Download In vivo correlates of infectious salmon anemia virus pathogenesis in

Journal of General Virology (2006), 87, 2645–2652
DOI 10.1099/vir.0.81719-0
In vivo correlates of infectious salmon anemia virus
pathogenesis in fish
Frederick S. B. Kibenge,1 Molly J. T. Kibenge,1 Dave Groman2
and Sandi McGeachy3
1,2
Department of Pathology and Microbiology1 and Aquatic Diagnostic Services2, Atlantic
Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown,
PEI C1A 4P3, Canada
Correspondence
Frederick S. B. Kibenge
[email protected]
3
New Brunswick Department of Agriculture, Fisheries and Aquaculture, Fredericton, NB,
Canada
Received 28 November 2005
Accepted 19 April 2006
The phenotypic correlates of pathogenicity for Infectious salmon anemia virus (ISAV) in salmonid
fishes have not been thoroughly studied to date. In this study, a comparison was made of 13
different strains of ISAV, isolated from different geographical regions between 1997 and 2004, for
their infectivity in three fish species [Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus
kisutch) and rainbow trout (Oncorhynchus mykiss)]. When the different virus isolates were
used at an approximate inoculum dose of 106 TCID50 in 0?2 ml per fish, it was found that the most
virulent strains had an acute mortality phase in Atlantic salmon that started at 10–13 days
post-inoculation and lasted for 9–15 days with a cumulative mortality of ¢90 %. These highly
pathogenic strains also caused low mortality in rainbow trout, albeit later in infection. Viruses with a
more delayed or protracted mortality phase resulting in cumulative mortalities of 50–89 % in
Atlantic salmon were considered to be of intermediate pathogenicity and isolates with cumulative
mortalities of ¡49 % were considered to be of low pathogenicity. On this basis, three of the ISAV
isolates showed a high-, eight an intermediate- and two a low-pathogenicity phenotype in Atlantic
salmon. Coho salmon were resistant to all ISAV isolates. These results confirmed that there is
variation in pathogenicity among ISAV strains for Atlantic salmon and rainbow trout, and that other
salmonid species such as coho salmon can carry highly pathogenic strains of ISAV without
showing signs of disease. The identified pathogenicity phenotypes may aid in the identification of
molecular markers of ISAV virulence.
INTRODUCTION
Infectious salmon anemia virus (ISAV) is a segmented,
negative-sense, single-stranded RNA virus of the family
Orthomyxoviridae, the only member of the genus Isavirus
(Kawaoka et al., 2005). It is a significant fish pathogen that has
caused and continues to cause severe economic losses to the
salmon-farming industry in the northern hemisphere.
Clinical infectious salmon anaemia (ISA) is characterized
by high mortality, with dead fish exhibiting exophthalmia,
pale gills, ascites and haemorrhagic liver necrosis, and renal
interstitial haemorrhage and tubular nephrosis (Evensen et
al., 1991; Thorud & Djupvik, 1988). The virus has also been
detected in diseased farmed coho salmon (Oncorhynchus
kisutch) in Chile in 2000 (Kibenge et al., 2001a), in apparently
normal seawater-farmed rainbow trout (Oncorhynchus
mykiss) in Ireland in 2002 (Siggins, 2002), in apparently
normal wild fish [sea trout and brown trout (Salmo trutta)
and Atlantic salmon (Salmo salar)] from the same geographical locations as Atlantic salmon farms with clinical ISA in
Scotland, UK (Cunningham et al., 2002; Raynard et al.,
0008-1719 G 2006 SGM
2001a), and in apparently normal non-salmonid marine fish
[pollock (Pollachius virens) and Atlantic cod (Gadus morhua)]
collected from cages with ISA-diseased salmon in Maine, USA
(MacLean et al., 2003). These findings support the observation that natural clinical disease from ISAV only occurs in
marine-farmed Atlantic salmon.
Under experimental conditions, ISAV may infect other fish,
resulting in asymptomatic, probably life-long, carriers of
the virus (Nylund et al., 1994, 1995, 1997, 2002; Rolland &
Nylund, 1999). Rainbow trout, brown trout and Arctic char
(Salvelinus alpinus) are reported to be resistant to experimental infection with the Scottish ISAV isolate 390/98 (Snow
et al., 2001). The pacific salmonid species chum (Oncorhynchus
keta), steelhead trout (O. mykiss), chinnok (Oncorhynchus
tshawytscha) and coho salmon were also found to be resistant to experimental infection with a Norwegian strain and
a Canadian strain of ISAV, even with doses as high as 108
TCID50 ml21 that induced 98 % cumulative mortality in
Atlantic salmon (Rolland & Winton, 2003).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
Printed in Great Britain
2645
F. S. B. Kibenge and others
It is assumed, but not yet proven, that there is variation
in pathogenicity among ISAV strains. As only samples
from clinical cases routinely receive appropriate diagnostic
attention, naturally avirulent ISAV strains may be rarely
isolated. Attempts to isolate virus from some natural ISA
outbreaks (Mjaaland et al., 2002) and from some ISAV RTPCR-positive fish (Devold et al., 2001; Kibenge et al., 2001a;
Nylund et al., 2002; Raynard et al., 2001a; Snow et al., 2001)
have not been successful. One possible explanation for this
failure is that the available fish cell lines are not sensitive
enough to grow low virus titres, in contrast to naı̈ve Atlantic
salmon. It is also not known whether the currently available fish cell lines are equally permissive to both pathogenic
and non-pathogenic ISAV strains. Mjaaland et al. (2002)
found no clear correlation between the replication properties of different ISAV isolates in SHK-1 cells and the development of cytopathic effects (CPE); however, the isolates
that replicated well showed CPE either early or later in the
infection.
Most experimental studies of ISAV infection in Atlantic
salmon reported to date have used single ISAV isolates and/
or different virus doses, making it difficult to extrapolate the
relative levels of pathogenicity among different ISAV strains.
These experiments investigated the effects of freshwater
versus seawater, different routes of infection, different virus
doses (Raynard et al., 2001b), the effects of mixed infection with togavirus-like virus (Kibenge et al., 2000a), the
effects of vaccination with inactivated whole ISAV (Jones
et al., 1999) and the relative resistance of pacific salmon
to ISAV infection (Rolland & Winton, 2003). One study
compared different ISAV isolates using MHC-compatible
Atlantic salmon half-siblings (Mjaaland et al., 2005), but
virus infection was by cohabitation transmission making it
difficult to standardize the virus dose and hence limiting
the range in death prevalence. Raynard et al. (2001b) showed
that there is a relationship between the onset of mortality
and the virus dose used to infect Atlantic salmon, with those
fish that received a higher viral dose starting to die earlier
than those given lower doses of the same virus.
The present study compared the pathogenicity of 13 different strains of ISAV isolated from different geographical
regions between 1997 and 2004, used at an equal virus dose,
for Atlantic salmon, coho salmon and rainbow trout in an
attempt to identify and characterize the correlates of ISAV
virulence. To our knowledge, this is the first systematic demonstration of virulence variation among ISAV
isolates in Atlantic salmon and including a second fish
species, Oncorhynchus spp., susceptible to clinical disease.
The rainbow trout infection phenotype of ISAV is presented
as a correlate of ISAV pathogenicity and may facilitate the
identification of ISAV virulence genes.
METHODS
Cells, viruses and virus culture. TO cells (Wergeland &
Jakobsen, 2001) were grown in Hanks’ minimum essential medium
(BioWhittaker) supplemented with 10 % fetal bovine serum, 2 mM
2646
L-glutamine,
1 % non-essential amino acids and 50 mg gentamicin
ml21. Cells were incubated at room temperature (24 uC) and the
monolayer cultures were used 24 h after seeding. All viruses used in
this study were received as SHK-1 cell line lysates except for isolate
NBISA01, which was a CHSE-214 cell line lysate. The geographical
origin, laboratory source and genotype of the 13 ISAV isolates used
in this study are summarized in Table 1. The viruses were propagated and titrated in TO cells, as described previously (Kibenge et al.,
2001b), prior to use in the in vivo infectivity assay.
In vivo infectivity assay. Specific-pathogen-free Atlantic salmon
parr (St John River stock) were obtained either from the Cardigan
Fish Hatchery, Cardigan, PEI, Canada, or from Atlantic Sea Smolt
Ltd, Souris, PEI, Canada. Rainbow trout were obtained from the
Dover Fish Hatchery Ltd, Murray River, PEI, Canada. Coho salmon
were from Aqua Health Ltd’s quarantine facility in Victoria, PEI,
Canada. The mean mass and length of the fish at the start of each
trial were approximately 10–20 g and 10 cm, respectively, in the
freshwater phase. For each trial, an opportune sample of five fish of
each species was screened for ISAV by virus isolation attempts from
tissue samples (kidney, heart, spleen and pyloric caeca) in the TO
cell line and monitoring for CPE and RT-PCR (Kibenge et al.,
2001a) to establish the ISAV-negative status of the stock before virus
challenge. The fish were maintained in the Aquatic Animal Facility
of the Atlantic Veterinary College (AVC) in 1 m diameter fibreglassreinforced plastic tanks using a freshwater flow-through system at a
temperature of approximately 11 uC. The Atlantic salmon, rainbow
trout and coho salmon were acclimatized in separate tanks in freshwater for 2–4 weeks at a water flow rate of 3?5 l min21. The experimental procedures used in this study were performed in accordance
with the guidelines of the Canadian Council of Animal Care (Olfert
et al., 1993).
The fish were divided into groups: the uninfected control group
consisted of 70–100 Atlantic salmon, 40 rainbow trout and 40 coho
salmon, whilst each of the infected groups consisted of 30–50 Atlantic
salmon, 10 rainbow trout and 10 coho salmon in each tank. For each
trial, the uninfected control tank was located in a separate ‘clean’ room
from the tanks with experimentally infected fish. For the experimental
infection, the challenge fish were removed from the stock-holding
tank and anaesthetized by immersion in an aerated solution of tricaine methanesulphonate (TMS-222; 100 mg l21). Each fish was then
challenged by the intraperitoneal route with 0?2 ml virus suspension
containing approximately 106?0 TCID50 ISAV and was then returned
to the respective study tank. In all trials, caution was taken to avoid
contact between the tanks for the different ISAV isolates. Fish were
fed once a day and the fish tanks were flushed once a day. All tanks
were checked twice daily for mortality and the fish were observed for
abnormal behaviour and external lesions for the duration of the study.
All dead fish were necropsied and, where necessary, tissues (kidney,
heart, spleen and pyloric caeca) were collected for virus detection
by RT-PCR and histopathology. At the termination of the study at
58–76 days post-inoculation (p.i.), all surviving fish were necropsied
and tissues (kidney, heart, spleen and pyloric caeca) were collected for
virus detection by RT-PCR.
RT-PCR. Total RNA was extracted from 300 ml clarified tissue
homogenates of pooled organ tissues (kidney, heart, liver and
spleen) of fish mortalities or hearts of the surviving fish using
TRIzol reagent (Invitrogen Life Technologies) following the manufacturer’s protocol. The RT-PCR primers and conditions and the
thermal cycler used to detect ISAV by RT-PCR have been described
previously (Kibenge et al., 2000b). PCR products were resolved by
electrophoresis on a 1 % agarose gel and visualized under 304 nm
UV light after staining with ethidium bromide (Sambrook et al.,
1989).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
Journal of General Virology 87
In vivo correlates of virulence of ISAV in fish
Table 1. Origin and genotype of the ISAV isolates used in this study
Isolate and geographical origin
Laboratory source*
and year of isolation
ISAV genotypeD
NBISA01, NB, Canada
RPC/NB 98-049-1, NB, Canada
RPC/NB 98-0280-2, NB, Canada
RPC/NB 01-0593-1, NB, Canada
RPC/NB 01-0973-3, NB, Canada
RPC/NB 02-0775-14, NB, Canada
RPC/NB 02-1179-4, NB, Canada
485/9/97, Norway
810/9/99, Norway
390/98, Scotland, UK
7833-1, Chile
U5575-1, NS, Canada
RPC/NB 04-085-1, NB, Canada
Aqua Health, 1998
RPC, 1998
RPC, 1998
RPC, 2001
RPC, 2001
RPC, 2002
RPC, 2002
B. Dannevig, 1997
B. Dannevig, 1999
A. McVicar, 1998
Aquatic Health, 1999
AVC, 2000
RPC, 2004
North American, HPR21
North American, HPR21
North American, HPR20
North American, HPR21
North American, HPR21
North American, HPR21
North American, HPR21
European, HPR14
European, HPR15
European, HPR7
North American, HPR21
European, HPR3
European, new HPR
*Sources of isolates were: Aqua Health Ltd, Charlottetown, PEI, Canada; RPC, Research and Productivity
Council, Fredericton, NB, Canada; B. Dannevig, National Veterinary Institute, Oslo, Norway; A. McVicar,
Fish Health Inspectorate, FRS, Marine Laboratories, Aberdeen, UK; Aquatic Health, Chile Ltd, Puerto Mont,
Chile; AVC, Atlantic Veterinary College, Regional Diagnostic Virology Laboratory, Charlottetown, PEI,
Canada.
DGenotypes of ISAV (F. S. B. Kibenge, M. J. T. Kibenge, Y. Wang, B. Qian, S. Hariharan & S. McGeachy,
unpublished results): HPR groups denote grouping based on amino acid sequences in the highly
polymorphic region (HPR) of the ISAV haemagglutinin–esterase gene, as reported by Nylund et al. (2003)
and Plarre et al. (2005).
RESULTS AND DISCUSSION
Performance of multiple experimental fish trials
and mortality
Most experimental studies of ISAV infection in Atlantic
salmon reported to date have used single ISAV isolates
and/or different virus doses, making it difficult to extrapolate the relative levels of pathogenicity among different ISAV
strains. In the present study, we wanted to compare the pathogenicity of several different strains of ISAV
isolated from different geographical regions, potentially
representing a wide range of virus virulence, in an attempt
to identify and characterize the correlates of ISAV virulence. Because of space limitations for Biosafety Level 2
pathogen studies, only two to four different strains of ISAV
could be investigated at any one time. Therefore, attempts
were made to use fish of the same genetic stock strain and
quality by purchasing fish for all of the trials from the
same source and using them at the same mass under a
standardized protocol (Kibenge et al., 2000a). A total of five
experimental trials (1–5) were performed in this study. These
trials were designed according to the fish infection model
originally used to reproduce ISA mortality and gross pathology experimentally at the AVC Aquatic Animal Facility
(Kibenge et al., 2000a), which is now used routinely (Moneke
et al., 2003).
http://vir.sgmjournals.org
The cumulative percentage mortalities from ISAV-injected
fish in the five trials are summarized in Table 2. The fish
mortality data from trials 3 and 5 were compromised
because of an aggressive fungal infection (in the control
Atlantic salmon group, control coho salmon group and
groups infected with ISAV isolates 810/9/99 and RPC/NB
98-0280-2 in trial 3, and in the control Atlantic salmon
group and the group infected with RPC/NB 01-0973-3 in
trial 5) that was successfully controlled with formalin bath
treatment. Fungal infections are not uncommon in fish
challenge trials (Rolland & Winton, 2003) and both prophylactic and therapeutic formalin bath treatments are routine additions to experimental protocols. No gross clinical
signs of ISA were observed in any of the fish and they were
not counted with the ISAV-induced mortalities. In addition, the Atlantic salmon that died at 7 days p.i. in the
group infected with isolate RPC/NB 01-0973-3 in trial 4
had no gross pathology lesions and this fish was also not
counted with the ISAV-induced mortalities. All groups
infected with ISAV experienced mortality among Atlantic
salmon, with cumulative percentage mortalities ranging
from 10 to 100 %, occurring between 10 and 41 days p.i.,
and exhibited gross lesions characteristic of ISAV infection
(Thorud & Djupvik, 1988). Some of the ISAV-infected
groups also experienced mortality in rainbow trout, with
cumulative percentage mortalities ranging from 10 to 50 %,
occurring between 13 and 40 days p.i. and exhibited gross
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
2647
F. S. B. Kibenge and others
Table 2. Cumulative percentage mortality among three different fish experimentally infected with 13 different strains of ISAV
ISAV strainD
NBISA01 (CHSE P3)
RPC/NB 98-049-1 (TO P3)
810/9/99 (TO P2)
810/9/99 (TO P4)
RPC/NB 02-1179-4 (TO P2)
390/98 (TO P3)
RPC/NB 98-0280-2 (TO P4)
7833-1 (TO P1)
U5575-1 (TO P1)
RPC/NB 02-0775-14 (TO P2)
485/9/97 (TO P4)
RPC/NB 01-0593-1 (TO P2)
RPC/NB 01-0973-3 (TO P3)
RPC/NB 01-0973-3 (TO P3)
RPC/NB 04-085-1 (TO P4)
Virus dosed
5?80
10
105?80
106?13
106?30
106?30
106?13
105?80
105?80
105?80
105?80
105?80
105?80
105?80
105?80
105?50
Trial
2
2
3
4
4
2
3
1
1
4
4
1
4
5
5
Atlantic salmon§
95?6
100
92?1
100
83?3
79?2
66?7
66?0
64?0
63?3
60?0
50?0
10?3
28?6
18?2
(10–19)
(11–23)
(11–26)*
(13–33)
(15–35)
(11–23)
(11–28)*
(13–25)
(18–39)
(15–26)
(18–41)
(15–30)
(22–38)*
(22–34)*
(21–28)
Rainbow trout§
50
10
10
20
10
0
10
0
0
0
0
0
10
(13–27)
(18)
(21)
(30–40)
(15)
(33)
(34)||
Coho salmon§
0
0
0
0
0
0
0
0
0
0
0
0
0
ND
ND
ND
ND
DThe cell type for propagation and passage number are given in parentheses.
dApproximate virus titre of residual inoculum expressed as TCID50 in 0?2 ml per fish.
§Percentage mortality, ranked based on the ability to kill Atlantic salmon and rainbow trout. Numbers in parentheses refer to time of duration of
mortality in days p.i. *Denotes adjusted percentage mortality data in infected groups with non-specific mortality occurring on day 6 postinjection, as explained in the text.
||One rainbow trout mortality (10 % mortality at 34 days p.i.): this fish had a spinal cord deformity that made it physically impaired and may
have contributed to its increased susceptibility to ISAV infection.
ND, Not done.
lesions consistent with ISA observed in Atlantic salmon,
which included moderate haemorrhage in pyloric caeca,
pale gills and dark liver. There was no mortality in the coho
salmon in any of the ISAV-injected groups during any of the
trials. No mortalities occurred in any of the control groups
during trials 1, 2 and 4.
of ISAV are impossible. Thus, we undertook a systematic
study in which all ISAV isolates were used at an equal virus
dose for Atlantic salmon, coho salmon and rainbow trout.
To quantify virus titres of the different viruses in order to
use equivalent amounts of virus, all of the viruses were
propagated and titrated in TO cells.
Pathogenicity of ISAV in different fish
Several theories have been put forward to explain the geographical and host origin, and therefore the virulence, of
ISAV in farmed fish (Devold et al., 2001; Mjaaland et al.,
2002; Nylund et al., 1995, 1997, 2003). For example, it was
suggested that ISAV may have been introduced to Norway
with the importation of rainbow trout from North America
(Krossøy et al., 2001). It has been suggested that there are
natural reservoirs for the virus, probably in fish occurring
in the coastal areas where ISA outbreaks frequently occur.
Thus, the virulence of ISAV is intrinsically linked to its
emergence as a pathogen in marine-farmed Atlantic salmon
(Nylund et al., 2003). The correlates of ISAV pathogenicity, however, are as yet poorly understood. For any virus,
virulence varies over a wide range: from strains that almost
always cause inapparent infections, to those that usually
cause disease, to those that usually cause death (Murphy
et al., 1999). As virus virulence is dependent on both virus
factors and host factors, and as there are no available
inbred Atlantic salmon lines, precise measures of virulence
2648
Fig. 1. Bar chart for total percentage mortality in the Atlantic
salmon and rainbow trout groups. ISAV isolates: A, NBISA01;
B, RPC/NB 98-049-1; C, 810/9/99; D, RPC/NB 02-1179-4;
E, 390/98; F, RPC/NB 98-0280-2; G, 7833-1; H, U5575-1;
I, RPC/NB 02-0775-14; J, 485/9/97; K, RPC/NB 01-0593-1;
L, RPC/NB 01-0973-3; M, RPC/NB 04-085-1.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
Journal of General Virology 87
In vivo correlates of virulence of ISAV in fish
Fig. 2. Graph of cumulative percentage mortality for the three
ISAV pathogenicity phenotypes in Atlantic salmon. High pathogenicity: NBISA01 and 810/9/99 (trial 4); intermediate pathogenicity: 390/98 and U5575-1; low pathogenicity: RPC/NB
01-0973-3 (trial 5) and RPC/NB 04-085-1.
The 13 ISAV isolates varied in their mortality patterns for
Atlantic salmon, reflecting a variation in their virulence, as
depicted in Figs 1 and 2 and summarized in Table 2. Thus,
the most virulent virus strains, NBISA01, RPC/NB 98049-1 and 810/9/99, had the highest mortality of >90 %.
Fish infected with these viruses had the most acute mortality phase in Atlantic salmon, starting between 10 and
13 days p.i. and lasting for only 9–15 days. The Atlantic
salmon mortality of the less-virulent ISAV isolates either
lasted longer and/or started later, portraying a protracted
mortality phase. For example, for isolates U5575-1 and 485/
9/97 with mortalities of 64 and 60 %, respectively, the first
mortality occurred at 18 days p.i. and the last at 39 and
41 days p.i., respectively. For isolates RPC/NB 01-0973-3
and RPC/NB 04-085-1, which were even less virulent with
mortalities of 28?6 and 18?2 %, respectively, the first mortality occurred later at 22 and 21 days p.i., respectively.
When the ISAV isolates were ranked based on their ability
to kill Atlantic salmon, the isolates with the highest cumulative percentage mortalities (i.e. >90 %) and some with
medium cumulative percentage mortalities (65–85 %) also
killed rainbow trout (Table 2). The cumulative percentage
mortality in rainbow trout was always lower than in Atlantic
salmon. In addition, the mortality phase in ISAV-infected
rainbow trout was protracted in that it started later and/
or lasted longer than in Atlantic salmon for the same virus
isolates. Thus, for isolate NBISA01, mortality in rainbow
trout started at 13 days p.i. (compared with 10 days p.i. in
Atlantic salmon) and stopped at 27 days p.i. (compared
with 19 days p.i. in Atlantic salmon) (Table 2). On the basis
of these data, the in vivo correlates of virulence of ISAV
include the following: (i) cumulative percentage mortality
in Atlantic salmon; the most pathogenic strains had mortality levels of >90 %; (ii) nature of the mortality phase
(i.e. time of onset and duration of mortality) in Atlantic
salmon; the most pathogenic strains had an acute mortality phase that started at 10–13 days p.i. and lasted for
9–15 days; and (iii) mortality in rainbow trout, with systemic haemorrhagic lesions that were consistent with those
typically seen in Atlantic salmon infected with ISAV. Previous analyses of natural ISA disease patterns (Mjaaland
et al., 2002) and experimental ISA disease in a cohabitant challenge model (Mjaaland et al., 2005) also suggest
a grouping of two main forms, acute and protracted. The
shorter duration of the mortality phase in the present study
would be analogous to acute disease, whilst the longer
duration of mortality would be analogous to protracted
disease. Table 3 summarizes a traceback on some of the
ISAV isolates used in the present study. Isolate RPC/NB
01-0973-3 was associated with an acute natural disease
pattern and isolate RPC/NB 04-085-1 was non-pathogenic
in natural infections, but both isolates had a protracted
experimental disease pattern. This indicates that the natural
disease may not necessarily correlate with the experimental
disease. However, of more significance is the fact that we
could separate ISAV isolates into those that killed rainbow
trout and those that did not, and this separation appeared to
correlate with ISAV pathogenicity in Atlantic salmon. Thus,
the rainbow trout infection phenotype might facilitate the
identification of ISAV virulence genes.
As ISAV-induced mortality in rainbow trout has never
been reported, let alone described, several rainbow trout
mortalities were examined for histopathological lesions.
Significant morphological changes were noted in six
tissues taken from the dead rainbow trout. The trunk
kidney showed a focal region of peritubular and interstitial
Table 3. Clinical history of the ISAV isolates
ISAV isolate
RPC/NB
RPC/NB
RPC/NB
RPC/NB
01-0593-1
01-0973-3
02-1179-4
04-085-1
Date site stocked
May
May
June
June
2000*
2001
2002
2002
Date site sampled
Date cage removed
Nature of outbreak
26 July 2001
29 November 2001
17 October 2002
9 February 2004
31 July 2001
10 December 2001
18 October 2002
Harvested at market mass
Acute
Acute
Acute
Non-clinicalD
*Multiple year site (older fish were ISA-positive; the class of 2000 was ISA-positive 4 months after stocking).
DMortality was below 0?05 % per day; identification at infection level as a result of management practice.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
2649
F. S. B. Kibenge and others
haemorrhage, with evidence of increased frequency of
erythrophagia (Fig. 3a). The pyloric caeca had multifocal
regions of mild congestion and haemorrhage within the
lamina propria vasculature (Fig. 3b). Affected heart tissue
contained moderate diffuse endocardial hyperplasia with
evidence of a mild to moderate mononuclear cell infiltrate
and phagocytosis of debris and effete erythrocytes (Fig. 3c).
The gill demonstrated moderate diffuse filamental arteriole congestion and lamellar telangiectasia. Changes in the
Fig. 3. Haemorrhagic lesions in tissues from rainbow trout that
died of experimental infection with ISAV. (a) Trunk kidney from
a rainbow trout inoculated intraperitoneally with ISAV, showing
mild interstitial haemorrhage and sinusoidal congestion. Stained
with haematoxylin and eosin (H&E). Bar, 79 mm. (b) Villi of the
pyloric caeca from a rainbow trout inoculated intraperitoneally
with ISAV. The arrows show congestion and haemorrhage
within the lamina propria. Stained with H&E. Bar, 32 mm. (c)
Endocardium from a rainbow trout inoculated intraperitoneally
with ISAV. The arrow shows endocardial inflammation. Stained
with H&E. Bar, 32 mm.
2650
spleen included mild diffuse sinusoidal congestion and
increased frequency of erythrophagia, and for the liver a
mild peribilliary cuffing by mononuclear leukocytes. These
lesions were consistent with those typically seen in Atlantic
salmon infected with ISAV and are consistent with a clinical diagnosis of ISA in rainbow trout. Moreover, since
histological analysis was performed only on the fish mortalities, we could not categorically rule out histopathological
lesions in any surviving fish during the mortality phase. It
is noteworthy that changes seen for the kidney (interstitial
haemorrhage), gill (filamental arteriole congestion), spleen
(diffuse sinusoidal congestion and erythrophagia) and pyloric caeca (congestion of the lamina propria vasculature)
were all consistent with lesions typically seen in Atlantic
salmon with ISA in both naturally acquired (Mullins et al.,
1998) and experimentally induced scenarios (Jones &
Groman, 2001). The finding of endocarditis, in association
with the above tissue changes was, however, unexpected,
as ISAV in Atlantic salmon typically does not produce
significant inflammation to heart tissues.
The results of virus detection by RT-PCR in tissues from the
fish mortalities sampled at the beginning, middle and end of
the mortality period are summarized in Table 4. Virus was
detected in fish tissues throughout the mortality period in
Atlantic salmon mortalities. Most mortalities that occurred
during the middle of the mortality period (15–22 days p.i.)
for all virus groups tested were positive for ISAV, whereas
at the end of the mortality period (23–42 days p.i.), only
about 50 % of the mortalities were positive for ISAV and
these were mainly from viruses of lower pathogenicity for
Atlantic salmon. In case of the rainbow trout mortalities,
virus was detected in fish tissues during the beginning and
middle of the mortality period only. The heart tissues from
all surviving fish were also tested for virus by RT-PCR. All
samples were negative except for one pool sample from
Atlantic salmon infected with ISAV isolate 485/9/97, indicating that most surviving fish were free of virus regardless
of the ISAV isolate used. As there was no mortality among
ISAV-injected coho salmon, there were no data on virus
presence in coho salmon tissues. However, it is unlikely
that this fish species does not get infected with ISAV. In a
preliminary study (data not shown), we observed histopathological lesions characteristic of ISA in the absence of
mortality in coho salmon. Moreover, others have detected
ISAV titres of 102?5–103 TCID50 g21 in coho salmon randomly sampled 13–15 days after injection with 108 TCID50
virus ml21 (Rolland & Winton, 2003), indicating that this
fish species undergoes asymptomatic infection.
From our research work, it is apparent that a combination
of virus virulence and host susceptibility determines the
outcome of ISAV infection in fish. Thus, a highly virulent
virus in a resistant host such as rainbow trout will have a
similar outcome to a virus of low pathogenicity in a highly
susceptible host such as Atlantic salmon. This presentation of ISAV infection in the three fish species is analogous
to that of avian influenza A viruses in domestic poultry and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
Journal of General Virology 87
In vivo correlates of virulence of ISAV in fish
Table 4. RT-PCR detection of ISAV in mortalities among fish experimentally infected with different strains of ISAV
Results are given as number of fish positive for ISAV/total number tested. –, No mortalities during period tested;
ISAV strain
NBISA01
RPC/NB 98-049-1
810/9/99 (trial 3)
RPC/NB 02-1179-4
390/98
485/9/97
RPC/NB 01-0973-3 (trial 4)
RPC/NB 01-0973-3 (trial 5)
RPC/NB 04-085-1
Mortality 10–14 days p.i.
Mortality 15–22 days p.i.
ND,
not done.
Mortality 23–42 days p.i.
Atlantic salmon
Rainbow trout
Atlantic salmon
Rainbow trout
Atlantic salmon
Rainbow trout
0/2*
0/1
2/2
–
1/1
–
–
–
–
1/1
–
–
–
–
–
–
1/2
2/2
5/5
6/6
4/4
4/4
0/1
–
1/2
1/1
1/1
–
ND
0/2
–
0/2
–
–
–
0/1
ND
0/1
–
–
–
0/4
0/4
4/9
4/8
ND
0/6
0/5
feral birds. Thus, it is evident from this study that other
species such as coho salmon can carry highly pathogenic
strains of ISAV without showing signs of disease. A similar
situation is known to occur with avian influenza A viruses.
For example, water fowl such as ducks can carry highly
pathogenic strains of avian influenza A virus without
showing signs of disease (Kawaoka et al., 1987). Moreover,
other wild birds are as susceptible as domestic poultry to
highly pathogenic avian influenza, but they can also carry
low-pathogenic strains of the virus, which, if introduced
into domestic poultry, can spread and mutate to the highly
pathogenic form. Thus, it is more likely that the emergence
of ISAV as a fish pathogen is a reflection of variation in
virus–host interactions similar to the situation with avian
influenza in poultry. For example, domestic poultry (order
Galliformes) are not the natural hosts of avian influenza
A viruses (Perdue et al., 1999; Suarez & Schultz-Cherry,
2000). However, people have altered the epidemiological
variables of avian influenza A viruses by creating new ecological niches via bird captivity and domestication, industrial agriculture, national and international commerce and
non-traditional raising practices (Swayne, 2000). In these
new ecosystems, pathogenic and non-pathogenic microorganisms can be transmitted within and between avian
species (or fish species, in the case of ISAV) and adapt to new
host species.
ACKNOWLEDGEMENTS
In conclusion, we have demonstrated experimentally that
the cumulative percentage mortality and the duration of
mortality in Atlantic salmon associated with a particular
virus strain are indicators of ISAV virulence. Whereas to
date ISAV has only been isolated from healthy rainbow
trout and is not known to cause natural clinical disease in
fish other than farmed Atlantic salmon, here we report the
first experimentally induced clinical disease associated with
high mortality due to experimental infection with ISAV in a
fish species other than Atlantic salmon. We identified three
ISAV isolates that were highly pathogenic in Atlantic salmon
and of low pathogenicity in rainbow trout. This rainbow
trout infection phenotype might facilitate the identification
of ISAV virulence genes.
Kawaoka, Y., Nestorowicz, A., Alexander, D. J. & Webster, R. G.
(1987). Molecular analyses of the haemagglutinin genes of H5 influ-
http://vir.sgmjournals.org
The assistance of Drs Emeka Moneke and Tomy Joseph and of Ms
Rebecca Marshall is appreciated. This work was supported by research
grants from the NSERC of Canada.
REFERENCES
Cunningham, C. O., Gregory, A., Black, J., Simpson, I. & Raynard,
R. S. (2002). A novel variant of infectious salmon anaemia virus
(ISAV) haemagglutinin gene suggests mechanisms for virus diversity.
Bull Eur Assoc Fish Pathol 22, 366–374.
Devold, M., Falk, K., Dale, O. B., Krossøy, B., Biering, E.,
Aspehaug, V., Nilsen, F. & Nylund, A. (2001). Strain variation,
based on the hemagglutinin gene, in Norwegian ISA virus isolates
collected from 1987 to 2001: indications of recombination. Dis Aquat
Org 47, 119–128.
Evensen, O., Thorud, K. E. & Olsen, Y. A. (1991). A morphological
study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar). Res Vet Sci 51, 215–222.
Jones, S. R. M. & Groman, D. B. (2001). Cohabitation transmission
of infectious salmon anaemia virus among freshwater-reared Atlantic
salmon (Salmo salar). J Aquat Anim Health 13, 340–346.
Jones, S. R. M., Mackinnon, A. M. & Salonius, K. (1999). Vaccination
of freshwater-reared Atlantic salmon reduces mortality associated
with infectious salmon anaemia virus. Bull Eur Assoc Fish Pathol 19,
98–101.
enza viruses: origin of a virulent turkey strain [published erratum
appears in Virology 1987; 159, 196]. Virology 158, 218–227.
Kawaoka, Y., Cox, N. J., Haller, O. & 8 other authors (2005).
Infectious salmon anaemia virus. In Virus Taxonomy. Classification
and Nomenclature of Viruses: Eighth Report of the International
Committee on Taxonomy of Viruses, pp. 681–693. Edited by C. M.
Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball.
New York: Elsevier, Academic Press.
Kibenge, F. S. B., Whyte, S. K., Hammell, K. L., Rainnie, D.,
Kibenge, M. T. & Martin, C. K. (2000a). A dual infection of infectious
salmon anaemia (ISA) virus and a togavirus-like virus in ISA of
Atlantic salmon Salmo salar in New Brunswick, Canada. Dis Aquat
Org 42, 11–15.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
2651
F. S. B. Kibenge and others
Kibenge, F. S. B., Lyaku, J., Rainnie, D. & Hammell, K. L. (2000b).
Nylund, A., Alexandersen, S., Rolland, J. B. & Jakobsen, P. (1995).
Growth of infectious salmon anaemia virus in CHSE-214 cells and
evidence for phenotypic differences between virus strains. J Gen Virol
81, 143–150.
Infectious salmon anaemia virus (ISAV) in brown trout. J Aquat
Anim Health 7, 236–240.
Kibenge, F. S. B., Garate, O. N., Johnson, G., Arriagada, R.,
Kibenge, M. J. T. & Wadowska, D. (2001a). Isolation and identifi-
Replication of the infectious salmon anaemia virus (ISAV) in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 20, 275–279.
cation of infectious salmon anaemia virus (ISAV) from Coho salmon
in Chile. Dis Aquat Org 45, 9–18.
Nylund, A., Devold, M., Mullins, J. & Plarre, H. (2002). Herring
Kibenge, F. S. B., Kibenge, M. J. T., McKenna, P. K., Stothard, P.,
Marshall, R., Cusack, R. R. & McGeachy, S. (2001b). Antigenic
Nylund, A., Kvenseth, A. M., Krossøy, B. & Hodneland, K. (1997).
(C. harengus): a host for infectious salmon anaemia virus (ISAV).
Bull Eur Assoc Fish Pathol 22, 311–318.
Nylund, A., Devold, M., Plarre, H., Isdal, E. & Aarseth, M. (2003).
variation among isolates of infectious salmon anaemia virus correlates with genetic variation of the viral haemagglutinin gene. J Gen
Virol 82, 2869–2879.
Emergence and maintenance of infectious salmon anaemia virus
(ISAV) in Europe: a new hypothesis. Dis Aquat Org 56, 11–24.
Krossøy, B., Nilsen, F., Falk, K., Endresen, C. & Nylund, A. (2001).
Care and Use of Experimental Animals, 2nd edn. Ottawa, ON,
Canada: Bradda Printing Services.
Olfert, E. D., Cross, B. M. & McWilliams, A. (1993). A Guide to the
Phylogenetic analysis of infectious salmon anaemia isolates from
Norway, Canada and Scotland. Dis Aquat Org 44, 1–6.
Perdue, M. L., Suarez, D. L. & Swayne, D. E. (1999). Avian influenza
MacLean, S. A., Bouchard, D. A. & Ellis, S. K. (2003). Survey of non-
in the 1990s. Avian Poult Biol Rev 11, 1–20.
salmonid marine fishes for detection of infectious salmon anaemia
virus and other salmonid pathogens. In International Response to
Infectious Salmon Anemia: Prevention, Control, and Eradication:
Proceedings of a Symposium, 3–4 September 2002, New Orleans, LA,
pp. 135–143. Technical coordinators: O. Miller & R. C. Cipriano.
Tech. Bull. 1902. Washington, DC: US Department of Agriculture,
Animal and Plant Health Inspection Service; US Department of the
Interior, US Geological Survey; US Department of Commerce,
National Marine Fisheries Service.
Plarre, H., Devold, M., Snow, M. & Nylund, A. (2005). Prevalence of
Mjaaland, S., Rimstad, E. & Cunningham, C. O. (2002). Molecular
diagnosis of infectious salmon anaemia. In Molecular Diagnosis of
Salmonid Fish. Reviews: Methods and Technology in Fish Biology and
Fisheries, pp. 1–22. Edited by C. O. Cunningham. London: Kluwer
Academic Publishers.
Mjaaland, S., Markussen, T., Sindre, H., Kjøglum, S., Dannevig,
B. H., Larsen, S. & Grimholt, U. (2005). Susceptibility and immune
responses following experimental infection of MHC compartible
Atlantic salmon (Salmo salar L.) with different infectious salmon
anaemia virus isolates. Arch Virol 150, 2195–2216.
Moneke, E. E., Kibenge, M. J. T., Groman, D., Johnson, G. R., Ikede,
B. O. & Kibenge, F. S. B. (2003). Infectious salmon anaemia virus
infectious salmon anaemia virus (ISAV) in wild salmonids in western
Norway. Dis Aquat Org 66, 71–79.
Raynard, R. S., Murray, A. G. & Gregory, A. (2001a). Infectious salmon
anaemia virus in wild fish from Scotland. Dis Aquat Org 46, 93–100.
Raynard, R. S., Snow, M. & Bruno, D. W. (2001b). Experimental
infection models and susceptibility of Atlantic salmon Salmo salar to
a Scottish isolate of infectious salmon anaemia virus. Dis Aquat Org
47, 169–174.
Rolland, J. B. & Nylund, A. (1999). Sea running brown trout: carrier
and transmitter of the infectious salmon anemia virus (ISAV). Bull
Eur Assoc Fish Pathol 18, 50–55.
Rolland, J. B. & Winton, J. R. (2003). Relative resistance of Pacific
salmon to infectious salmon anaemia virus. J Fish Dis 26, 511–520.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Siggins, L. (2002). Salmon virus detected in Clew Bay fish farm. The
Irish Times, 12 August 2002. Available at: www.ireland.com
Snow, M., Raynard, R. S. & Bruno, D. W. (2001). Comparative suscep-
RNA in fish cell cultures and in tissue sections of Atlantic salmon
experimentally infected with infectious salmon anaemia virus. J Vet
Diagn Invest 15, 407–417.
tibility of Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus
mykiss) and brown trout (Salmo trutta) to the Scottish isolate of
infectious salmon anaemia virus. Aquaculture 196, 47–54.
Mullins, J. E., Groman, D. & Wadowska, D. (1998). Infectious
Suarez, D. L. & Schultz-Cherry, S. (2000). Immunology of avian
influenza virus: a review. Dev Comp Immunol 24, 269–283.
salmon anaemia in salt water Atlantic salmon (Salmo salar L.) in
New Brunswick, Canada. Bull Eur Assoc Fish Pathol 18, 110–114.
Murphy, F. A., Gibbs, E. P., Horzinek, M. C. & Studdert, M. J.
(editors) (1999). Pathogenesis of viral diseases: representative model
diseases. In Veterinary Virology, 3rd edn, pp. 161–176. San Diego, CA:
Academic Press.
Swayne, D. E. (2000). Understanding the ecology and epidemiology
of avian influenza viruses: implications for zoonotic potential. In
Emerging Diseases of Animals, pp. 101–130. Edited by C. C. Brown &
C. A. Bolin. Washington, DC: American Society for Microbiology.
Thorud, K. & Djupvik, H. O. (1988). Infectious anaemia in Atlantic
Nylund, A., Alexandersen, S., Løvik, P. & Jakobsen, P. (1994). The
salmon (Salmo salar L.). Bull Eur Assoc Fish Pathol 8, 109–111.
response of brown trout (Salmo trutta L.) to repeated challenge
with infectious salmon anaemia (ISA). Bull Eur Assoc Fish Pathol 14,
167–170.
Wergeland, H. I. & Jakobsen, R. A. (2001). A salmonid cell line (TO)
2652
for production of infectious salmon anaemia virus (ISAV). Dis Aquat
Org 44, 183–190.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:13:30
Journal of General Virology 87