments consistently
reveals two genotypes that are designated with respect to
their geographic origin, European and North American,
hereafter designated Genotypes I and II, respectively.
Sequence variation in a 35-amino acid highly polymorphic region (HPR) on the HE glycoprotein stalk has
allowed the separation of ISAV isolates into different HPR
groups, with the HPR0 group consisting of the non-cultivable, non-pathogenic viruses detectable only by RT-PCR
[18] whereas deletion in the HPR of ≥ 13 amino acids (or
if less, with deletion or mutation of the motif at amino
acid positions 352FNT354) lead to increased pathogenicity,
and ability to replicate in cell culture with production of
CPE and consequent virus isolation [19].
This study reports the diagnostic findings of the first
occurrence of ISA in the Southern hemisphere, and the
first characterization the ISAV isolated from the affected
marine-farmed Atlantic salmon in Chile.
Results
Signalment
In mid-June 2007 following recovery from an outbreak of
Pisciricketsiosis, a severe increase in mortality was
recorded in 2 cages on an Atlantic salmon grow-out site
located in central Chiloé in Region X of Chile (Fig. 1A)
[see Additional file 1]. The moribund fish were lethargic
and had dark integument. By the time the fish were
removed from the cages 4 weeks later, the cumulative
mortality had reached 70% in one cage and 82% in the
second cage. Concomitantly, in other sites of the same
area, high mortalities attributed to amoebas and flexibacPage 2 of 13
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(A)
(B)
Figure 1
Geographical
location and mortality pattern of the first ISA outbreak in Chile
Geographical location and mortality pattern of the first ISA outbreak in Chile. (A)Geographical location of the
Atlantic salmon marine farm site: Lemuy Island, Central Chiloé, Region X, Chile. (B)Weekly percent cummulative mortality up
to the time of total harvest of the Atlantic salmon marine farm with ISA outbreak. Net Pen A and Net Pen B represent mortality in the 2 cages with ISA. FARM corresponds to the total mortality on the farm up to the time of harvest.
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teriosis were reported, which added to the diagnostic confusion. The total cumulative mortality on the index farm
was only 13.6% at the time of harvest due to company
decision to cull all remaining cages independently of their
clinical status (Fig. 1B).
Gross pathology
The external gross findings noted on Atlantic salmon that
were necropsied included exophthalmia, periocular
haemorrhage and darkening of the integument. Figure 2A
summarizes the frequency of the external lesions noted
from 100 affected fish at necropsy. Periocular haemorrhage and paleness of the gills were observed in 38% of
the fish, whereas 25% presented with exophthalmia [see
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Additional file 2] and jaundice in the ventral zone (Fig
2B). The presence of haemorrhages in the central zone
was observed in 19% of the fish (Fig. 2B). Of the 100 fish
that were examined, 63% were also infested with Caligus
sp. At necropsy, the internal gross findings included haemorrhaging in the visceral adipose tissue, liver, stomach and
gut, with the liver and spleen of some fish appearing dark
(Fig. 2B). Some fish had hydropericardium [see Additional file 3], and enlarged spleen and kidney. Figure 2C
summarizes the frequency of the internal gross lesions
noted from 100 affected fish which were necropsied in
this outbreak. The most common lesions were haemorrhagic enteritis in 64%, haemorrhages in pyloric caeca in
(A)
(B)
(C)
Figurelesions
Gross
2
in affected Atlantic salmon from the ISA outbreak
Gross lesions in affected Atlantic salmon from the ISA outbreak. (A) Frequency of the external gross lesions in
affected Atlantic salmon from the ISA outbreak. Percentage of fish with a specified lesion among 100 fish necropsied. (B) Common gross lesions seen at necropsy: Top panel – Atlantic salmon with exophthalmia and pale gills. Middle panel – Atlantic
salmon with petechial haemorrhages on the abdomen. Bottom panel – Atlantic salmon with very dark liver and haemorrhages
on the visceral adipose tissue. (C) Frequency of the internal gross lesions in affected Atlantic salmon from the ISA outbreak.
Percentage of fish with a specified lesion among 100 fish necropsied.
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43%, and haemorrhages in visceral adipose tissue in 31%
of the cases [see Additional files 4 and 5].
Histopathology
Diffuse congestion of lamellar capillaries, with marked
infiltration of the filamental subcutis by eosinophillic
granular leucocytes was evident in gill tissue. The kidney
showed mild diffuse sinusoidal congestion with evidence
of increase in circulating granulocytes and intravascular
erythrophagia. The intestine and pyloric caeca were the
most severely affected, showing marked congestion of the
lamina propria vasculature with an associated mixed leucocytic infiltration of submucosa and localized perivascular and intra-lumenal haemorrhage (Fig. 3A). The liver
was affected by multifocal to coalescing regions of sinusoidal congestion and peliosis, with mild adjacent hepatocellular necrosis (Fig. 3B). Changes in the spleen
included marked sinusoidal congestion and intravascular
erythrophagia (Fig. 3C). Mucosal epithelium in the most
severely affected regions was denuded. There were no significant morphologic changes noted in the heart, stomach
or body wall skeletal muscle. Morphologic diagnoses
included: a) necrotizing and congestive hepatitis, subacute, multifocal to bridging, moderate to marked; b)
ulcerative mixed leucocytic and congestive enteritis, with
marked intra-lumenal haemorrhage, subacute, moderate
to marked; and d) congestive splenitis and interstitial
nephritis, with intravascular erythrophagia, moderate to
marked.
Virus antigen detection
Sections of the kidney, spleen, heart, gills and intestines
were subjected to immunohistochemistry staining with
anti-ISAV monoclonal antibody P10 (Aquatic Diagnostics
Ltd, Stirling, Scotland). ISAV proteins were detected in all
the organs tested. Figure 4A shows positive staining of
endothelial cells of the heart. Homogenates of pooled
spleen and kidney tissues used for virus isolation were
also tested in a rapid kit made by Aquatic Diagnostics,
based on immunochromatography with anti-ISAV monoclonal antibody. The samples taken from affected fish
were positive for ISAV. The infectious haematopoietic
necrosis virus (IHNV) ELISA kit and viral haemorrhagic
septicaemia virus (VHSV) ELISA kit (Bio-X Diagnostics,
Jemelle, Belgium) were used to rule out the presence of
IHNV and VHSV in the tissue samples.
Virus isolation
Homogenates of pooled spleen and kidney tissues inoculated on Chinook salmon embryo (CHSE-214), Salmon
head kidney (SHK-1) and Epithelioma papulosum cyprini
(EPC) cell lines produced CPE between 4 and 7 days post
inoculation during primary isolation. The CPE, which was
reproduced on subsequent passage on fresh cell line monolayers, was characterized initially by cell vacuolization
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and rounding and then detachment from the substrate
involving the whole cell monolayer. The presence of ISAV
in the cell lysates was confirmed using RT-PCR and an
indirect immunofluorescent antibody test (IFAT) with
anti-ISAV monoclonal antibody P10 (Aquatic Diagnostics
Ltd) including appropriate controls. Figure 4B shows positive IFAT of a CHSE-214 cell culture inoculated with a tissue sample from affected fish. Similar fluorescence was
seen with EPC cell cultures with CPE (data not shown).
Neither CPE nor presence of ISAV was detected by RT-PCR
or IFAT in inoculated cultures of Bluegill Fry (BF2) cell
line.
RT-PCR
Tissue samples shipped in RNA later® (Ambion Inc., Foster
City CA) to the Regional Diagnostic Virology Laboratory,
AVC, UPEI, Canada, were positive for ISAV by RT-PCR and
negative for both IHNV and VHSV by nested RT-PCR.
Sequence analysis
The nucleotide sequences in this report, which are all of
ISAV RNA segment 5, are available through GenBank
http://www.ncbi.nlm.nih.gov/Genbank, accession nos.
EU130923, EU486160, and EU486161. A partial
sequence of ISAV RNA segment 6 was generated by the
Norwegian Veterinary Institute from duplicate samples of
the same ISA outbreak, and appears in GenBank, accession no. AM941715. Tables 1 [see Additional file 6] and 2
[see Additional file 7] show the percent sequence identities between the new Chile ISAV and selected ISAV isolates
from Europe and North America. Comparison of the RNA
segment 5 sequences of the Chilean ISAV with ISAV
strains of Genotype I (European genotype) showed nucleotide sequence identities ≥ 95.9% whereas with ISAV
strains of Genotype II (North American genotype), it was
≤ 74.7% [see Additional file 6]. Comparison of the segment 6 sequence of the Chilean ISAV with ISAV strains of
Genotype I showed sequence identities ≥ 95.7% whereas
with ISAV strains of Genotype II, it was ≤ 79.4% [see Additional file 7]. Thus the new Chile ISAV involved in this
outbreak belongs to Genotype I (the European genotype).
Alignment of the amino acid sequences of RNA segment
5, which encodes the F glycoprotein, revealed that the new
Chile ISAV has a small insert of 11 amino acids (or 33
nucleotides) relative to ISAV strains from Europe and
North America near the putative proteolytic cleavage site
of the precursor F0 protein (Fig. 5). This insert has 100%
sequence identity with RNA segment 2 of Genotype I.
ISAV RNA segment 2 encodes the PB1 polymerase [22].
Alignment of the amino acid sequences of RNA segment
6, which encodes the HE glycoprotein, revealed that the
new Chile ISAV belongs to HPR7b of the European genotype as described by Nylund et al. [23] and Plarre et al. [24]
(Fig. 6) and reviewed by Rimstad et al. [18].
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(A)
(B)
(C)
Figure 3 lesions in affected Atlantic salmon from the ISA outbreak
Microscopic
Microscopic lesions in affected Atlantic salmon from the ISA outbreak. (A) Histologic section of intestine of Atlantic
salmon. H&E staining. (→) indicates mucosal ulceration and haemorrhage. (B) Histologic section of liver of Atlantic salmon
from the ISA outbreak H&E staining. (→) indicates hepatocellular necrosis, with marked haemorrhage and sinusoidal congestion/peliosis. (C) Histologic section of spleen of Atlantic salmon from the ISA outbreak H&E staining. (→) indicates endothelial
erythrophagia.
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(A)
(B)
Figure
ISAV
antigen
4
staining of samples from the ISA outbreak
ISAV antigen staining of samples from the ISA outbreak. (A) Immunohistochemical staining of histologic section of
heart of Atlantic salmon from the ISA outbreak. Dark brown colour indicates positive staining in endothelial cells with antiISAV monoclonal antibody. (B)Virus isolation in CHSE-214 cells: ISAV-infected cells (250×) showing fluorescent staining following IFAT with anti-ISAV monoclonal antibody.
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ISAV isolate (GenBank Acc. No.)
Norway MR60/01 (AY853944)
Norway MR46/99 (AY853962)
Norway SF70/02 (AY853938)
Norway SF57/00 (AY853939)
Norway MR62/01 (AY853937)
Norway SF71/02 (AY853936)
Norway MR61/01 (AY853935)
Chile U24636
(EU130923)
Norway SK779/06(EU118819)
Norway SF83/04 (AY744392)
Can RPC/NB 04-085-1 (EF432567)
Norway SF14/95 (AY853925)
Norway N32/98 (AY853921)
Can NovaScotia NS2003(AY853919)
Norway 810/9/99 (EF217313)
Scotland 390/98(AF429988)
Norway 485/9/97(EF217315)
Can NovaScotiaU5575-1(EF217314)
Can NBISA01(DQ465044)
Can RPC/NB 98-049-1(DQ465043)
Can RPC/NB 02-0775-14(DQ465045)
Chile 7833-1(DQ465046)
Can RPC/NB 02-0775-14(DQ440507)
Can RPC/NB 01-0593-1(DQ440508)
Can RPC/NB 01-0973-3(DQ465047)
Can RPC/NB 98-0280-2(DQ440506)
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Predicted amino acid sequence
aa inserted at cleavage site
& in-vivo/in-vitro virulence
250
260
270
280
290
300
310
320
330
RANLANQHGWSKYSFGHSVHKLSN-----------QRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------QRAPGGVLLTETITFPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RASLANQHGWSKYSF--------N-----------QRAIPRTGYVRSA-FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RASLANQHGWSKYSF--------N-----------QRAIPRTGYVRSA-FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RASLANQHGWSKYSF--------N-----------QRAIPRTGYVRSA-FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RASLANQHGWSKYSF--------N-----------HRAIPRTGYVRSA-FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RASLANQHGWSKYSF--------N-----------HRAIPRTGYVRSA-FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RAGLANQHGWSKYNF--------NKGKSANDIISDQRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RAGLANQHGWSKYNF--------N-----------QRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RAGLANQHGWSKYNF--------N-----------QRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------Y-----------PRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RADLANQHGWSKYNF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RAGLANQHGWSKYNF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
RANLANQHGWSKYSF--------N-----------LRA-----------FPGEEFIKCCGFTLGIGGAWFQAYLNGEVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
KANFVNKHGWSKYNF--------N-----------LRG-----------FPGEEFIKCCGFTLGVGGAWFQAYLNGMVQGD
8 (IN1)*
11 (IN2)*
10 (IN3)*
10 (IN3)*
10 (IN3)*
10 (IN3)*
10 (IN3)*
11 (IN4)
0 (ISAV HPR0)**
0 (low virul. ISAV)***
0 (low virul. ISAV)
0*
0*
0
0 (high virul. ISAV)
0
0
0
0 (high virul. ISAV)
0 (high virul. ISAV)
0
0
0
0
0
0
Figure 5 of amino acid sequences in the proteolytic cleavage site of the precursor F0 protein (modified from Kibenge et al
Alignment
Alignment of amino acid sequences in the proteolytic cleavage site of the precursor F0 protein (modified from
Kibenge et al. [19]). The amino acid sequence corresponding to the fusion protein of the Chilean ISAV in this disease outbreak is highlighted in yellow. The designation of amino acid inserts IN1, IN2, and IN3 are as reported by Devold et al. [21].
The unique 11-amino acid insert found in the Chilean ISAV in this disease outbreak is designated IN4. Other sources of information are indicated as *Devold et al. [21], **Markussen et al. [20], and ***Plarre and Nylund (2004; SF83/04, GenBank Accession No. AY744392). It has been suggested by Markussen et al. [20] that ISAV isolate SF83/04 represents a mixed virus
infection of HPR0 and another HPR group, which might explain the confusion in the virulence designation.
Discussion
The present work constitutes the first report of a case of
ISA in farmed Atlantic salmon in Chile. The outbreak was
caused by ISAV of Genotype I (the European genotype).
The gross and microscopic lesions observed in this outbreak are consistent with the classical ISA described in
Norway [2], Canada [3], Scotland [4], Faeroe Islands [5]
and United States [6]. In particular, the liver congestion
and necrosis, as well as the renal, splenic and enteric congestion/haemorrhage in this outbreak are morphologically consistent with lesions pathognomonic for ISA.
However, where these lesions differed from other presentations of the disease were primarily in the kidney, where
in the Canadian situation there is profound interstitial
haemorrhage, and in the heart, in the Chilean ISA outbreaks there is hydropericardium and severe myocarditis.
Previously, such heart lesions have been shown to be
prominent only in experimental infections of rainbow
trout with ISAV [28].
The affected fish group in the Chilean ISA outbreak was
recovering from Piscirickettsiosis. This particular group
had been treated several times against Rickettsia at the
farm site, and on this occasion the antibiotic treatment
which was administered by injection effectively prevented
mortality due to Piscirickettsiosis. Although the pathologic findings in this outbreak were in the range of what is
considered as typical ISA, it cannot be categorically stated
what contribution, if any, the Piscirickettsiosis disease
contributed to the pathology observed.
The virus responsible for the outbreak caused CPE and
was recovered using CHSE-214, SHK-1 and EPC cell lines,
and confirmed as ISAV by means of RT-PCR and IFA testing. This is the first report of primary isolation of ISAV
using EPC cell line, which is a non-salmonid cell line
derived from a skin tumour of carp (Cyprinus carpio L.)
[29] and never before reported to be permissive to ISAV.
Most recently, EPC cell line was also reported to be permissive to ISAV of North American genotype [30]. Moreover rapid sequencing of virus-specific RT-PCR products
amplified from the fish tissues identified the virus as
belonging to the European genotype. Up until now, only
some strains of ISAV of the North American genotype [3133] but not the European genotype [32] have been known
to replicate and cause CPE in the CHSE-214 cell line, a
property that is not associated with the antigenic subtype
of ISAV nor with the HPR group of the HE gene [34]. Thus,
the Chilean ISAV has cell line specificities that are different from those of common European Genotype ISAV isolates from Europe and North America.
Now that we have ISAV isolates of both genotypes from
Europe, North America, and South America, we should
remove the stigmatizing labeling of ISAV genotypes by
geographic reference. We therefore propose to designate
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HPR
Group
ISAV isolate(GenBank Acc. No.)
HPR0 Norway farmed salmonSK779/06 (EU118820)
HPR0
Scotland wild salmon(AJ440971)
HPR0 Can Nova Scotia farmed salmon(AY646058)
HPR0
Can NB farmed salmon(AY646060)
HPR0
USA Maine farmed salmon(AY973194)
Can RPC/NB 04-085-1(AY963263)
HPR9
Norway 47/99 SF(AF364888)
HPR12
Norway N5/89(AY127882)
HPR3
Can Nova Scotia U5575-1(AF294881)
HPR2
Scotland 1490/98(AF391126)
PR2 Can F1-97(AF427045)
HPR4
Norway T10/93(AF302801)
HPR5
Norway MR14/95(AF364873)
HPR8
Norway 48/99 SF(AF364878)
HPR11
Norway 54/00 SF(AF364884)
PR12 Norway 97/09/393(AF427070)
HPR6
Norway ST25/97(AF364885)
HPR14
Norway 485/9/97(AY378181)
HPR10
Norway MR52/00(AF364892)
HPR13
Faroe Islands 1173/01/12(AJ440970)
HPR1
Norway H1/87(AF364893)
Norway Glesvaer/2/90(AF283998)
HPR7
Scotland 390/98(AF283997)
HPR7
Chile 1065 (AM941715)
HPR15
Norway 810/9/99(AF378180)
PR16 Norway 94/09/579(AF427073)
HPR20
Can RPC/NB 98-0280-2(AF294870)
HPR21
Can NBISA01(AF283996)
HPR21
Can RPC/NB 01-0593-1(AY062033)
HPR21
Can RPC/NB 01-0973-3(AY963266)
HPR21
Can RPC/NB 98-049-1(AF294876)
HPR21
Chile 7833-1(AF294879)
HPR21
Can RPC/NB 02-0775-14(AY963265)
HPR21
Can RPC/NB 02-1179-4(AY963264)
Can NB1330-2(AY646063)
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Predicted amino acid sequence
330
340
350
360
370
SKLQRNITDVKIRVDAIPPQLNQTFNTNQVEQPANSVLSNIFISMGVAGF
SKLQRNITDVKIRVDAIPPQLNQTFNTNQVEQPANSVLSNIFISMGVAGF
....RNITDVKIRVDAIPPQLNQTFNTNQVEQPATSVLSNIFISMGV...
....RNITDVKIRVDAIPPQLNQTFNTNQVEQPATSVLSNIFISMGV...
SKLQRNITDVKIRVDAIPPQLNQTFNTNQVEQPSTSVLSNIFISMGVAGF
SKLQRNITDVKIRVDAIPPQLNQTFNTN-------------FISMGVAGF
SKLQRNITDVKIRVDAIPPQLNQTFNT-----------------MGVAGF
SKLQRNITDVKIRVDAIPPQLNQT---------------NIFISMGVAGF
SKLQRNITDVKIRVDAIPPQLNQT-----------------FISMGVAGF
SKLQRNITDVKIRVDAIPPQLNQT--------------------MGVAGF
SKLQRNITDVKIRVDAIPPQLNQT-------------L-------GVAGF
SKLQRNITDVKIRVDAIPPQL-----------------SNIFISMGVAGF
SKLQRNITDVKIRVDAIPPQL---------------------ISMGVAGF
SKLQRNITDVKIRVDAIPPQL------------------------GVAGF
SKLQRNITDVKIRVDAIPP-------------------RNIFISMGVAGF
SKLQRNITDVKIRVDAI-------FNTNQVEQPATSVLSNIFISMGVAGF
SKLQRNITDVKIRVDAI-----------QVEQPATSVLSNIFISMGVAGF
SKLQRNITDVKIRVDA-----------NQVEQPATSVLSNIFISMGVAGF
SKLQRNITDVKIK------------------QPATSVLSNIFISMGVAGF
SKLQRNITDVK-------------------EQPANSVLSNIFISMGVAGF
SKLQRNITDVK---------------------PATSVLSNIFISMGVAGF
SKLQRNITDVK-------------TCNICVEQ----HL--I-ISMGVAGF
SKLQRNITDVK-----------------------TSVLSNIFISMGVAGF
SKLQRNITDVK-----------------------TSVLSNIFISMGVAGF
SKLQRNITDV--------------------E---TSVLSNIFISMGVAGF
S-------------DA--------FNTNQVEQPATSVLSNIFISMGVAGF
GKLGRNITDVNNRVDAI-------LGVNQVEQPSTSVPSNIFISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNIFISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNIFISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNIFISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNILISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNIFISMGVAGF
GKLGRNITDVNNRVDAIPPQL-----------------SNIFISMGVAGF
GKLGRNITDVNNRVAAIPPQL-----------------SNIFISMGVAGF
....RNITDVNNRVDAIPPQLNQT--------------------MGV...
_
__
____________
HPR
aa deleted in HPR
Virus isolated?
0
No*
0
No**
0
No CPE in SHK***
0
No CPE in SHK***
0
No
13
Yes
17
Yes
15
Yes
17
Yes
20
Yes
20
Yes****
17
Yes
21
Yes
23
Yes
19
Yes
7
No****
11
Yes
11
Yes
18
Yes
19
Yes**
21
Yes
20 (FNT ĺ TCN)
Yes
23
Yes
23
Yes
23
Yes
12 (no 333NIT335)
No****
7 (FNT ĺ LGV)
Yes
17
Yes
17
Yes
17
Yes
17
Yes
17
Yes
17
Yes
17
Yes
20
Yes***
Figure
Alignment
from
Kibenge
6 of amino
et al acid sequences in the highly polymorphic region (HPR) of the HE genes of various strains of ISAV (modified
Alignment of amino acid sequences in the highly polymorphic region (HPR) of the HE genes of various strains
of ISAV (modified from Kibenge et al. [19]). The amino acid sequence corresponding to the HE-HPR of the Chilean ISAV
in this disease outbreak is highlighted in yellow, and identifies it as HPR7. Sequences that are not determined are indicated by
dots, and amino acid (aa) deletions in the HPR are indicated by dashes. The HPR groups identified are as reported by Nylund et
al. [23] and Plarre et al. [24]; other sources of information are indicated as *Markussen et al. [20], **Cunningham et al. [25],
***Cook-Versloot et al. [26], and ****Mjaaland et al. [27].
the European genotype as Genotype I and the North
American genotype as Genotype II. Such designation is
consistent with our present knowledge, which is not sufficient to explain the anomalous geographic distribution of
the two genotypes of ISAV.
The Chilean ISAV responsible for the ISA outbreak is
unique in that it has a 33-nucleotide (or 11-amino acid)
insert in the middle of RNA segment 5 which encodes the
F glycoprotein. This insert has 100% sequence identity
with RNA segment 2 of ISAV of Genotype I, and probably
arose through a non-homologous recombination
between the F and PB1 genes of the same virus. To date,
there have been only 8 other ISAV isolates with inserts in
RNA segment 5 [20,21]. All these isolates are Norwegian
ISAV isolates; seven of them were recovered between 1999
and 2002 [21] and one was recovered in 2006 [20]. Seven
of these isolates had inserts from different parts of RNA
segment 5 while in one isolate, the insert was shown to
come from RNA segment 3, which encodes the ISAV
nucleoprotein. Therefore, this Chilean ISAV is the first
known isolate to have an insert in RNA segment 5 coming
from RNA segment 2. Overall, this information indicates
that this Chilean ISAV is different from common Genotype I ISAV isolates in Europe. The location of the insert in
the Chilean ISAV RNA segment 5 is also unique in that it
occurs right in the middle of the 265YP266 motif, which was
recently shown to be a marker for reduced virulence [19].
The RNA segment 5 of ISAV belonging to HPR0, a nonpathogenic virus, has 265NQ266 at this site but without a
sequence insertion [20], which would be similar to the
Chilean ISAV prior to the non-homologous recombination event between the F and PB1 genes. Such recombination events are well known in avian influenza virus (AIV),
involving an insertion in the haemagglutinin (HA) gene
of AIV near the cleavage site of the protein and leading to
emergence of new virulent strains [35-38]. For example,
the avian influenza disease outbreak that occurred in
Chile in 2002 was attributed to the non-homologous
recombination between the HA and nucleoprotein genes
of H7N3 AIV [38].
The Chilean ISAV described here clearly differs from the
7833-1 isolate (of Genotype II) from coho salmon in
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BMC Veterinary Research 2008, 4:28
Chile [12] in segment 5 and the partial segment 6 amino
acid sequences. Thus the 7833-1 virus, which is now wide
spread in the Atlantic salmon industry in Chile, is unlikely
to have been the source of this new virus. It is interesting
that the alignments on RNA segment 6 showed the Chilean ISAV to belong to HPR7b that has previously been
found in Scotland and Norway. However, all ISAV isolates
of HPR7b sequenced to date on RNA segment 5 do not
have the 11-amino acid insert found in the Chilean ISAV.
Comparison of the RNA segment 5 sequence of this Chilean ISAV with the HPR0 virus strain SK779/06 [20]
showed a nucleotide sequence identity of 98.3% and
amino acid sequence identity of 97.1%. In contrast, ISAV
strain 390/98, which is also of HPR7 but from Scotland,
when compared to the HPR0 virus showed a nucleotide
sequence identity of 98.4% and amino acid sequence
identity of 99.1% [see Additional file 6]. This suggests that
the Chilean ISAV is distinct from common Genotype I
ISAV isolates from Europe and North America. The location of the 11-amino acid insert at a motif in the F glycoprotein which is associated with virus virulence, may
account for the elusive nature of this Chilean virus. It is
therefore necessary to sequence the whole genome of this
virus and clarify the epidemiology of this ISA outbreak so
as to establish the origin of this virus, its virulence characteristics and the risk factors associated with its presentation and dissemination, in order to institute adequate
strategies for the control and prevention of ISA in the
Chilean salmon industry.
Conclusion
In conclusion, the present work constitutes the first report
of a case of ISA in farmed Atlantic salmon in Chile. The
clinical signs and lesions are consistent with the classical
descriptions of the disease in marine-farmed Atlantic
salmon in the Northern hemisphere. The outbreak was
caused by ISAV of Genotype I of HPR 7b but distinct from
common European ISAV isolates from Europe and North
America.
Methods
Field sampling
Atlantic salmon with mean weight of 3.9 Kg, held in seawater rearing cages, were necropsied and submitted for
diagnostic testing. Moribund fish were submitted for laboratory analysis to the Biovac S.A. laboratory in Puerto
Montt, Chile, where a full necropsy was conducted and
samples were collected for histological evaluation, virus
isolation, and immunohistochemistry and molecular
biology analysis.
Histology
Tissue samples for histological analysis were collected in
10% buffered formalin. They were then processed using
standard procedures and the sections were stained with
Haematoxylin & Eosin (H&E), in order to describe the sig-
http://www.biomedcentral.com/1746-6148/4/28
nificant morphological changes. Tissues from 9 fish were
additionally submitted to Aquatic Diagnostic Services at
the Atlantic Veterinary College in Prince Edward Island,
Canada, for histological evaluation.
Virus antigen detection assays
The presence of virus antigens in the fish tissues utilized
three different types of assays: immunohistochemistry,
immunochromatography, and antigen ELISA. The immunohistochemistry analyses were carried out on waterproofed tissue sections of 5 μm thickness, using anti-ISAV
monoclonal antibody P10 (Aquatic Diagnostics Ltd, Stirling, Scotland) with the immunohistochemistry kit from
Vector Laboratories. Immunochromatography utilized
tissue homogenates applied to a rapid kit with anti-ISAV
monoclonal antibody according to the manufacturer's
procedures (Aquatic Diagnostics Ltd). The IHNV ELISA kit
(Bio-X Diagnostics) and VHSV ELISA kit (Bio-X Diagnostics) were used to rule out IHNV and VHSV in the tissue
samples. The ELISA procedures followed the manufacturer's protocols.
Virus isolation
Homogenized pools of spleen and kidney were inoculated on monolayers of CHSE-214, EPC, SHK-1 and BF2
cell lines following standard protocols in the OIE Aquatic
Manual [39].
RT-PCR and nucleic acid sequencing
Total RNA was extracted from the same homogenates of
pooled spleen and kidney tissues used for virus isolation,
using EZNA® kits (Omega Biotech) or the RNeasy® kit
(Qiagen) according to the manufacturer's recommended
protocol. For tissues preserved in RNA later® (Ambion
Inc), the tissues were first washed three times with phosphate buffered saline (PBS) and then homogenized using
a tissue homogenizer (Brinkman, Ontario, Canada) prior
to total RNA extraction using the RNeasy® kit (Qiagen).
For the detection of ISAV in tissue homogenates, ISAVspecific primers and conditions described by Devold et al.
[40] and Mjaaland et al. [41] for RNA segment 8, and the
primers described in the OIE Aquatic Manual [39] for
RNA segment 6 were used. Briefly, amplification was performed using 50 μl reaction mixture utilizing EzrTth RTPCR kit (Applied Biosystems, Montreal, Quebec) as follows: EzrTth DNA polymerase – 1 μl, ISA forward primer0.4 uM, ISA reverse primer- 0.4 uM, Mn-2.5 mM, dNTPs200 uM each, 5× PCR buffer-10 μl, ddH2O-19 μl and 5 μl
RNA template. The amplification was performed in Perkin
Elmer Gene Amp PCR System 2400 using the following
conditions: 1 cycle at 53°C for 45 min, one cycle at 94°C
for 2 min, 35 cycles at 94°C for 15 s, 60°C for 15 s and
72°C for 15 s and 1 cycle at 72°C for 7 min before holding the reactions at 4°C.
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BMC Veterinary Research 2008, 4:28
Samples were tested for IHNV and VHSV using nested RTPCR with primers and conditions described by Batts and
Winton [42] and [43], respectively, with minor modifications. The RT-PCR was performed using 50 μl reaction
mixture utilizing EzrTth RT-PCR kit (Applied Biosystems)
as follows: EzrTth DNA Polymerase-2.5 units, forward
primer-0.4 uM, reverse primer -0.4 uM, Mn-2.5 mM,
dNTPs -200 uM each, 5× PCR buffer-10 μl, ddH2O-19 μl
and 5 μl RNA template. For IHNV, the primers consisted
of IHNV 1F primer and IHNV 1R primer whereas for
VHSV they were VHSV 1F primer and VHSV 1R primer.
The amplification was performed in Perkin Elmer Gene
Amp PCR System 2400 using the following conditions: 1
cycle at 50°C for 15 min, 1 cycle at 95°C for 2 min, 25
cycles at 95°C for 30 s, 50°C for 30 s and 72°C for 60 s,
1 cycle at 72°C for 1 min and 1 cycle at 72°C for 7 min
before soaking at 4°C. The second amplification was done
using AmpliTaq Gold (Applied Biosystems) utilizing 50 μl
mixture as follows: AmpliTaq Gold Polymerase-2.5 units,
IHNV 2F primer – 0.4 uM, IHNV 2R primer-0.4 uM for
IHNV or VHSV 2F primer – 0.4 uM, VHSV 2R primer-0.4
uM for VHSV, Mg-2 mM, dNTPs -200 mM each, 10× PCR
buffer – 5 μl, ddH2O- 33.5 μl and 2 μl RT-PCR mixture.
The following cycling conditions were used: 1 cycle of
95°C for 2 min, 25 cycles at 95°C for 30 s, 50°C for 30 s
and 72°C for 60 s and 1 cycle at 72°C for 7 min before
holding the reactions at 4°C.
http://www.biomedcentral.com/1746-6148/4/28
MJTK performed the RT-PCR and sequence analysis of
RNA segment 5 and edited the manuscript. DBG performed the histological evaluation and edited the manuscript. CVY performed the RT-PCR for ISAV, IHNV and
VHSV, provided total RNA of U24636 and U24637 to the
OIE Ref Lab, and edited the manuscript. HG performed
RT-PCR on tissue homogenates and cell culture lysates. AL
and MC isolated the virus in cell culture. FA coordinated
all the MH laboratory procedures. MI made the veterinary
investigation of the increased mortality in the outbreak.
MJ coordinated the MH laboratory investigation and
wrote the manuscript. FSBK coordinated the viral analysis
and DNA sequence analysis of RNA segment 5, and
helped to write the manuscript. All authors read and
approved the final manuscript.
Additional material
Additional File 1
Additional clinical signs in affected Atlantic salmon (Salmo salar) from
the 2007 infectious salmon anaemia (ISA) outbreak in Chile. A marinefish cage on the Atlantic salmon grow-out site showing market-size Atlantic salmon affected in the ISA outbreak.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S1.doc]
Additional File 2
Additional clinical signs in affected Atlantic salmon (Salmo salar) from
the 2007 infectious salmon anaemia (ISA) outbreak in Chile. Affected
Atlantic salmon with exophthalmia from the ISA outbreak.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S2.doc]
For DNA sequencing of ISAV RT-PCR products, total RNA
was extracted from 300 μl of tissue homogenate using Trizol LS® (Invitrogen). The RT-PCR was carried out using the
Titan® One Tube RT-PCR (Roche Diagnostic), in a PTC200 DNA Engine Peltier thermal cycler (MJ Research, Inc.)
using oligonucleotide primers and cycling conditions as
previously described [19,44]. The PCR products were then
either directly sequenced or they were cloned into the
pCRII vector using a TOPO TA cloning kit (Invitrogen) in
preparation for nucleotide sequencing. Plasmid DNA for
sequencing was prepared as described before [45]. DNA
sequencing was performed as previously described [19] by
ACGT Corporation (Toronto, Ontario, Canada). DNA
Sequencing was done either directly on RT-PCR products
or on plasmid DNA containing the cloned RT-PCR products obtained from reactions using total RNA from tissue
samples. Sequence analysis used the BLAST programs
[46], the Sequence Manipulation suite version 2 [47], and
the FASTA program package for personal computers [48].
Additional clinical signs in affected Atlantic salmon (Salmo salar) from
the 2007 infectious salmon anaemia (ISA) outbreak in Chile. Fluid
drawn with a 3-ml syringe from the pericardial sac of affected Atlantic
salmon with Hydropericardium from the ISA outbreak.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S3.doc]
Authors' contributions
Additional File 5
MGG made the veterinary investigation of the outbreak,
performed the necropsy and histological analysis, coordinated the laboratory investigation, and helped to write the
manuscript. AA coordinated the laboratory investigation
and performed the immunohistochemistry analysis.
Additional File 3
Additional File 4
Additional clinical signs in affected Atlantic salmon (Salmo salar) from
the 2007 infectious salmon anaemia (ISA) outbreak in Chile. Affected
Atlantic salmon with abdominal jaundice from the ISA outbreak.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S4.doc]
Additional clinical signs in affected Atlantic salmon (Salmo salar) from
the 2007 infectious salmon anaemia (ISA) outbreak in Chile. Affected
Atlantic salmon with haemorrhages in stomach from the ISA outbreak.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S5.doc]
Page 11 of 13
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BMC Veterinary Research 2008, 4:28
Additional File 6
http://www.biomedcentral.com/1746-6148/4/28
12.
Table 1. The data provided represent percent sequence identities of the
viral Fusion (Segment 5) gene of Chilean ISAV and selected isolates of
Genotype I (European) and Genotype II (North American).
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S6.doc]
13.
Additional File 7
14.
Table 2. The data provided represent percent sequence identities of the
viral Haemagglutinin-Esterase (Segment 6) gene of Chilean ISAV and
selected isolates of Genotype I (European) and Genotype II (North American).
Click here for file
[http://www.biomedcentral.com/content/supplementary/17466148-4-28-S7.doc]
15.
16.
17.
18.
Acknowledgements
Funding for this study was provided by Marine Harvest S.A., Puerto Montt,
Chile, Biovac S.A., Puerto Montt, Chile, and OIE Reference Laboratory for
ISA, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada.
19.
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