Journal of Fish Diseases 2007, 30, 631–636
Short communication
Fish genotype significantly influences susceptibility of
juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), to
waterborne infection with infectious salmon anaemia virus
S Biacchesi1, M Le Berre1, S Le Guillou2, A Benmansour1, M Brmont1, E Quillet2
and P Boudinot1
1 UR892, Unité de Virologie et dÕImmunologie Moléculaires, INRA, Jouy-en-Josas, France
2 UR544, Unité de Génétique des Poissons, INRA, Jouy-en-Josas, France
Keywords: genetic susceptibility, infectious salmon
anaemia virus, rainbow trout, waterborne infection.
The aetiological agent of infectious salmon anaemia
(ISA) is an orthomyxo-like virus with a negativestranded RNA genome consisting of eight segments
(Dannevig, Falk & Namork 1995; Falk, Namork,
Rimstad, Mjaaland & Dannevig 1997; Mjaaland,
Rimstad, Falk & Dannevig 1997). The ISA virus
(ISAV) constitutes the only member of the Isavirus
genus (Kawaoka, Cox, Haller, Hongo, Kaverin,
Klenk, Lamb, McCauley, Palese, Rimstad & Webster 2005). ISA was first observed in Norway in
Atlantic salmon, Salmo salar L. (Thorud & Djupvik
1988). The disease has caused considerable economic loss to the Norwegian salmon farming
industry and has also been reported from Scotland
(Rowley, Campbell, Curran, Turnbull & Bryson
1999) and from the eastern coast of North America
(Bouchard, Keleher, Opitz, Blake, Edwards &
Nicholson 1999; Lovely, Dannevig, Falk, Hutchin,
MacKinnon, Melville, Rimstad & Griffiths 1999).
Clinical signs of ISA include anaemia and leucopenia, exophthalmos, haemorrhages, ascites, petechiae in viscera, and congestion of the liver, spleen
and gut (Evensen, Thorud & Olsen 1991). The
cumulative mortality in infected salmon farms may
2007 The Authors.
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Correspondence P Boudinot, UR892, Unité de Virologie et
dÕImmunologie Moléculaires, INRA, F-78352 Jouy-en-Josas,
France
(e-mail: pierre.boudinot@jouy.inra.fr)
631
reach 100% over several months. Natural outbreaks
of ISA have been observed only in Atlantic salmon,
but the virus was detected from apparently normal
individuals in other species including coho salmon,
Oncorhynchus kisutch (Walbaum), (Kibenge,
Garate, Johnson, Arriagada, Kibenge & Wadowska
2001) and brown trout, Salmo trutta L. (Raynard,
Murray & Gregory 2001). ISAV was also isolated
from coho salmon showing jaundice in a Chilean
fish farm (Kibenge et al. 2001), and ISAV was
subsequently considered as one of the aetiological
agents (Smith, Larenas, Contreras, Cassigoli, Venegas, Rojas, Guajardo, Perez & Diaz 2006). No
mortality was recorded in experimentally infected
adult Arctic char, Salvelinus alpinus (L.), rainbow
trout, Oncorhynchus mykiss (Walbaum), brown
trout, chum, Oncorhynchus keta (Walbaum), chinook, Oncorhynchus tshawytscha (Walbaum), and
coho salmon, and steelhead trout, O. mykiss (Snow,
Raynard & Bruno 2001; Rolland & Winton 2003).
When injected with ascitic fluid from infected
salmon, sea trout, S. trutta, did not develop clinical
signs of ISA apart from an abnormal haematocrit
(Nylund & Jakobsen 1995). However, ISAV could
replicate in this species, and blood samples were
successfully used to infect Atlantic salmon. Furthermore, it was recently reported that the injection
of ISAV NBIS A01 or 810/9/99 induced low
mortalities in juvenile rainbow trout (Kibenge,
Kibenge, Groman & McGeachy 2006). These
observations suggested that ISAV is able to replicate
Journal of Fish Diseases 2007, 30, 631–636
S Biacchesi et al. Susceptibility of young rainbow trout to ISAV waterborne infection
in different salmonids, but that only Atlantic
salmon is highly sensitive to natural infection.
We performed experimental infections with
ISAV using juvenile rainbow trout. ISAV isolate
Glesvaer/2/90 (Dannevig et al. 1995) was amplified
in vitro by four passages in SHK cells and either one
(ISAV P5) or two (ISAV P6) additional passages in
TO cells (Wergeland & Jakobsen 2001) at 15 C.
Virus titres were determined by plaque assay on TO
cells under agarose medium overlay and plaques
were visualized 5 days later by immunofluorescence
(IF) with an anti-ISAV nucleoprotein monoclonal
antibody (Bio-X Diagnostics, Jemelle, Belgium).
Naive juvenile rainbow trout (Drennec strain; mean
weight 1.2 g) in groups of 14 or 15 were inoculated
intraperitoneally, under light anaesthesia, with
0.05 mL of L15 medium containing 5 ·
106 PFU of ISAV P5 or P6, respectively. The two
groups were then transferred to aquaria (10 C) and
mortality was recorded every day. As shown in
Fig. 1a, mortalities in both groups started at day 6
post-injection and reached 80% 3–4 days later.
During this acute phase, the moribund fish displayed typical pathological signs of ISA including
exophthalmos, pale gills and abdominal congestion.
The last mortality was recorded on day 20 postinjection and the final cumulative mortalities
reached in this experiment were 80% (ISAV P5)
and 100% (ISAV P6). Our observations confirm
that juvenile rainbow trout are susceptible to ISAV
Glesvaer/2/90 injection as recently reported for
other ISAV strains (Kibenge et al. 2006).
Waterborne infections were then performed
with groups of 100 juvenile trout incubated with
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the virus in a 3 L bath at 10 C for 2 h. Rainbow
trout from either the Drennec strain (Assay no. 1,
mean weight 2.8 g) or the INRA synthetic strain
(Assay no. 2, mean weight 0.4 g) were incubated
with 5 · 104 and 105 PFU mL)1 of ISAV P6
virus, respectively. As shown in Fig. 1b, mortality
started on day 14 post-infection in both experiments and reached a final cumulative mortality of
26–28%. No additional mortality was recorded
after day 60 post-infection in either experiment.
As observed after ISAV injection, the moribund
fish displayed typical signs of ISA during the acute
phase of the disease (day 14–day 23), including
exophthalmia, pale gills, presence of ascites, darkening and congestion of the spleen and haemorrhages along the digestive tract (Fig. 2). Four
individuals from Assay no. 1 were sacrificed on
day 2, 4, 7, 9, 14 and 18 post-infection and virus
isolation was assayed from serum, spleen, kidney
and intestine samples. No virus could be isolated
from the harvested organs at days 2, 4 and 7 postinfection. On day 9 post-infection, virus-induced
cytopathic effect was observed with the serum of
an asymptomatic fish and the presence of ISAV
was confirmed by IF assay after two passages in
TO cells. Fourteen days post-infection, the virus
was isolated at high titre from organ samples
harvested from a fish displaying clinical signs of
ISA. The virus titre in the serum of this fish was
higher than 106 PFU mL)1, and a titre higher
than 5 · 104 PFU g)1 was found in the intestine
and in a mixture of spleen and head kidney.
Finally, on day 18 post-infection, two of the four
sampled fish had high virus titres in their serum.
Figure 1 (a) Naive juvenile rainbow trout (Drennec strain, mean weight 1.2 g) infected by intra-peritoneal injection with 5 · 106 PFU
per animal in 50 lL of infectious salmon anaemia virus (ISAV) obtained after five (ISAV P5) or six (ISAV P6) successive passages of the
Glesvaer/2/90 virus strain in cell culture. (b) Waterborne infection of juvenile trout. Fish from the Drennec strain (n = 76 due to
sequential harvesting during infection; mean weight = 2.8 g) were used for Assay no. 1, while fish from the INRA synthetic strain
(n = 100; mean weight = 0.4 g) were used for Assay no. 2. Animals were infected by immersion in an aqueous suspension of
5 · 104 PFU mL)1 (Assay no. 1) or 105 PFU mL)1 (Assay no. 2) of ISAV P6. In both series of experiments, mortalities were recorded
each day and are expressed as a percentage of cumulative mortality.
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Journal of Fish Diseases 2007, 30, 631–636
S Biacchesi et al. Susceptibility of young rainbow trout to ISAV waterborne infection
(a)
(b)
Figure 2 (a) Clinical signs of infectious salmon anaemia (ISA) in experimentally infected rainbow trout (clone A22, mean weight 6.2 g)
by the waterborne route (bottom): note pale gills, dark liver, moderate splenomegaly and scarce petechiae. The upper fish is a mockinfected control. (b) Exophthalmos in diseased fish.
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No mortality was recorded in mock-infected fish
groups. Since high titres of virus were recovered
from the organs and serum of fish exposed to
waterborne infection, the mortality can be unambiguously attributed to the ISAV infection. To our
knowledge, this is the first record of significant
ISAV-induced mortality in an Oncorhynchus species after waterborne infection. This susceptibility
can be explained by the use of juvenile animals. In
previous reports adult rainbow trout infected with
ISAV, either by injection or by waterborne
infection (Nylund, Kvenseth, Krossøy & Hodneland 1997; Hjeltnes unpublished, cited in Nylund
& Jakobsen 1995) did not develop any clinical
sign of ISA except abnormal haematocrit and no
mortality was recorded. These results indicate that
ISAV may represent a threat to the rainbow trout
industry, especially in regions where both salmon
and trout are reared.
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To investigate the influence of the genotype on
the rainbow trout susceptibility to ISAV infection,
we performed waterborne infections using six fish
homozygous clones. Those clones were recently
established from the INRA synthetic strain, and
displayed a wide variety of susceptibility to rhabdovirus infection (Quillet, Dorson, Le Guillou,
Benmansour & Boudinot 2006). For each
clone, two groups of 40 juvenile animals (mean
weight: 0.56–0.90 g) were infected by immersion
in an aqueous suspension of ISAV P6 (5 ·
104 PFU mL)1) in 3 L for 2 h (Fig. 3a). A large
range of survival rates was observed among clones
(0–95%), indicating a considerable variability of
response to ISAV infection. The clone B3 was
almost fully resistant to ISAV, and a low mortality
of 15–22.5% was recorded for clones B57 and A36,
respectively. Interestingly, B57 animals never displayed any external sign of disease, while A36
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S Biacchesi et al. Susceptibility of young rainbow trout to ISAV waterborne infection
Figure 3 Sensitivity of different homozygous rainbow trout clones to infectious salmon anaemia virus (ISAV). (a) Waterborne infection
of naive juvenile rainbow trout clones (n = 40 in duplicates; mean weights: clone B45 = 0.80 g, clone A22 = 0.87 g, clone A3 = 0.86 g,
clone A36 = 0.56 g, clone B57 = 0.90 g, clone B3 = 0.87 g). Fish were infected by immersion in an ISAV P6 aqueous suspension of
5 · 104 PFU mL)1. Bars indicate standard error calculated from the two replicates performed for each clone (only the bottom of each
error bar is shown). (b) Waterborne infection of naive juvenile rainbow trout clone A22 (n = 42, mean weight = 6.5 g). Mortalities were
recorded each day and expressed as a percentage of cumulative mortality.
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exhibited very clear exophthalmia, a swollen belly
and pin point lateral petechiae, but generally
survived. This observation suggests that the mechanisms of resistance to ISAV are diverse, and
probably differ between these two genotypes. In
contrast, clones B45 and A22 were highly susceptible and showed mortalities close to 100% as well
as typical signs of the disease. The phase of acute
mortality occurred between days 15–23 post-infection for B45 and days 19–30 for A22. The kinetics
of the infection seemed to be slower for clone A22.
Finally, clone A3 showed a medium susceptibility
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with a 50% final cumulative mortality. To test
whether older fish were still susceptible to ISAV, a
waterborne challenge was performed as previously
described on 42 A22 animals of 6.5 g average
weight (Fig. 3b). Interestingly, these animals remained highly sensitive to the infection and a
cumulative mortality of 93% was recorded. These
results provide the first evidence that resistance to
ISAV in rainbow trout depends on the genotype, as
in Atlantic salmon (Gjøen, Refstie, Ulla & Gjerde
1997; Kjøglum, Grimholt & Larsen 2005). In
salmon, the variability of susceptibility was used to
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S Biacchesi et al. Susceptibility of young rainbow trout to ISAV waterborne infection
identify associations between susceptibility and
MHC haplotypes (Grimholt, Larsen, Nordmo,
Midtlyng, Kjoeglum, Storset, Saebo & Stet 2003;
Kjøglum, Larsen, Bakke & Grimholt 2006).
A wide range of susceptibility to rhabdoviruses
was previously recorded between these homozygous
rainbow trout clones (Quillet et al. 2006). The
susceptibility to ISAV did not clearly correspond to
the susceptibility to viral haemorrhagic septicaemia
virus (VHSV). For example, clones A36 and B57
were both rather resistant to ISAV, while A36 was
highly susceptible to VHSV and B57 was fully
resistant. Thus, the susceptibility to isaviruses and
rhabdoviruses may be determined by different
mechanisms.
This study has shown that juvenile rainbow trout
are sensitive to waterborne infection with ISAV,
which may represent a threat to the trout industry.
It has also shown a wide range of genetic susceptibility among haplotypes. Thus, rainbow trout is a
valuable model for further research on ISAV.
Gjøen H., Refstie T., Ulla O. & Gjerde B. (1997) Genetic
correlations between survival of Atlantic salmon in challenge
and field tests. Aquaculture 158, 277–288.
Acknowledgements
Kjøglum S., Larsen S., Bakke H.G. & Grimholt U. (2006) How
specific MHC class I and class II combinations affect disease
resistance against infectious salmon anaemia in Atlantic salmon (Salmo salar). Fish & Shellfish Immunology 21, 431–441.
This research was supported by the Intramural
Research Programme of the Animal Health Department, INRA. We thank Franck Tiquet and Eric
Letellier (Unité Expérimentale Piscicole, INRA,
France) for taking care of experimental fish,
Alpharma Inc. (Oslo, Norway) for providing the
TO cells, Jeannette Castric (AFSSA, Plouzané,
France) for providing an initial inoculum of
ISAV Glesvaer/2/90 and P. de Kinkelin for
discussions.
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Received: 20 January 2007
Revision received and accepted: 18 April 2007