fish

149
© CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:
Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
4
Other Viral Diseases and Agents of
Cold-water Fish: Infectious Salmon
Anaemia, Pancreas Disease and Viral
Erythrocytic Necrosis
B.H. Dannevig
1,2
and K.E. Thorud
2
1
Department of Morphology, Genetics and Aquatic Biology, Norwegian
College of Veterinary Medicine, PO Box 8146 Dep., 0033 Oslo, Norway;
2
National Veterinary Institute, PO Box 8156 Dep., 0033 Oslo, Norway.
INFECTIOUS SALMON ANAEMIA
Introduction
Infectious salmon anaemia (ISA)* is a viral disease of farmed Atlantic salmon
(Salmo salar L.) associated with high mortalities and is of great economic
significance for the Norwegian fish farming industry. The first recorded
outbreak of ISA occurred in late 1984 in a hatchery on the west coast of Norway.
Infected fish were lethargic and severely anaemic. Other typical signs were
ascites, petechiae in internal organs and haemorrhagic liver necrosis (Thorud,
1991). During the following year, a disease with similar clinical and
pathological signs occurred in smolt and adult Atlantic salmon in sea water
farms that had received smolts from the hatchery that experienced the initial
outbreak. Subsequently, the disease spread to fish farms near processing plants
where fish from affected farms had been slaughtered. The development of the
disease and the pattern of spread were consistent with a contagious disease.
Transmission experiments verified the infectious nature of the disease
(Thorud and Djupvik, 1988; Thorud, 1991). A viral aetiology was suggested as
filtration of tissue homogenates from moribund fish through a 220 nm filter did
not reduce infectivity. No ISA infectivity could be detected following ether
treatment, indicating an enveloped virus as the causal agent (Thorud, 1991). No
pathogenic bacteria were isolated from diseased fish and, although infectious
pancreatic necrosis (IPN) virus (IPNV) was isolated from ISA-infected fish, no
aetiological connection between IPN and ISA could be demonstrated (Melby
*Infectious salmon anaemia (ISA) is the designation of the disease recommended by
the Office International des Epizooties (OIE) Fish Disease Commission.
150
B.H. Dannevig and K.E. Thorud
and Falk, 1995). Early attempts to isolate the ISA virus (ISAV) in commercial
fish cell lines were not successful. Isolation of the causal virus was achieved
approximately 10 years after the first outbreak of ISA, using a new cell line
(SHK-1) established from Atlantic salmon head kidney (Dannevig et al., 1995).
The disease and agent
Species of fish affected and geographical distribution of the
organism and disease
Natural outbreaks of ISA have not been recorded in countries other than Norway
and they only occur in fish that have been transferred to sea or exposed to sea
water in hatcheries. However, farmed Atlantic salmon parr kept in fresh water
also developed ISA following experimental infection (Dannevig et al., 1993).
Wild Atlantic salmon are susceptible to ISA and show the same clinical signs
(see below) as farmed fish, after intraperitoneal injection of infective material
(Nylund et al., 1995a). Replication of ISAV has been demonstrated in experi-
mentally infected freshwater brown trout, in anadromous sea trout (Salmo trutta
L.) and in rainbow trout (Oncorhynchus mykiss) (Thorud and Torgersen, 1994;
Nylund and Jakobsen, 1995; Nylund et al., 1995b; Nylund et al., 1997).
However, none of these species develop any clinical signs of ISA. The disease
has not been detected in marine fish.
The number of ISA-affected fish farms increased steadily from 1988 and
reached a peak in 1991, when 80 new cases were reported in both southern and
northern Norway. Thereafter, the frequency of new cases decreased and, from
1994, only a few cases have been recorded per year. A seasonal variation in
outbreaks of ISA has been observed, with the main peak of new cases occurring
from May to July and a minor peak in November.
General signs of the disease
Acute outbreaks of ISA are usually associated with high mortality. Infected fish
appear lethargic and may keep close to the walls of the net-pen. In terminal
stages, diseased fish are often at the bottom of the cage. The most prominent
external signs are gill pallor and haemorrhage in the anterior eye chamber.
Exophthalmia is often seen (Fig. 4.1). The major internal gross pathological
changes are ascites, darkening and extensive congestion of liver and spleen,
petechiae in visceral fat and, in some cases, congestion of the foregut (Fig. 4.2).
A fibrinous layer may cover the liver capsule. The stomach may be distended by
a serous/viscous fluid, and streaky haemorrhages in the mucosa of the stomach
can be seen. Occasionally, haemorrhages in skeletal muscle occur.
Liver lesions are the major histopathological finding, with typical multi-
focal haemorrhagic necroses, which, in the terminal stages, may become
confluent (Evensen et al., 1991). The areas around the large veins appear to be
intact, giving the lesions a zonal appearance (Fig. 4.3). Necrotic areas are easily
distinguished from normal liver tissue and are characterized by eosinophilia and
dark and swollen hepatocytes, with pyknotic nuclei. Karyorhexis is seldom seen.
The lesions result in an extensively congested liver with dilated sinusoids and, in
151 Other Viral Diseases and Agents of Cold-water Fish
Fig. 4.1. ISA-diseased fish with exophthalmia and skin oedema.
Fig. 4.2. Gross pathology of ISA in the terminal stage. Note pale gills, dark and enlarged liver,
enlarged spleen, petechiae in visceral fat, congested gut and ascites.
152
B.H. Dannevig and K.E. Thorud
later stages, the appearance of blood-filled spaces (Evensen et al., 1991).
Moribund fish are severely anaemic and haematocrit values below 5% are
common. Circulating erythrocytes show several abnormalities, such as reduced
size, cytoplasmic vacuolation, nuclear disintegration and cell fragmentation.
Infected fish also develop lymphocytopenia and thrombocytopenia. The propor-
tion of immature erythrocytes increases and erythroblasts appear in the circulation.
There is variation in the severity of the pathological changes. A dark liver is
usually associated with a low haematocrit. However, not all ISA-diseased fish
have dark livers. The liver can also appear pale or yellow with haemorrhagic
spots. Extensive congestion of the gut may not be associated with a dark liver
and low haematocrit.
Infectious salmon anaemia may also appear in a chronic form, with diffuse
signs that can be difficult to interpret. In chronic infections, the liver may appear
yellow and the anaemia may not be as severe as in acute outbreaks. Furthermore,
there is less ascitic fluid, but haemorrhages in the skin and swim-bladder and
oedema in the scale pockets and swim-bladder are more frequent than in acutely
diseased fish.
Infectious salmon anaemia virus – general characteristics
and taxonomic position
Infectious salmon anaemia virus was first detected in endothelial cells of cardiac
blood-vessels, using electron microscopy (Hovland et al., 1994). In this study
and in later reports, the authors demonstrated that the ISAV is enveloped and is
Fig. 4.3. Liver from ISA-moribund fish showing multifocal haemorrhagic necroses. The affected
areas have become confluent to give the changes a zonal appearance, leaving areas around large
veins intact.
153 Other Viral Diseases and Agents of Cold-water Fish
slightly pleomorphic, with a diameter of approximately 100 nm. The virus is
released from the cells by budding, which apparently occurs without destroying
the host cell (Fig. 4.4) (Hovland et al., 1994; Nylund et al., 1995c, 1996). The
virus contains granules of 10–12 nm in diameter (Hovland et al., 1994). It
replicates in endothelial cells of several organs, and these cells seem to be the
primary target cells in Atlantic salmon. However, replication of ISAV in
leucocytes has been demonstrated both in vivo (Dannevig et al., 1994; Hovland
et al., 1994; Nylund et al., 1995c, 1996) and in vitro (Dannevig and Falk, 1994;
Sommer and Mennen, 1996).
Isolation and purification of ISAV from infected tissues have been
unsuccessful. The availability of cell cultures that could support growth of the
virus has thus been crucial. Because of the leucotropic property of the ISAV, it
seemed reasonable to initiate cell lines originating from cultures of adherent
head-kidney leucocytes. One of these cell lines, SHK-1, was shown to be
susceptible to the virus (Dannevig et al., 1995). Following inoculation of SHK-
1 cells with ISA-infective material, a cytopathic effect (CPE) is observed which
cannot be ascribed to other viruses, and Atlantic salmon develop ISA after
injection with infected cell cultures. Virus titres higher than 10
7
tissue culture
infectious dose at 50% end-point

(TCID
50
) ml
–1
have been obtained after
repeated passages in SHK-1 cells.
Enveloped virus particles of 100–120 nm budding from the cell membrane
can be seen in electron micrographs of ISAV-infected cell cultures (Dannevig et
al., 1995). Granules are observed in the particles. The morphology of in vitro
Fig. 4.4. Transmission electron micrographs of ISAV in vivo. (a) Virus particles budding from
cardiac endothelial cells of ISA-infected Atlantic salmon. (b) Higher magnification of virus. Note
the presence of an envelope and granules (× 55,000). (Photo: Are Nylund, Department of Fisheries
and Marine Biology, University of Bergen, Norway.)
(a) (b)
154
B.H. Dannevig and K.E. Thorud
replicated virus is similar with that observed in vivo (Hovland et al., 1994).
Electron micrographs of negatively stained purified virus preparations show that
the ISAV is covered with surface projections about 10 nm in length (Dannevig
et al., 1995; Falk et al., 1997) (Fig. 4.5).
Recent studies have shown that ISAV is a ribonucleic acid (RNA) virus with
a single-stranded genome, consisting of eight segments, and preliminary results
suggest a negative polarity (Mjaaland et al., 1997). Peak buoyant density after
isopyknic centrifugation in sucrose gradients is approximately 1.18 g ml
–1
.
Infectious salmon anaemia virus is sensitive to high temperature and acid, as
infectivity of virus supernatants is lost after heating at 56°C for 5 min or
exposure to acid medium (pH 4 or lower) for 30 min. Furthermore, ISAV
haemagglutinates erythrocytes from several fish species, including Atlantic
salmon, and receptor-destroying activity has been demonstrated (Falk et al.,
1997). Thus, the morphological, functional and genomic properties of ISAV are
consistent with those of a member of the Orthomyxoviridae (Falk et al., 1997;
Mjaaland et al., 1997).
Fig. 4.5. Negative-stained micrograph of ISAV purified from medium of infected cell cultures
(bar=100 nm). (Photo: Ellen Namork, National Institute of Public Health, Oslo, Norway.)
155 Other Viral Diseases and Agents of Cold-water Fish
Diagnostic methods
Diagnosis of ISA is based on typical pathological changes, including macro-
scopic signs and histological and haematological findings. A dark liver is a
necessary finding, while other macroscopic signs are considered to support the
diagnosis. The presence of multifocal, haemorrhagic liver necrosis with a ‘zonal’
appearance and haematocrit values below 10% confirms the diagnosis. Leuco-
penia, erythrocytic degeneration and erythroblastosis are supportive findings.
This approach has been satisfactory for diagnosis of ISA in acute outbreaks.
However, it has limitations in the diagnosis of subacute and chronic forms of
ISA and of carriers. Polyclonal and monoclonal antibodies against ISAV have
recently been produced (Falk and Dannevig, 1995a; Falk et al., 1997). These
antibodies have proved useful for the detection of viral antigens in ISAV-
infected SHK-1 cells, using an indirect immunofluorescence antibody technique
(IFAT). One of the monoclonal antibodies reacts with ISAV antigens in frozen
tissue sections of both experimentally and naturally infected Atlantic salmon.
Thus, by applying IFAT on tissue sections, suspected cases can be verified. It
should be noted that a negative reaction does not imply that ISAV is not present,
since strain variation of ISAV may exist. However, in cases where the diagnosis
cannot be confirmed by the required pathological changes, a positive IFAT will
confirm suspected cases.
Parts of the genome have been sequenced and a reverse-transcriptase
polymerase chain reaction (PCR) method has been established (Mjaaland et al.,
1997). Evaluation of the different methods for detection of ISAV in experi-
mentally and naturally infected fish is in progress.
Control and treatment
Transmission of the disease
Horizontal transmission has been demonstrated in cohabitation experiments,
indicating that water-borne transmission is important for the spread of ISA
(Thorud and Djupvik, 1988; Thorud, 1991). The virus may be shed into the
water by various routes, such as skin, mucus, faeces and urine (Totland et al.,
1996). The authors suggest that the most likely route of virus entry is through the
gills and skin injuries. Transmission by coprophagy has also been proposed
(Nylund et al., 1994a).
No significant loss in virus titre was observed after incubation of virus
supernatants for 14 days at 4°C and 10 days at 15°C (K. Falk, Oslo, Norway,
1996, personal communication), but the survival time of ISAV in full sea water
seems shorter. Transmission experiments have shown that infectivity of tissue
preparations is retained for at least 20 h in sea water at 6°C (Nylund et al.,
1994a). Comparable experiments have shown that infectivity is retained for at
least 48 h at 0°C, 24 h at 10°C and 12 h at 15°C (Y. Torgersen, Oslo, Norway,
1996, personal communication).
The spread of ISA between fish farms mainly occurs by the purchase of
infected smolts, transport of infected adult fish between fish farms and release of
156
B.H. Dannevig and K.E. Thorud
untreated water into the sea from nearby processing plants (Vågsholm et al.,
1994). Furthermore, the sea louse (Lepeophtheirus salmonis) has been suggested
as a possible vector for ISAV, as Atlantic salmon exposed to sea lice and
removed from ISA-infected fish develop ISA (Nylund et al., 1993, 1994a).
Whether this route of transmission represents a passive transfer of virus or is due
to active replication of virus in the sea lice has not yet been clarified. There is no
evidence for vertical transmission of ISAV.
As ISAV replicates in sea trout, it has been discussed whether this species
could be a marine reservoir of the virus. In Norway, sea trout are abundant in
fjords and coastal areas, i.e. in the vicinity of the fish farms, while the feeding
areas of wild Atlantic salmon are distant from the coast in the North Atlantic.
Infectious salmon anaemia virus seems to persist in infected fish for at least 7
months (Nylund et al., 1995b). The possibility therefore exists that outbreaks of
ISA in farmed Atlantic salmon may cause a persistent ISAV infection in sea
trout. The migration behaviour of sea trout may explain the appearance of
disease in fish farms located far from ISA-affected farms.
Treatment and protection
There is no known treatment for ISA. Following the high incidence, with more
than 50 outbreaks of ISA per year, in the late 1980s, government restrictions on
the management of farmed Atlantic salmon within regions were introduced in
1990/91. The transfer of live fish between regions and the use of untreated sea
water in hatcheries were prohibited. Also, water from processing plants was
treated. A significant reduction in the number of new outbreaks has been
reported since these regulations have been in force. Only a few, sporadic
outbreaks of ISA occurred in 1995 and 1996, and the disease is not regarded as
endemic at this time.
Pathogenesis and immunity
The late stages of ISA are characterized by severe anaemia. During early
investigations, the development of pathological changes in naturally infected
fish was related to a low haematocrit. In fish with haematocrit values below 25,
the plasma levels of intracellular enzymes, such as alanine aminotransferase,
lactate dehydrogenase and aspartate aminotransferase, increase considerably.
These changes indicate liver damage. The osmoregulatory ability of diseased
fish is gradually impaired, as indicated by high plasma osmolality and a high
concentration of plasma electrolytes in individuals with a haematocrit between
30 and 20. The concentration of other plasma components, such as total protein,
albumin, triacylglycerol and cholesterol, decreases in fish with similar
haematocrit values. A further decrease in haematocrit (below 20) is not
accompanied by additional changes in these parameters (Thorud, 1991).
Leucopenia develops concomitantly with the anaemia, while the proportion of
immature erythrocytes increases.
The most prominent lesions of the internal organs are in the liver, where
congestion, enlargement and darkening appear at early infection. In fish from a
157 Other Viral Diseases and Agents of Cold-water Fish
natural outbreak, gross liver lesions were demonstrated in 80% of individuals
with a haematocrit between 26 and 30%, while no lesions were observed in the
spleens from the same fish (Evensen et al., 1991). Splenic lesions, characterized
by congestion and increased erythrophagocytosis, develop in individuals with
haematocrit values of 25% or lower. The severity of organ lesions increases and
peritoneal petechiae and ascites appear more frequently with decreasing
haematocrit. Histologically, congestion of the liver is seen in early infections,
with dilatation of sinusoids, degeneration and necrotic hepatocytes in later
stages (haematocrit <10%). Ultimately, the lesions develop into coalescent
haemorrhagic necroses. According to Evensen et al. (1991), hypoxia due to
anaemia cannot fully explain the development of liver lesions.
Transmission experiments have been crucial in providing insights into the
pathogenesis of ISA. Following an intraperitoneal injection of ISA-infective
tissue homogenate, mortality is observed 2–3 weeks postinfection (p.i.) and may
reach 50–100% within the subsequent week. Mortality of cohabitants may
appear, with a delay of 10–12 days compared with mortality in the intra-
peritoneally infected fish.
Electron-microscopic studies of experimentally infected Atlantic salmon
show that pathological changes in the liver first occur in the perisinusoidal
macrophages (PSM) (Speilberg et al., 1995). At 4 days p.i., which is approx-
imately 2 weeks before haematocrit values decrease, cytoplasmic vacuolization
of PSM is obvious. The vacuolization progresses with time and leads to
enlargement of these cells. At this stage, degeneration of the sinusoidal
endothelium also occurs. These changes may impede the sinusoidal blood flow,
with resulting congestion, which further develops to ischaemic hepatocellular
necrosis. Degeneration of the sinusoidal endothelium may be ascribed to the
enlargement of PSM or, more probably, to replication of ISAV, since liver
endothelial cells are also target cells for the virus (Nylund et al., 1995c). There is
no evidence that ISAV replicates in hepatocytes.
Histochemical changes in the spleen and kidney also appear early in an ISA
infection. Erythrophagocytosis in sinusoidal macrophages of the red pulp in the
spleen and clustering of immunoglobulin positive cells in the head kidney are
early events (Falk et al., 1995).
The kidney is one of the first internal organ to become infected. Infectious
salmon anaemia infectivity of kidney tissue homogenate and of isolated head-
kidney leucocytes is detectable 7 days after intraperitoneal injection (Dannevig
et al., 1994). Infectivity of plasma appears later and peaks 2–3 weeks p.i. At this
time, ISA infectivity is present in most internal organs, which is consistent with
endothelial cells as the major site of ISAV replication (Hovland et al., 1994;
Nylund et al., 1995c).
Field experience indicates protective immunity in Atlantic salmon that have
survived a natural outbreak of ISA. This has been verified experimentally.
Atlantic salmon presmolts that have survived an experimental ISA infection are
significantly less susceptible to a secondary infection (Falk and Dannevig,
1995b). An anti-ISAV response is also seen in brown trout after repeated
challenges (Nylund et al., 1994b). Humoral factors seem to be involved in the
immune response against ISA, as passive immunization with ISA-convalescent
158
B.H. Dannevig and K.E. Thorud
antiserum reduces ISA mortality after infection (Falk and Dannevig, 1995b).
This is partly ascribed to the neutralizing activity of the convalescent antiserum,
with the immunoglobulin fraction beeing the active component.
Early changes in leucocyte function of ISA-infected fish may indicate the
initiation of an immune response. The response of head-kidney leucocytes to the
T-cell mitogen phytohaemagglutinin (PHA) increases 1 week p.i., while it is
nearly abolished in the later stages of ISA (Dannevig et al., 1993). This variation
in response to PHA may indicate a virus-induced activation of lymphoid cells in
the head kidney, which results in the inhibition of leucocyte function, perhaps
because ISAV infects these cells. The ISA-induced changes in spleen and head
kidney may also result from an activation of the immune system. Later in the
course of infection, cells associated with the ellipsoids of the spleen show
reactivity for immunoglobulin and complement factor C3, indicating immune
complex trapping (Falk et al., 1995).
The role of cell-mediated immunity in the anti-ISA response is not clear.
Little is known about cell-mediated immunity in teleosts, and the elucidation of
antiviral cytotoxic cells in fish is only beginning (Hogan et al., 1996).
Topics for further study
Development of reliable specific diagnostic methods is one of the major areas
for further research on ISA. Diagnosis of acutely diseased fish is done with IFAT
on frozen tissue sections. However, survivors may harbour the virus for long
periods, as has been shown for sea trout. An antigen detection method for
identification of subacute cases and virus carriers should be given high priority.
Strain variations of ISAV have to be investigated before fish populations can be
screened for ISAV.
PANCREAS DISEASE
Introduction
Pancreas disease (PD) is a disease of farmed Atlantic salmon (S. salar L.)
characterized by extensive necrosis of the exocrine pancreas (Munro et al.,
1984; McVicar, 1987). A moderate mortality is usually associated with
outbreaks of PD, but this may vary between geographical regions. In Ireland,
mortality rates from 10 to 50% have been reported (Menzies et al., 1996).
Survivors often fail to grow and may die several months after the primary
outbreak (Munro et al., 1984). Various secondary pathological conditions
associated with the disease may contribute to mortality.
A nutritional aetiology was initially considered, as vitamin E and selenium
deficiencies were present in diseased fish (Ferguson et al., 1986a), but the course
and pattern of spread of the disease indicated an infectious aetiology (McVicar,
1987). This was later confirmed in transmission experiments (McVicar, 1990;
Raynard and Houghton, 1993). A viral aetiology was suggested, as infective
159 Other Viral Diseases and Agents of Cold-water Fish
material was inactivated after treatment with chloroform (Murphy et al., 1995)
and the spread of the infectious agent through the circulatory system was typical
of a viraemia (Houghton, 1995). No pathogenic bacteria were isolated from PD-
affected fish. Although numerous attempts to propagate the causal agent in cell
culture had failed, Nelson and coworkers recently isolated a toga-like virus from
Atlantic salmon with clinical PD (Nelson et al., 1995). Reinjection of the virus
into experimental fish caused development of the disease and they proposed that
the virus be termed salmon pancreas disease virus (SPDV).
The disease and agent
Species of fish affected and geographical distribution of the
organism and disease
Pancreas disease typically infects farmed Atlantic salmon during their first year
in sea water, with the peak prevalence occurring from late July to early
September (McVicar, 1987). In France, the disease also affects sea-water-reared
brown trout (S. trutta), but the clinical signs are not as clear-cut as in Atlantic
salmon (Boucher et al., 1995). Recently, the outbreak of a disease with PD-like
clinical signs and pathology in sea-water-reared rainbow trout (O. mykiss) has
been observed in Norway (A.B. Olsen, Bergen, Norway, 1996, personal
communication). The susceptibility of sea-water-reared rainbow trout, brown
trout and Atlantic salmon has been compared. Following experimental
transmission, rainbow trout and brown trout develop pathological changes
typical for PD. However, the lesions are less severe than those observed in
Atlantic salmon, and brown trout are less susceptible than rainbow trout
(Boucher et al., 1995). Experimental induction of the disease in Atlantic salmon
parr in fresh water has also been achieved (McLoughlin et al., 1995).
An infectious disease of fresh-water-reared rainbow trout, known as
sleeping disease, is characterized by lesions similar to those in PD-infected fish.
A similar or identical aetiology for PD and sleeping disease has been suggested,
since survivors of PD-infected fish are protected against sleeping disease and
vice versa (Boucher and Baudin Laurencin, 1996).
Pancreas disease was first recorded in 1976 in Scotland (Munro et al.,
1984). Later, a similar disease was reported to affect fish farms in North America
(Kent and Elston, 1987), Norway (Poppe et al., 1989), Ireland (Murphy et al.,
1992) and France and Spain (Raynard et al., 1992).
General signs of the disease
Early clinical signs of PD are sluggish swimming activity and a sudden decrease
in feeding. Infected fish become dark and may rapidly lose weight (Fig. 4.6). The
most prominent post-mortem findings are petechial haemorrhages in periacinar
fat, absence of food in the gut and reduced body fat. The most typical and
consistent microscopic lesion is exocrine pancreatic necrosis, which results in an
almost complete loss of acinar tissue (Munro et al., 1984; McVicar, 1987) (Fig.
4.7). Fibrotic tissue may replace the degenerated acinar tissue, but in recovering
fish, a regeneration of exocrine tissue seems to take place (Munro et al., 1984).
160
B.H. Dannevig and K.E. Thorud
Fig. 4.6. Normal (upper) and PD-affected (lower) Atlantic salmon of the same age. (Photo:
Trygve Poppe, Department of Morphology, Genetics and Aquatic Biology, Norwegian College of
Veterinary Medicine, Oslo, Norway.)
Fig. 4.7. Pancreas from PD-affected fish showing extensive acinar necrosis to the left and less
affected tissue to the right. (Photo: Trygve Poppe, Department of Morphology, Genetics and
Aquatic Biology, Norwegian College of Veterinary Medicine, Oslo, Norway.)
161 Other Viral Diseases and Agents of Cold-water Fish
Severe cardiac as well as skeletal myopathy is often associated with high PD
mortality (Ferguson et al., 1986a, b; McVicar, 1987; McCoy et al., 1994; Rodger
et al., 1994). The major cardiac lesions are characterized by necrosis of the
ventricular myocardium, which affects both spongy and compact layers. The
striation of the muscle may disappear, with an increased eosinophilia. The atrial
myocardium seems to be less affected (Ferguson et al., 1986b). Similar lesions
are found in the skeletal muscle, usually affecting the red fibres along the lateral
line. However, these muscle lesions are not found in all cases (McVicar, 1987;
Rodger et al., 1994) and may represent secondary effects – for example, as a
response to changes in the nutritional status of the fish due to reduced feeding.
Furthermore, vitamin E deficiency has been observed in PD-diseased fish
(Ferguson et al., 1986a; Raynard et al., 1991) and it was suggested that this
deficiency could induce myopathy. However, other reports do not support this
hypothesis, as pancreatic lesions and myopathy occur simultaneously, even with
high tissue vitamin E concentrations (McCoy et al., 1994). Low plasma levels of
vitamin E also occur in Atlantic salmon suffering from IPN (Taksdal et al.,
1995). Thus, vitamin E deficiency cannot be used to differentiate IPN from PD.
An increased concentration of lipase in plasma occurs in early PD-affected
fish (Grant et al., 1994), while the levels of the digestive enzymes trypsin and
chymotrypsin in pyloric caecum tissue decline significantly during the
development of the disease, in both experimentally and naturally infected
Atlantic salmon (Pringle et al., 1992). The presence of digestive enzymes in the
plasma can be explained by leakage of enzymes from acinar cells, due to the
pathological changes in the tissue.The decrease in enzyme levels in digestive
tissues correlates well with the histological changes and may be used as an early
indicator of reduced feeding and exocrine pancreatic dysfunction.
The incubation period is 6 days at 11–12°C in experimentally infected fish
(McVicar, 1990). The incubation period and transmission rate of PD in fish
farms are not known, but an outbreak in a population of farmed Atlantic salmon
usually lasts a minimum of 5 weeks (Murphy et al., 1992).
Salmon pancreas disease virus – general characteristics and
taxonomic position
Conclusive evidence of a viral aetiology of PD was recently provided by Nelson
et al. (1995), who isolated a virus, SPDV, from naturally infected Atlantic
salmon. Atlantic salmon postsmolts develop PD following intraperitonal
injection of supernatant from infected cell cultures (McLoughlin et al., 1996).
The clinical signs and microscopic lesions in these fish are indistinguishable
from those in naturally infected fish. The successful growth of the virus was
performed in chinook salmon embryo (CHSE-214) cells after cocultivation with
kidney tissues from infected fish. No CPE was seen in the primary cell culture,
but distinct CPE appeared after passages to new cultures.
The SPDV is a spherical, enveloped virus with a diameter of approximately
65 nm (Nelson et al., 1995). Although the viral nucleic acid has not yet been
identified, Nelson et al. (1995) suggested that SPDV is an RNA virus, due to its
resistance to inhibition by 5-bromo-2′-deoxyuridine. Haemagglutinating
activity of SPDV has not been demonstrated. The buoyant density of SPDV in
162
B.H. Dannevig and K.E. Thorud
caesium chloride (CsCl) gradients is approximately 1.20 g ml
–1
. Based on these
characteristics and on virus morphology, Nelson et al. (1995) suggested that
SPDV is a member of the Togaviridae.
Diagnostic methods
The diagnosis of PD is based on clinical signs and the characteristic pancreatic
lesions (Fig. 4.7). The lesions may be difficult to distinguish from those in fish
suffering from IPN, but the absence of IPNV in pancreatic tissue would support
a positive PD diagnosis in acute cases. Furthermore, in PD-affected fish, there is
a more extensive loss of exocrine pancreatic tissue than in IPN-affected fish.
Monitoring plasma lipase levels as well as other pancreatic enzymes may
represent a non-lethal method that could support a diagnosis of PD (Grant et al.,
1994). No method is currently available to identify SPDV in cells or tissue.
However, since SPDV can be grown in cell culture, detection of neutralizing
antibodies in serum is possible (McLoughlin et al., 1996). The presence of such
antibodies would be highly indicative of PD.
Control and treatment
Transmission of the disease
Field data have provided evidence for horizontal water-borne transmission of
PD (McVicar, 1987) and this has been demonstrated experimentally (Raynard
and Houghton, 1993). The disease seems to persist in farms where PD has been
introduced, as annual outbreaks have frequently been observed (McVicar, 1987).
There is no evidence for vertical transmission and the natural reservoir of the
virus is unknown.
Treatment and protection
Pancreas disease cannot be treated by medication, because of its viral aetiology.
Fish farmers usually stop feeding affected fish as soon as the disease is
recognized. Reduced feed intake appears to reduce the severity of an outbreak,
which supports the finding that feeding is necessary for disease development in
transmission experiments. No vaccine has been developed, but, since the causal
virus can be propagated in cell culture, the production of a protective vaccine
seems feasible. No benefit was detected in PD-infected Atlantic salmon fed
mammalian pancreatic enzymes (Rodger et al., 1995). Protection currently
relies on zoosanitary precautions and restrictions on the purchase of live fish.
Pathogenesis and immunity
Pancreas disease affects many organs and the interpretation of the histological
changes can often be complicated. Studies of pathogenesis have been difficult
since no specific methods for the detection of the viral agent are available. The
163 Other Viral Diseases and Agents of Cold-water Fish
studies that have been performed have been based on histological methods and
transmission experiments.
According to Munro et al. (1984), the pathogenesis of PD in Atlantic salmon
can be divided into three phases with respect to histological changes in the
pancreas: the preacute, the acute with pancreatic necrosis and the postacute
phase. The preacute phase, which occurs within 4–6 weeks after sea transfer, is
characterized by a predominance of pancreatic acinar type B cells, which have a
densely basophilic cytoplasm and contain few zymogen granules. Vacuolation of
the acinar cells can also be observed. There are no changes in haematocrit
values, erythrocyte morphology or serum proteins in the preacute phase.
The acute phase occurs approximately 3 months after sea transfer, but may
also be seen later. This phase is characterized as described above in ‘General
signs of the disease’. The most prominent lesion is the massive loss of pancreatic
acinar cells. Erythrocyte morphology and haematocrit values are normal, but
leucocyte hyperplasia in the head kidney and a drop in serum protein
concentration can be observed. The disease seems to affect nearly all fish in a
population over a 5-week period after the initial outbreak.
In the postacute phase, a large proportion of fish from affected populations
show normal or recovering pancreas, while, in a minor proportion of the fish,
fibrotic tissue has replaced the exocrine pancreatic tissue. Fish that show no
recovery of pancreatic tissue may gain weight after some months, but most of
them are unlikely to recover and are usually culled.
The development of myopathy has been studied in Atlantic salmon by
monitoring pathological changes over the 6 months following sea transfer
(Murphy et al., 1992). Cardiomyopathy is observed in fish with both normal and
affected pancreas before the appearance of clinical signs, but the prevalence of
heart lesions is highest among fish with PD. Skeletal myopathy of white fibres
occurs in both normal and PD-infected fish, whereas red fibre changes appear
only in fish in the chronic stage or in the recovery stage of the disease (Murphy
et al., 1992).
The pathogenesis of PD in experimentally infected freshwater Atlantic
salmon parr is similar to that in naturally infected sea-water postsmolts
(McLoughlin et al., 1995). Parr that received an intraperitoneal injection of PD-
infective tissue material developed pancreatic and cardiac lesions that were
indistinguishable from those in naturally infected fish. Cardiac lesions were also
seen in experimentally infected fish.
Blood plasma of Atlantic salmon is infectious from day 1 after intra-
peritoneal injection of PD-infective kidney homogenate (Houghton, 1995).
Leucocytes of blood and spleen and kidney tissue become infective from
approximately day 3 p.i. and the appearance of infectivity in cells and organs is
temperature-dependent. The infectivity remains high until pancreatic lesions are
observed, thereafter the plasma, leucocytes and kidney are not infective.
Evidence for a protective immune response against PD has been reported, in
both naturally (Murphy et al., 1995) and experimentally (Houghton, 1994)
infected farmed Atlantic salmon. In the latter case, PD-infected fish acquire a
resistance to reinfection that lasts at least 9 months after the primary infection.
Humoral factors seem to be involved in the immune response, as passive
164
B.H. Dannevig and K.E. Thorud
immunization with serum from recovered fish protects parr against challenge
with PD-infective material (Murphy et al., 1995). Neutralizing activity in serum
from experimentally infected fish collected at day 10 p.i. has been demonstrated
when tested against SPDV in cell culture (McLoughlin et al., 1996). Antisera
collected from experimentally infected Atlantic salmon up to 15 weeks p.i.
neutralizes PD-infective material when tested in transmission experiments
(Houghton and Ellis, 1996). Neutralizing activity is found in sera from fish
infected both by injection and by cohabitation. Also, passive immunization of
fish with antisera collected 8 weeks p.i. results in 100% protection against the
disease. These results indicate that protection against PD may be mediated, at
least in part, by neutralizing serum factors. It remains to be shown that the
neutralizing activity is associated with serum immunoglobulins.
Topics for further study
Diagnostic methods that can distinguish PD from IPN should be given high
priority, as well as methods that can be used for the detection of virus carrier fish.
Since SPDV can be grown in cell culture, antibody-based diagnostic methods
should be a reality within the near future. Studies on immune mechanisms
involved in PD and the development of a protective vaccine are relevant topics
for further research.
VIRAL ERYTHROCYTIC NECROSIS
Introduction
Viral erythrocytic necrosis (VEN) is a disease of marine fish characterized by
erythrocytic degeneration and the presence of cytoplasmic inclusion bodies in
circulating erythrocytes (Evelyn and Traxler, 1978; Reno et al., 1978). A similar
condition has been reported in amphibians and reptiles. For a long time, the
inclusions were suspected to be caused by blood parasites, but evidence of a viral
aetiology was first reported in reptiles (Stehbens and Johnston, 1966). In the first
report on erythrocytic inclusion bodies in fish, the condition was termed ‘piscine
erythrocytic necrosis (PEN)’ (Laird and Bullock, 1969). The viral nature of the
disease in fish was later demonstrated (Appy et al., 1976; Walker and Sherburne,
1977). The causal virus has been referred to as erythrocyte necrosis virus (ENV)
(Haney et al., 1992).
The disease has now been reported in more than 20 species of marine and
anadromous fish, in both wild and cultured populations (Smail, 1982). The
disease is not usually associated with high mortality, but it has been reported to
occur epizootically in Pacific herring (Meyers et al., 1986). Transmission
experiments have verified the infectious nature of the disease. Blood smears
from affected fish reveal a single inclusion body in the cytoplasm of erythro-
cytes, visible by light microscopy. A virus belonging to the icosahedral
cytoplasmic deoxyribovirus (ICDV) group and presumptively classified as an
165 Other Viral Diseases and Agents of Cold-water Fish
iridovirus has been associated with the inclusion bodies (Appy et al., 1976; Reno
et al., 1978).
Viral erythrocytic necrosis is not considered to be a severe or economically
important fish disease. The current research on VEN is low compared with that
on other viral diseases in fish and most of the information was published in the
late 1970s and 1980s.
The disease and agent
Species of fish affected and geographical distribution of the
organism and disease
A wide variety of marine fish species from the northern parts of the Atlantic and
Pacific Oceans are affected. These include Atlantic cod (Laird and Bullock,
1969; Appy et al., 1976; Reno and Nicholson, 1981), blenny (Johnston and
Davies, 1973), Atlantic (Reno et al., 1978) and Pacific (Meyers et al., 1986)
herring and the Pacific salmonids, chum, pink, coho, chinook salmon and
steelhead trout (Evelyn and Traxler, 1978; Rohovec and Amandi, 1981; Bell and
Traxler, 1985). Viral erythrocytic necrosis has also been observed in the southern
Pacific Ocean, namely in coho salmon in Chile (Reyes and Campalans, 1987). It
has been reported in cultured eel in Taiwan (Chen et al., 1985), and is suspected
to affect several marine species in coastal waters off Portugal, including Madeira
(Eiras, 1984; Eiras et al., 1996). Thus, VEN has a wide geographical distribution
and is not confined to special fish groups occupying particular ecological niches.
Experimental induction of VEN in Atlantic cod (Reno et al., 1986) and in
several Pacific salmonids – Atlantic salmon, rainbow trout, brown trout and
brook trout (MacMillan and Mulcahy, 1979) – has been described. The trans-
mission experiments have confirmed field observations that the susceptibility to
VEN in salmonids is age- and species-dependent. For example, juveniles <1 g
are more susceptible than larger fish. No signs of VEN are observed in large
coho salmon and rainbow trout 6 months after infection. Furthermore, in
experiments with juveniles, erythrocytic inclusion bodies characteristic of VEN
appear earlier in chum salmon, brown trout and brook trout (2 days p.i.) than in
pink, coho, sockeye and chinook salmon and rainbow trout (5–7 days)
(MacMillan and Mulcahy, 1979).
General signs of the disease
Viral erythrocytic necrosis appears to be a chronic disease and, in most cases, no
external signs occur. Post-mortem findings ascribed to VEN are pale gills and
pale internal organs. Histologically, there may be increased haemopoietic
activity in the kidney of naturally infected chum salmon (Evelyn and Traxler,
1978).
The characteristic histological finding in VEN-affected fish is rounded
cytoplasmic inclusions (0.8–4 µm) in circulating erythrocytes (Laird and
Bullock, 1969; Evelyn and Traxler, 1978). A single inclusion body is usually
seen in each cell and it appears pink- or magenta-coloured in Giemsa-stained
blood smears (Fig. 4.8). The proportion of erythrocytes with inclusion bodies in
166
B.H. Dannevig and K.E. Thorud
naturally infected fish varies from about 35% in cod (Reno et al., 1985) to 60–
80% in herring (Reno et al., 1985; Meyers et al., 1986).
The cytoplasmic inclusions seen in Giemsa-stained blood smears of Pacific
salmon are assumed to be virion aggregates (see below) and viral precursor
material (viroplasm) (Appy et al., 1976; Evelyn and Traxler, 1978). In Atlantic
cod, the inclusions are frequently surrounded by punctiform bodies less than 0.5
µm in diameter. Non-membrane bound electron-dense viroplasms are often
associated with virions (Nicholson and Reno, 1981). Two types of inclusions are
seen in electron micrographs of infected erythrocytes from Pacific herring (Reno
et al., 1978). One (measuring 1.5 µm) is non-membrane-bound and appears to be
associated with virus, while the other type (diameter from 0.5 to 3 µm) is
membrane-bound but not virus-associated.
Other typical findings are degenerative changes of the erythrocytes. In
Pacific salmon, nuclear displacement and cytoplasmic vacuolation of the
erythrocytes may occur (Evelyn and Traxler, 1978). In cod and herring, the
nuclear changes also include chromatin margination, karyohexis, pyknosis and
karyolysis (Nicholson and Reno, 1981).
The infection results in moderate anaemia in cod and herring (Reno et al.,
1985), while a more severe anaemia (haematocrit values < 5) develops in
experimentally infected Pacific salmonids (MacMillan and Mulcahy, 1979).
Haemolytic anaemia, with haemosiderosis and erythroblastosis, has been
decribed in moribund Pacific herring (Meyers et al., 1986). Erythroblastosis can
also be observed in experimentally infected Atlantic cod (Reno et al., 1986) and
chum salmon (MacMillan and Mulcahy, 1979).
Fig. 4.8. Blood smears from VEN-infected Atlantic herring showing single, rounded
cytoplasmic inclusions in erythrocytes.
167 Other Viral Diseases and Agents of Cold-water Fish
Viral erythrocytic necrosis mortality is usually low, but may increase in
association with vibriosis or bacterial kidney disease (Evelyn and Traxler, 1978).
High mortality associated with epizootics of VEN has only been reported in
Pacific herring (Meyers et al., 1986). Experimentally infected chum salmon had
increased mortality, due to vibriosis, decreased tolerance to depletion of oxygen
and reduced osmoregulatory ability in sea water (MacMillan et al., 1980).
However, no increased mortality was observed in experimentally infected
Atlantic cod, although a reduced resistance to stress was reported (Nicholson
and Reno, 1981).
The prevalence of VEN varies from 1% to 90%, depending on species and
geographical location of fish. The prevalence was 1.5% and 3% in North
Atlantic cod and North Pacific herring, respectively (Reno and Nicholson, 1981;
Traxler and Bell, 1988). In another study, up to 60% of Pacific herring were
infected, and young fish had a higher prevalence than adult fish within a region
(MacMillan and Mulcahy, 1979). During a 5-month holding period, the
prevalence of VEN in Pacific herring increased from 3% to 70% (Traxler and
Bell, 1988). More than 80% of a chum and pink salmon population may be
infected (Evelyn and Traxler, 1978).
Erythrocyte necrosis virus – general characteristics and
taxonomic position
Attempts to cultivate ENV in established fish cell lines have not been succesful.
Incorporation of radiolabelled thymidine into erythrocytes from infected cod has
been shown to be higher than in cells from uninfected fish, which could
indicate that the virus replicates in erythrocytes (Reno and Nicholson, 1980).
Furthermore, the inclusions bodies have reactivity with deoxyribonucleic acid
(DNA)-specific stains, such as bisbenzamide (Reno and Nicholson, 1980) and
Feulgen stain (Evelyn and Traxler, 1978; Reno et al., 1978), indicating that the
virus genome may consists of DNA.
Electron micrographs of erythrocytes from VEN-infected fish show
hexagonal or pentagonal non-enveloped virus particles, with an electron-dense
core in the cytoplasm (Fig. 4.9). The diameter of the virus particles varies
between fish species, from 145 nm in herring (Reno et al., 1978) to 180 nm in
salmonids (Evelyn and Traxler, 1978) and to 300 nm in cod (Reno and
Nicholson, 1981). From its ultrastructural appearance and the presence of DNA,
ENV has tentative been classified as an iridovirus (Appy et al., 1976; Reno et al.,
1978; Reno and Nicholson, 1981).
Diagnostic methods
The presence of inclusion bodies in the erythrocytes of Giemsa-stained blood
smears would indicate VEN infection. The eosinophilic staining and the
morphology of the inclusions, often single in each cell, together with erythrocyte
degeneration, are considered as consistent findings. However, confirmation
should be made using electron microscopy to verify the presence of hexagonal
virus particles in the cytoplasm.
168
B.H. Dannevig and K.E. Thorud
Control and treatment
Transmission of the disease
Viral erythrocyte necrosis has only been recognized in marine fish and Pacific
salmon reared in sea water. The disease can be experimentally transmitted from
Pacific herring to chum salmon by injection. Water-borne transmission has been
demonstrated in chum salmon and brook trout (MacMillan and Mulcahy, 1979).
Therefore, a marine reservoir for ENV has been suggested. The high prevalence
of VEN in the progeny of infected adults may indicate vertical transmission
(Schieve et al., 1988).
Treatment and protection
There is no treatment against the infection. The demonstration of increased
susceptibility to stress and secondary infections in VEN-affected fish makes it
advisable to avoid stressors. By heating to 60°C for 15 min, VEN-infective
material is inactivated (Evelyn and Traxler, 1978).
Pathogenesis and immunity
The progression of VEN infection has been studied in experimentally infected
fish, but only in chum salmon and Atlantic cod.
In chum salmon juveniles (0.5 g), circulating mature erythrocytes are
infected within 2 days after inoculation with VEN-infective, sonicated, whole
Fig. 4.9. Transmission electron micrograph of VEN-infected erythrocytes of Atlantic herring
showing cytoplasmic hexagonal virions.
169 Other Viral Diseases and Agents of Cold-water Fish
blood (MacMillan et al., 1989). Single, acidophilic cytoplasmic inclusions,
often adjacent to the nucleus, are present in 40–60% of the erythrocytes after 2
days. The percentage of infected mature erythrocytes increases with time,
reaching 80% after 2 weeks and 90% after 1 month. Concomitantly, haematocrit
values decrease to approximately 4%. While the number and proportion of
mature erythrocytes decrease with time, the proportion of circulating
erythroblasts increases. Inclusion bodies are present in 80% of the erythroblasts
1 month p.i. The virus infects the erythroblasts in the kidney before they are
released to the circulation, since cytoplasmic inclusions are seen in all stages of
morphologically identifiable erythroblasts in the pronephros 1 month p.i.
(MacMillan et al., 1989). The occurrence of circulating multinucleate, giant
erythroblasts, abnormal erythroid cell maturation and phagocytosis of abnormal
erythroblasts by macrophages indicates both virally mediated erythroid cell
destruction and ineffective erythropoiesis. Increased coagulation time also
occurs in VEN-affected chum salmon fry.
In larger chum salmon (75–150 g), an experimentally induced VEN
infection also results in haematological changes, such as reduced number of
erythrocytes, increased proportion of immature erythrocytes and extensive
leuocytosis (Haney et al., 1992). The course of haematological changes
indicates that the peak of infection occurs between 3 and 4 weeks p.i., while
recovery starts after 5 weeks. In the same study, it is concluded that a VEN
infection does not cause development of a stress response. The mean cortisol
level in infected fish was only slightly elevated during a 5-week period, and no
increase in plasma glucose and lactate or reduction in liver glycogen could be
observed. Plasma proteins and osmolality were unchanged (Haney et al., 1992).
In contrast to chum salmon, no inclusion bodies were seen in mature
erythrocytes of Atlantic cod early in experimental VEN infection (Reno et al.,
1986). Circulating immature erythrocytes are the first cells to become infected in
cod, and cytoplasmic inclusions appear approximately 40 days p.i. The
proportion of infected immature erythrocytes increases and may reach 100% 3
months after infection. Subsequently, the infection in immature eryothrocytes
decreases while infected erythrocytes increase to approximately 40–60%. A
significant erythroblastosis occurs, but, in the cod, there is no evidence for
infection of newly generated erythrocytes (Reno et al., 1986). The infection
seems to follow the same temporal pattern in naturally and experimentally
infected cod.
No changes of plasma electrolytes occur in VEN-infected cod, suggesting
that the infection has no impact on the osmoregulatory capacity (Reno et al.,
1985). Erythrocytes from infected fish lyse more rapidly that those from
uninfected fish when kept in isotonic media (Nicholson and Reno, 1981), but an
increase in fragility in hypotonic media has not been demonstrated (Reno and
Nicholson, 1980; Haney et al., 1992).
Immunity to VEN has not been described.
170
B.H. Dannevig and K.E. Thorud
Topics for further study
Erythrocyte necrosis virus is one of several intraerythrocytic viruses found in
mature erythrocytes of salmonids and marine fish (Leek, 1987; Hedrick et al.,
1990; Thorud et al., 1990; Pintó et al., 1991). To distinguish between the
different infections and to screen populations for different viruses, specific
diagnostic tools, such as antibodies or genetic probes, must be available. It is
difficult to evaluate the economic impact of the disease on both wild and farmed
salmonids and on populations of marine fish until specific diagnostic tests have
been developed.
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