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Health
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New Diseases – Phenomena Developing into Problems
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Virulece Mechanisms
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The Fish Immune System
Atle Lillehaug1) and Aud Skrudland2)
1) National Veterinary Institute, 2) Norwegian Food Safety Authority
New Diseases – Phenomena Developing into
Problems
Overall losses in fish farming include mortality, escape, predation and sorting
out of fish at slaughter, in addition to other unregistered demise of fish. These
losses represent substantial reductions in profit. The overall proportion of fish
lost has been quite stable during the last 15 years. In salmonids, it is assumed that
“new” diseases are a major cause of losses, including pancreas disease (PD), heart
and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS),
epiteliocystis / proliferative gill inflammation (PGI), and disease caused by
Parvicapsula pseudobranchiola. In marine fish aquaculture, bacteria such as
Vibrio anguillarum and Aeromonas salmonicida create problems, as does infection with nodavirus. Parasites such as “the Scottish louse” (Caligus elongatus)
and other lice (Caligus curtus, Argulus sp.), as well as gill worms, flagellates, ciliates, microsporidiae, nematodes, and different gyrodactylus species, can turn
problematic for marine fish production.
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Measures for fish health
The state of fish health in Norwegian aquaculture
is considered substantially improved during recent years, compared to the situation in the late
1980s and the early 1990s. One yardstick for
demonstrating an improved health situation is the
consumption of antibacterial drugs in fish. The
number of outbreaks of ISA is another health
parameter used.
Using losses of fish as a measure, statistics from
Kontali Analyse AS illustrate that after a reduction in overall losses during the early 1990s, the
percentages of lost fish have been quite constant
or slightly increasing, as can be seen in Figure 1.
A great proportion of both governmental and private research funding for aquaculture purposes
has been applied in the area of fish health and
closely related topics. However, this effort has
not resulted in a reduction of losses.
There are no statistics describing the different
causes of overall losses. The organisation of the
aquaculture industry in big companies may lead
to more secrecy relating to the causes of losses,
and fear of negative reactions from the market
may contribute to reduced openness in health
problems. Some diseases must be reported in accordance with Norway’s Food Safety Act, and all
outbreaks should be registered.
Phenomena developing into problems
One can assume that a predominant cause of
losses is “new” diseases such as CMS, HSMI,
PD, etc. These disease conditions seem to have
gained increased importance in recent years, developing from phenomena into problems, and
they will be described in more detail in this chapter. In general, most infections causing disease
outbreaks under aquaculture conditions develop
from rare phenomena in wild fish. This has been
the case for major bacterial infections, such as
vibriosis and furunculosis. But “new” diseases
have also been discovered in farmed fish, such as
coldwater vibriosis, IPN and ISA, which have
never been described in wild fish. These infections do, in all probability, also have reservoirs in
wild stocks of fish or other feral organisms. In
aquaculture, a great number of individuals are
Overall losses in Norwegian Atlantic salmon production 1986–2005,
Loss %
expressed as per cent of total number of fish per generation
40 %
35 %
30 %
25 %
20 %
15 %
10 %
5%
0%
86G 87G 88G 89G 90G 91G 92G 93G 94G 95G 96G 97G 98G 99G 00G 01G 02G 03G
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Figure 1. The graph shows the progression of losses calculated per generation of Atlantic salmon from 86G to 03G in the
period 1986–2005. The figures represent percentages lost from the total number of smolt put to sea. 92G designates fish
put to sea in the calendar year 1992, minus the number of fish of that generation slaughtered. Loss includes mortality,
escape, predation, culled fish at slaughter, and any unrecorded losses.
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157
concentrated in a limited volume of water, and
the conditions may be optimal for infectious
agents to flourish and spread. The examples already mentioned have been thoroughly described
earlier, and will not be dealt with in this chapter.
Vibrio infections and furunculosis in salmonids
are effectively controlled by use of vaccines, resulting in limited use of antibiotics in Norwegian
fish farming for many years. However, the number of prescriptions for antibacterials now seems
to be increasing; these are mainly associated with
the cultivation of cod larvae. Hence, in this context, we can observe that new infections, and,
moreover, new variants of well-known disease
agents, may develop from phenomena into problems.
Pancreas disease (PD)
Pancreas disease is a viral disease first described
in Scotland by Munro et al. (1984). Later, PD has
been reported in North America, and the disease
is particularly significant in Ireland. In Norway,
PD has been present as a disease problem since
the second half of the 1980s (Poppe et al. 1989),
with a geographic localisation in the county of
Hordaland and the southern parts of Sogn og
Fjordane. Today, PD is probably one of the diseases inflicting the greatest losses to the salmo-
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Figure 2. Atlantic salmon surviving an outbreak of
pancreas disease may suffer from reduced growth and
have poor quality at slaughter. The two fishes are from the
same population. (Photo: T.T. Poppe)
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nid fish farming industry in the western parts of
Norway. In 2003, the first outbreaks were reported in Troms and Finnmark, probably as a result
of spreading from the western parts of the country with infected smolt. From 2002 to 2005, the
number of farms experiencing outbreaks per year
has increased from 12 to 35. In 2004, 44 outbreaks were reported, and the same year the disease has spread to the counties of Nordland and
Rogaland.
Pancreas disease affects both Atlantic salmon
and rainbow trout in the seawater phase; usually
outbreaks occur half a year or more after the fish
have been transferred to sea. A typical disease
outbreak has chronic characteristics with symptoms including loss of appetite and low mortalities, but a long duration of an outbreak may result
in high overall mortality numbers. Losses of several hundreds of tonnes of fish have been reported in one single farm location. Moreover,
surviving fish may have reduced growth rate, and
fish slaughtered after an outbreak may have reduced meat quality due to muscle damage.
The development of the disease first results in
necroses in pancreas, caused by the virus, which
has given rise to the name of the condition. Next,
damage develops in the heart muscle, and later
even in skeletal muscle. In Norway, pathological
changes have even been seen in the kidney and
spleen late in the course of the disease.
Diagnostic examinations for PD should be done
on fish showing clinical signs of disease (Taksdal
et al., manuscript). Post mortem may demonstrate mucous intestinal contents, caused by anorexia, and signs of circulatory disturbances may
be seen, such as oedema in scale pockets, protruding eyes and liquid contents in the abdominal
cavity. Petechial haemorrhages in the adipose tissue surrounding pancreas may be found. Early in
the course of the disease, typical acute changes in
pancreas may be found in histological sections,
and positive immunohistochemical staining for
the PD virus may be demonstrated. Next, inflammatory cells and tissue damage can be found in
the heart muscle, and later in skeletal muscle.
It has been difficult to isolate the PD virus in cell
culture. However, the methods have been improved. Moreover, the virus can be detected by
use of PCR. Demonstration of specific anti-PD
virus antibodies in fish blood is crucial in the diagnosis of pancreas disease, but such tests can
only be employed some time after the infection
has been established. Antibodies may be detected for a long time after infection, and may be
used to find out if a fish population has been infected with the PD virus.
Causality
Three different but closely related PD viruses
have been described. Salmon pancreas disease
virus (SPDV) is the cause of PD in Atlantic salmon in Scotland and Ireland (Nelson et al. 1995).
Hodneland et al. (2005) describe a Norwegian
strain called salmonid alphavirus (NSAV) causing disease both in Atlantic salmon and rainbow
trout. A third variant of the virus gives rise to
“sleeping disease” in rainbow trout in fresh water
in Europe (Welsh et al. 2000). It has been suggested to unite the three types in a species named
“Salmonid alphavirus”.
Experience indicates that PD contamination is
connected to specific locations, such that outbreaks often reappear when fish are transferred
to sea at a site where the disease has been seen
earlier. Spread of the infection to new areas
seems to be connected to movements of infected
fish. A vaccine against pancreas disease is being
tested.
Current topics for further research
Studies which have contributed to create knowledge on PD, including the development of diagnostic methods, have been carried out in
cooperation between research institutions in
Norway, Ireland and Scotland (Taksdal et al.
manuscript). The cooperation is being continued
in new research projects aimed at elucidating the
geographical distribution of the pertinent virus
types, and identifying risk factors for acquiring
infection and for disease outbreaks, as well as
further characterisation of virus isolates and validation of diagnostic methods.
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Figure 3. Fish suffering from heart and skeletal muscle inflammation. The heart is visibly paler than normal.
(Photo: T. Taksdal)
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Figure 4. The histological section to the left shows normal, red skeletal muscle, and to the right is a section of muscle from
an Atlantic salmon with heart and skeletal muscle inflammation. The muscle is infiltrated with inflammatory cells, and
many of the muscle cells shrink and degenerate. (Photo: R.T. Kongtorp)
Heart and skeletal muscle
inflammation (HSMI)
HSMI was found for the first time during routine
diagnostic investigations of farmed Atlantic
salmon in 1999; the disease has since created increasing problems for the Norwegian aquaculture industry. Most of the outbreaks are occurring
in Central Norway, but HSMI is reported in most
fish farming coastal areas (Kongtorp et al.
2004a). No cases have been confirmed outside
Norway, but outbreaks have been suspected in
Scotland (Ferguson et al. 2005).
HSMI affects Atlantic salmon in seawater. Outbreaks usually occur between five and nine
months after sea transfer of the smolt, and body
weights are usually below 1.5 kg (Kongtorp et al.
2004a). Results from field studies and challenge
trials indicate that close to 100 per cent of the individuals in a farm can be affected, although not
all fish show obvious symptoms (Kongtorp et al.
2004b; Kongtorp et al. 2006). During an HSMI
outbreak, lethargic fish and increased mortality
may be observed for one to six months. Mortality
numbers vary from almost negligible to nearly
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20 per cent in some pens. Stressing of the fish
seems to result in increased mortality (Kongtorp
et al. 2004a). Results of a study of the development of a disease outbreak indicate that HSMI
can act as a sub-clinical inflammation in the heart
several months prior to the onset of increased
mortalities in the farm, and heart injuries may be
observed for a long period after mortality numbers have normalised (Kongtorp et al. 2006).
The diagnosis is based on clinical signs, including increased mortality and increasing numbers
of lethargic individuals, together with observations during autopsy and histopathology. Typical
findings are coagulated blood in the pericardium,
pale and flabby heart, liquid contents in the abdominal cavity, yellowish or spotted liver with a
fibrinous veil covering the capsule, tiny haemorrhages in the liver parenchyma, and a swollen
spleen (Kongtorp et al. 2004a).
Histological characteristics of HSMI include severe inflammation and necroses in the heart and
in the red skeletal muscle. In the heart ventricle,
all layers are involved. Usually, an extensive infiltration of inflammatory cells is observed in the
epicardium, together with inflammation and necroses in both the compact and spongy layers of
the heart muscle. The atrium is usually less affected, and in bulbus arteriosus (a pear-shaped
expansion of the first part of the aorta, made up
of elastic tissue) no inflammation has been observed in connection with HSMI. There are inflammatory reactions and necroses in red
skeletal muscle, but the white muscle is rarely affected. Necroses may be found in the liver, probably as a result of impaired blood circulation.
Some individuals even have oedematous stretches in the spleen, and other organs may show different levels of circulation disturbances
(Kongtorp et al. 2004a).
Causality
Challenge trials have demonstrated that the disease can be transmitted both by intraperitoneal
injection of heart tissue homogenate from sick
fish, as well as by cohabitation with injected fish
(Kongtorp et al. 2004b). A viral cause of HSMI
is suspected. However, isolation and characterisation of an etiological agent has not been published yet.
Due to the contagious characteristics of HSMI, it
is important to prevent further spread of the disease by measures such as disinfection of equipment and fallowing of contaminated sites
between fish generations. Due to the high infection rate in affected populations, together with a
prolonged course of the disease, any transfer of
fish during the seawater phase will involve a risk
of spread of the disease. This is also the case for
the introduction of new populations to sites already inhabited by salmon.
Current topics for further research
A study of the inflammatory reactions in fish
with HSMI is in progress, aiming at an improved
understanding of the development of the disease.
Figure 5. Atlantic salmon dead from cardiomyopathy
syndrome. Coagulated blood can be observed in the
pericardium, causing a so-called heart tamponade.
(Photo: T.T. Poppe)
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Important aims for future research will be to
identify the etiological agent and to develop diagnostic tools in order to improve the sensitivity
and specificity of the diagnosis. It is also crucial
to develop further competence regarding pathogenesis, applying pathological and microbiological studies. An epidemiological project is
necessary in order to map geographic distribution and spread of disease, together with risk factors for acquiring the disease. Such knowledge is
basic for the implementation of control measures
and eradication strategies for HSMI.
Cardiomyopathy syndrome
(CMS)
Cardiomyopathy syndrome is a serious heart
condition, mainly causing disease in farmed Atlantic salmon late in the seawater phase, when
the fish is approaching slaughter 14–18 months
after sea transfer. At this time, the monetary
value of the fish is greatest. CMS was first
observed in Norway in 1985 (Amin & Trasti
1988). Later, it has been diagnosed in Scotland
(Rodger & Turnbull 2000) and on the Faeroe
Islands, and there have been suspected cases in
Canada (Brocklebank & Raverty 2002). Similar
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disease conditions have been observed in wild
salmon as well (Poppe & Seierstad 2003).
In Norway, CMS occurs along the entire coastline, but is most prevalent in Central Norway.
The disease is rare in the counties of Troms and
Finnmark (Østvik & Kjerstad 2003). There has
been an increase in the annual number of diagnosed outbreaks, from 25 in 1998 to 101 in 2002
(Brun et al. 2003), and statistics from diagnostic
work at the National Veterinary Institute demonstrate a lasting high prevalence in recent years.
A clinical outbreak can last from one to six
months, even longer. Typically, an outbreak arises more than one year after sea transfer (mean:
400 days), at a fish body weight of 2–3 kg. According to a study by Østvik & Kjerstad (2003),
mean total mortality during an outbreak was six
per cent. In most cases, only some of the net pens
in a farm were affected by the disease outbreak.
Still, up to 80 per cent of the total losses in affected farms were caused by CMS. The course of the
disease can go in one of two directions: either
sudden onset of mortalities in a population of apparently healthy fish (Skrudland et al. 2002), or
moderately increased mortalities over several
months (Brun et al. 2003). During a prolonged
course of the disease, abnormal swimming
movements and anorexia may be observed prior
to deaths. Due to the high value of each fish dying from CMS, the economic losses may be significant (Brun et al. 2003; Østvik & Kjerstad
2003).
Losses may be limited by avoiding stresses such
as sorting and moving of fish, delousing or other
handling, when CMS is suspected or diagnosed
in the farm. Emergency slaughtering may contribute to the limitation of losses (Skrudland et al.
2002).
Autopsy of fish dying from CMS will typically
reveal haemorrhages in the skin, and protruding
scales and eyeballs. Fluid contents in the abdominal cavity and fibrinous coverings on the liver
surface can be seen. In the heart, the atrium and
the adjacent great veins are usually dilated and
enlarged, and the pericardium may contain blood
coagula (Bruno & Poppe 1996). Sometimes, the
wall of the atrium may have ruptured (Amin &
Trasti 1988). The pathological changes described may also be studied in live, anaesthetised
fish by use of ultrasound examinations. In histological sections, CMS may be characterised by
inflammatory reactions and degenerations in the
spongy layers of the heart muscle (Ferguson et
al. 1990). Studies have documented that the
pathological changes develop over several
months, and in early stages the changes are de-
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Figure 6. The histological section to the left shows a normal gill filament, and to the right is a filament with proliferative
gill inflammation. Thickened lamellae with infiltrations of inflammatory cells can be seen. (Photo: A. Kvellestad)
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marcated. However, gradually the changes become generally distributed, and the inflammatory reactions and necroses in the muscular cells
may weaken the wall of the atrium and lead to a
rupture. Necroses may be observed even in the
liver (Ferguson et al. 1990). In other organs, e.g.
the gills and spleen, congestion of blood can be
seen, probably as a consequence of a general circulatory failure (Amin & Trasti 1988).
Causality
An infection hypothesis of CMS is based on the
findings of inflammatory reactions and necroses
in the heart muscle. Other theories have been
launched, relating to malnutrition, auto immune
reactions or environmental influences. However,
alternative causal relations (other than an infectious agent) have received little attention in research projects.
As early as in the first description of the condition, Amin & Trasti (1988) suggested a viral aetiology, based on the detection of inclusion bodies
in muscle cells adjacent to damaged cells. Later
studies, however, have not succeeded in cultivating any virus or in demonstrating viral particles
by use of electron microscopy (Rogder & Turnbull 2000; Ferguson et al. 1990). Still, Grotmol et
al. (1997) reported the detection of nodaviruslike particles in heart muscle of fish suffering
from CMS, both by use of immunohistochemistry against nodavirus, as well as by demonstrating virus-like particles 25 nm in size with
electron microscopy. It could not be concluded,
however, that the virus particles were the cause
of the disease, and similar findings have not been
reported from other outbreaks of CMS.
Transmission of the disease by injection of cellfree material based on heart, liver and kidney tissue from sick fish has been reported (Watanabe
et al. 1995; Nylund 2001), supporting a theory of
viral aetiology. In an epidemiological study,
Brun et al. (2003) found a connection between
outbreaks of CMS and earlier cases of infectious
pancreas necrosis (IPN) in the same farm. Inflammation of the heart muscle is also found in
connection with “erythrocytic inclusion body
syndrome” (EIBS) (Rodger & Richards 1998).
Recently, it was reported that a variant of the PD
virus, Norwegian salmonid alphavirus, had been
isolated from CMS fish (Hodneland et al. 2005).
It is not known whether this was merely a coincidental finding.
Current topics for further research
There are many unanswered questions related to
CMS, particularly connected to the aetiology, the
elucidation of the pathogenesis, and the mapping
of risk factors. An approach to these problems
can include observing groups of fish during the
entire seawater phase – preferably even during
outbreaks of CMS – and making clinical records
and taking samples in order to study pathological
changes and examine for microorganisms. Detection of microorganisms should be followed by
characterisation studies and challenge trials, in
order to try to reproduce the disease condition.
Possible connections between CMS and other
diseases, including IPN, should be studied further, and other risk factors should be identified in
epidemiological studies. Based on the results of
pathological and microbiological studies, better
diagnostic tools should be developed.
Epitheliocystis / proliferative gill
inflammation (PGI)
Comprehensive gill problems in Atlantic salmon, resulting in impaired growth and significant
mortalities, have been observed to varying degrees along the entire coast since the 1980s.
Some fish health services do report increasing
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163
problems in recent years (Myklebust & Holm
2005).
Outbreaks typically occur the first autumn in seawater, a few months after sea transfer. Mortalities
may vary from negligible to more than 20 per
cent. Even higher mortality numbers have been
reported in some outbreaks (Myklebust & Holm
2005). In addition, there are losses due to reduced growth in survivors. Fish suffering from
such gill problems may also be more vulnerable
to other disorders. Hence, the overall economic
impact of the disease is substantial.
So far, diagnosis of PGI has mainly been based
on clinical symptoms and histological findings,
the latter revealing intracellular accumulations of
rickettsia- or clamydia-like organisms, termed
“epitheliocysts” in the gills. These formations
give rise to the term “epitheliocystis” for this
condition. Typical findings in histology from
gills are inflammation and increased numbers of
surface cells; in recent years the disease has been
called “proliferative gill inflammation” (PGI).
So far, it has not been possible to cultivate the microorganisms in question, so challenge experiments have not been possible. Based on molecular
characterisations of the organism, a new species
has been suggested: Candidatus pisciclamydia
salmonis (Draghi et al. 2004). A paramyxovirus
has been isolated from the gills of sick post-smolt,
Atlantic salmon paramyxovirus (ASPV) (Kvellestad et al. 2003), and the virus has been detected
in fish suffering from clinical outbreaks in the
field (Kvellestad et al. 2005). However, it has not
been possible to reproduce the disease in challenge trials (Fridell et al. 2004). Therefore, the etiology for PGI is not fully elucidated, but the
pathogenesis probably includes an interaction between clamydia, ASPV and unfavourable environmental conditions (Holm et al. 2005).
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No effective treatment has been identified for
PGI. Bathing in different solutions has been tested, as well as antibiotics added to feed, but no
evident effect has been demonstrated (Myklebust
& Holm 2005).
The Research Council of Norway is supporting
two ongoing projects aimed at identifying risk
factors for outbreaks of PGI and developing
diagnostics for the disease condition.
Parvicapsula pseudobranchiola
During the spring and early summer of 2002, disease outbreaks in Atlantic salmon were recorded
in five sea farms in Northern Norway (Karlsbakk
et al. 2002; Sterud et al. 2003). Mortality numbers in the farms varied from three to 50 per cent.
A single-celled parasite, Parvicapsula pseudobranchiola, was found to be the cause of deaths.
The diagnosis was based on histology. The parasite is a myxosporidium, and the life cycle probably includes a switch between Atlantic salmon
(intermediate host) and an invertebrate (end
host). There is no known treatment for the disease.
Disease caused by Parvicapsula has previously
been reported in salmon in North America (Jones
et al. 2004). The discovery of a new parasite in
Norway attracted considerable attention, particularly in relation to a possible risk for important
wild stocks of Atlantic salmon. During 2003, the
parasite was found in more than 30 fish farming
sites from the county of Møre og Romsdal in
Central Norway, and northwards. In 2004, the
parasite was even found in Sunnhordaland. The
findings in 2002 resulted in the employment of
new routines for histological investigations of
diagnostic material. The increased numbers of
identifications of Parvicapsula in recent years
should be interpreted in this context.
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Figure 7. Vibrio anguillarum-isolates from Atlantic cod examined for antibiotic resistance. The plates from left to right
show a resistant, an intermediate and a sensitive isolate, respectively. (Photo: D.J. Colquhoun)
The Research Council of Norway has funded a
project to build up knowledge regarding the parasite, its spread, life cycle and ability to cause
disease. Such knowledge will be important in
order to evaluate different preventive measures.
Bacterial infections in marine
fish
Great effort goes into developing the cultivation
of marine fish species as an aquaculture industry.
Production is increasing, particularly for Atlantic
cod, and even for halibut. The range of diseases
is reminiscent of what was seen in salmonids
during the 1980s and early 1990s: bacterial infections are dominating these relatively early
phases of developing production of marine fishes. In 2005, a new bacterial species was found
and isolated from cod with granulomatous formations in different organs. The bacterium belongs to the genus Francisella (Olsen et al.
2006), and the disease is already of concern for
the cod farming industry.
Vibriosis
Globally, vibriosis is probably the most common
systemic bacterial infection in farmed fish. The
disease is caused by the Gram-negative bacterium usually known as Vibrio anguillarum. The
species has been reclassified in the genus
Listonella (MacDonald & Colwell 1985), but its
taxonomic transfer from genus Vibrio has not
been fully recognised (Austin et al. 1995) and is
still a subject of discussion (Thompson et al.
2004).
Vibriosis has caused sporadic mortalities in feral
fish, but the disease is primarily a problem under
marine aquaculture conditions in temperate
zones, including fish farming in brackish water.
Outbreaks are most common during relatively
high water temperatures in summer. Sørensen &
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165
Larsen (1986) originally suggested 10 different
serovariants of the bacterium. In addition, a number of sub-groups have been identified (Rasmussen 1987; Bolinches et al. 1990; Tiainen et al.
1997). Vibriosis in cod in Norway is caused
solely by V. anguillarum serovars O2α and O2β,
the latter being dominating.
Vibriosis is the most important bacterial infection in the production of cod fry in Norway, and
the majority of prescriptions of antibiotics for
cod fry are against this disease. Vibriosis has also
been seen in coalfish. The pathological findings
usually include ulcers and haemorrhages in the
skin, particularly in the head region, and at the
bases of the fins. With a more chronic disease
course, bloodshot, protruding eyeballs may be
seen.
Losses due to vibriosis can be limited by vaccination. While the vaccines used today are without adjuvants, and do not offer full protection
against disease, experiments with adjuvanted injection vaccines have shown promising results
(Mikkelsen et al. 2004). These vaccines cannot,
however, be applied to small fishes. Results from
experiments with fry have demonstrated better
protection of fish of two grams weight, compared
to those vaccinated at one gram weight. Fish vaccinated at five grams are protected for at least six
months (Schrøder et al. 2006). Vibrio bacteria
are usually sensitive to antibiotics, and oxolonic
acid is usually the drug of choice. However, resistant isolates have been identified (Colquhoun
et al. 2005).
Ongoing studies (Colquhoun, underway) indicate that Norwegian V. anguillarum O2β are
quite homogenous, while there are greater variations between O2α isolates. These differences
may be of importance for the design of vibriosis
vaccines intended for cod. Studies are underway
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of genetic differences between isolates of V. anguillarum O2α and O2β from different European countries. Their results will hopefully
provide important knowledge of the population
structure of V. anguillarum. Further research
should aim at improved vaccines on the one
hand, and at the optimisation of fry cultivation
systems on the other, in order to reduce the infection pressure.
Aeromonas salmonicida
A. salmonicida can cause serious systemic infections in many fish species around the world. The
bacterial species can be divided into five subspecies, but in connection with fish diseases, isolates
are categorised into two main groups; subspecies
salmonicida, and “atypical” isolates. Isolates
causing disease outbreaks in farmed marine species usually belong to the atypical group (Bergh
et al. 2001; Lund et al. 2002; Gudmundsdóttir et
al. 2003), but infections caused by subsp. salmonicida do occur as well.
In Norwegian aquaculture, A. salmonicida causes problems primarily in halibut, Atlantic cod,
wolffish and turbot. Typical external symptoms
are seen as skin lesions, including ulcers (Wiklund & Dalsgaard 1998). Acute mortality can be
observed, but more common is a moderately increased level of mortalities over time. Fish may
be covertly infected with the bacterium, showing
no signs of disease. One may expect that an increase in the farming of marine fish species in the
North Atlantic region can be followed by problems with different variants of A. salmonicida.
The reservoir for infection is supposed to be infected fish, wild or in farms. Outbreaks are probably activated by stressors such as high fish
densities, handling of fish, or poor water quality.
Losses during an outbreak may be reduced by
keeping proper routines. Treatment with antibi-
otics is usually effective, but the disease may reappear after some time, and A. salmonicida has
the ability to quite frequently develop strains
showing multi-resistance against several antibiotics (L’abée-Lund & Sørum 2001).
Vaccination against atypical furunculosis in marine fish species has not offered protection comparable to that achieved against furunculosis in
salmonids. Levels of protection seem to vary
with the bacterial strain causing disease (Lund et
al. 2003), and differences in the bacterial surface
A-layer protein seem to be of importance. Variation in the A-layer reflected in DNA of the isolates is a promising epidemiological marker for
virulence (Colquhoun, underway). Ongoing research activities are to a great extent focusing on
selection of isolates for vaccine development as
well as epidemiological typing systems based on
molecular biological characteristics.
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Figure 8. Cod louse (Caligus curtus) situated on the skin of
an Atlantic cod. (Photo: T.T. Poppe)
VER (Johansen et al. 2004b). However, the virus
was somewhat different from the one causing
disease in halibut. Disease is also reported in cod,
the virus being quite similar to the halibut virus
(Starkey et al. 2001).
Nodavirus
Nodavirus is the cause of viral encephalopathy
and retinopathy (VER) in halibut fry (Grotmol et
al. 1997; Grotmol et al. 1999). The disease
caused substantial losses throughout the 1990s,
but in more recent years, reported outbreaks have
been few. It can be assumed that general hygienic
management procedures implemented in the
farming of halibut have had a positive effect.
Infection of larger fish does not seem to cause
disease (Grove et al. 2002); however, they can
become carriers of the virus. Although they show
no obvious symptoms, growth rate may be affected (Johansen et al. 2004a). The virus may be
transmitted to spotted wolffish, resulting in a disease condition resembling that seen in halibut
(Johansen et al. 2003; Sommer et al. 2004). A
nodavirus has been isolated from turbot, as well,
in connection with a disease outbreak similar to
Methods for cultivating the virus in cell culture
have been established (Dannevig et al. 2000) and
antibodies have been produced and are being utilised in immunohistochemistry (Johansen et al.
2002). An ELISA to monitor the immune response in fish has been established (Grove et al.
2003), and PCR methods have been developed
for the detection of virus (Grotmol et al. 2000;
Grove et al. 2003). Experimental vaccines have
been designed and tested in turbot, and a vaccine
based on a recombinant capsid protein gave protection (Sommerset et al. 2005). Further studies
concerning the pathogenesis of VER are needed,
as is the development of better diagnostic tools.
Parasites in marine fish
When a fish species is kept in monoculture for
farming purposes, the conditions are ideal for the
Thematic area: Health
167
multiplication of parasites, particularly for species which do not need intermediate hosts. The
parasites get access to a great number of hosts
living in high population densities, compared to
wild populations. Therefore, it is of great importance to know which parasite species may be
found in wild stocks of the same fish species, and
to follow the development of parasites burdening
farmed fish. The multiplication of parasites in
aquaculture may also affect wild fish, due to increased infection pressure.
The “Scottish louse”, Caligus elongatus, constitutes a particular challenge; this louse can be a
parasite on many different fish species, including
Atlantic salmon, Atlantic cod (Karlsbakk et al.
2001), and halibut. In a research project, lice
sampled from different fish species are compared
in order to reveal any differences in morphology,
genetic material or specific preferences for different fish species. There are even other “lice” in
cod, such as Caligus curtus, which attack only
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cod, and the “fish louse”, genus Argulus (Schram
et al. 2005). All these species are parasitic crustaceans.
Other parasites in cod include the gill worm
(Lernaeocera branchialis, copepod) (Lysne &
Skorping 2002), the flagellate Spironucleus
torosa in the intestine, and the microsporidium
Pleistophora gadi in the muscle. Several ciliates
(e.g. Trichodina), flagellates (e.g. Ichthyobodo)
and myxosporidia (e.g. Pleistophora) can infest
different marine fish species, such as cod, halibut
and wolffish. The nematodes include species
such as Anisakis and Pseudoterranova, giving
rise to intermediate stages of the worm in muscle
and other organ systems (Hemmingsen &
MacKenzie 2001), and they may cause health
problems in humans (zoonoses). Monogenean
ectoparasites also need investigating, including
various species of gyrodactylus, which may
cause problems in farmed cod.
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171
Øystein Evensen1), Nina Santi1), Ann-Inger Sommer2) and Siri Mjaaland1)
1) Norwegian School of Veterinary Science, 2) Fiskeriforskning (Norwegian Institute of Fisheries and
Aquaculture Research)
Virulence Mechanisms
Virulence is the ability of infectious agents to cause disease in the infected animal. This is a trait that has developed over millions of years in close interaction
between the host and the agent. The relationship that has developed over millions of years of co-existence has resulted in a situation where there is on one side
a balance between the agent’s ability to spread within a susceptible population
and on the other side the outcome of an infection both at the level of the individual and the host. Rapid viral replication and spread within the host will result
in severe damage to internal organs and rapid death, which will limit the ability
of the agent to spread and survive within the population. It is anticipated that
agents will trade off virulence for persistence and less-efficient spreading mechanisms in lower vertebrates as well as mammals.
This chapter will describe characteristics of two fish viruses and present results
from recent years’ research into infectious pancreatic necrosis virus (IPNV) and
infectious salmon anaemia virus (ISAV). For the IPN virus, a good understanding
has been gained of the association between a strain’s amino acid residues at
defined positions in the VP2 protein and the virulence of the strain, defining a
virulence signature. Similarly, defined mutations in the same amino acid residues can easily be associated with loss of virulence. For the ISA virus it has been
shown there are large variations in some of the surface molecules between
strains, although it has not been possible to associate these differences with
virulence traits. It has also been shown that viral proteins can counteract the
defence mechanisms of the host.
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Definitions
Viral replication
Virulence is used as a term to describe the relative pathogenicity of the relative ability (capacity) of the agent to cause tissue damage/
disease. While virulence traits in bacteria are
often associated with different toxins and the
level at which they are produced, virulence in
viruses is often less well defined. This chapter
will focus on virulence traits of viruses for the
reason that it has been the main topic of the
Aquaculture programme for this reporting
period. In no way does this mean that the virulence traits of fish pathogenic bacteria are all
known and well described. Many factors contribute to the overall pathogenicity of a virus,
such as the host cell entry mechanisms, replication capacity, ability to counteract or resist the
defence mechanisms of the cell, and the ability
to cause temporary or permanent damage to the
host through cell lysis, production of toxic substances, cell transformation, and production of
antiviral substances by the infected cell in response to an infection. In addition, induction of
structural changes in the cell nucleus and the
cytoplasm are observed. A virus can infect the
host through damaged skin or an insect bite (in
fish it has been proposed that the salmon louse
– Lepeophteirus salmonis – can act as a carrier
and vector of infectious agents). However, in
both lower and higher vertebrates the mucosal
surfaces are considered a prime port of entry of
pathogens; because of this, pathogenic viruses
have developed the ability to exist in company
with the normal flora, both viruses and bacteria.
Replication of a virus can occur in the mucosal
lining, i.e. this site is the primary port of infection (as seen for koi herpes virus) but more
often the mucosal lining is the port of entry of
systemic infection.
The speed at which replication takes place plays
a key role in determining virulence. Fast replication will frequently be associated with disease symptoms and clinical disease. The ability
of the virus to replicate in vivo will depend on
the ability of the virus to replicate under the
conditions “provided” by the infected cell plus
the extent to which the virus can resist the defence mechanisms of the cell aimed at preventing replication and eliminating the virus. A
fine-tuned balance has developed over years of
co-existence and relates to different stages of an
infection: cell adherence, penetration, release of
the virus particle to the inner compartment of
the cell, access to energy, synthesis of components of importance during early stage of replication, synthesis of virus proteins and genome
replication, and assembly and release from the
cell.
Tissue damage
A successful infection will often result in cell
lysis, typical of cytolytic viruses. Typically,
membrane integrity is lost, followed by leakage
of enzymes and cell damage. Apoptosis is another classical cellular response to a virus infection and because of this many viruses have
developed strategies whereby they counteract
the induction of apoptosis. Virus proteins interact at different levels of the apoptosis “machinery” and the details are largely unknown for
many viruses; the consequence to the host cells
is a delay in the onset of apoptosis, which allows the virus to produce more progeny before
the cell machinery shuts down. Non-cytolytic
viruses, on the other hand, will often cause cell
damage through immunological defence mechanisms, typically through an expression of
capsid or membrane components of the virus
being expressed in the cell membrane of the infected cell. Antibodies in combination with
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173
complement can result in cytolysis while cytotoxic T cells can induce cell lysis through direct
interaction.
An important virulence trait of many viruses is
the ability to circumvent the immune responses
of the host. Different viruses have developed
different strategies; inhibition of presentation of
virus proteins via MHC molecules is one, yet
another is the ability of the virus to mutate single amino acids in immunodominant virus proteins.
Cellular effects
A virus infection can have toxic effects on the
infected cells. Examples are formation of syncytia, fusion of chromosomes (polykaryocytosis) and so on. Virus infections can also result in
cell transformation (pre-cancerous stages) and
suppression of immune responses as inhibition
of interferon responses. Cytoplasmic and nuclear inclusion bodies are structural cell changes
observed in association with virus infections.
In the ensuing paragraphs you will find a presentation of virulence mechanisms of infectious
pancreatic necrosis virus and infectious salmon
anaemia virus. IPNV is a small virus, 60 nm in
diameter with an icosahedral symmetry in the
capsid (Figure 1). The genome consists of two
double-stranded RNA molecules which encode
five virus proteins (VP1–5). VP2 and possibly
VP5 carry virulence traits. VP2 is exposed on
the virus surface and is important for the binding to and uptake in target cells. The VP2 protein also carries serotype specific and
neutralising epitopes. VP5 can promote the
ability of the virus to replicate and it has been
shown that certain strains of IPN virus have an
anti-apoptotic effect.
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145x100//Kap10-fig01.eps
Figure 1. Electron micrograph of IPN virus (negative
staining; Trygve Eliassen).
Methods relevant to studies of viral
virulence mechanisms
Both biotechnological methods and fish challenge studies are important tools when investigating the virulence mechanisms of IPNV. It
has been known for a while that some strains
cause higher mortality, and that passage in cell
culture can alter the virulence characteristics of
the virus.
Virulence differences among virus stains can be
revealed through experimental challenge of
salmonid fish. A number of factors influence
the mortality caused by IPN, both in field outbreaks and under experimental conditions. Accordingly, virulence comparison of IPNV
strains must be coordinated, and performed in a
controlled environment to attain credible results. All conditions, such as the number of fish,
virus dose, challenge time, temperature, tanks,
and water flow, must be equal for all experimental groups. By challenging susceptible rainbow trout fry, early investigators detected
virulence differences among IPNV strains of
145x100//Kap10-fig01.eps
Figure 2. The reverse genetics technique for production of recombinant IPN virus. First, a CDNA copy of the viral genome is
cloned into a plasmid vector. In front of the genomic sequences is a short sequence recognised by a RNA polymerase (T7).
This enables in vitro synthesis of RNA copies of viral genomic segments A and B. When this copy RNA is transfected into a
susceptible cell culture, new viral proteins and genomic segments are made, and later assembled into new viral particles.
Mutations introduced in the cDNA copy of the viral genome will be expressed as alteration in the proteins of the new,
recombinant virus isolate.
the same serotype, and mapped virulence traits
to RNA-segment A.
Sequencing and subsequent comparisons of the
viral genomes of different IPNV strains can reveal differences and lead to identification of molecular motifs involved in virulence. However,
the strains may differ in more than one way, making it hard to decide which motif is most significant for the virulence traits of the virus.
The reverse genetics technique can be applied
to gain a more detailed map of the virulence
markers (Figure. 2). By this technique it is pos-
sible to alter the viral proteins by introducing
specific mutations of the viral genome. It is, for
instance, possible to change one amino acid residue of a highly virulent strain to match the corresponding amino acid of a low virulent strain.
The resulting recombinant viral strain can be
further tested to confirm the role of this residue
as a virulence marker. The reverse genetics
technique provides a unique tool for studies of
the viral life cycle, viral assembly, the role of viral proteins in pathogenicity, and the interplay
of viral protein with components of the host’s
immune response. A reverse genetics technique
has been developed for the IPN virus as well.
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175
Identified virulence mechanisms of the
IPN virus
When Atlantic salmon fry are challenged with
different field strains of IPNV, great variation in
the pathogenicity of the isolates is displayed.
Some of the strains cause high mortality and severe lesions in the pancreas and liver of infected
fish, while other strains cause low mortality and
no detectable tissue damage. The same strains
cause varying mortality in experimental challenge of Atlantic salmon post-smolt as well.
The virulence was also tested after 10 passages
in cell culture.
From these studies, several molecular motifs associated with IPNV virulence were identified.
Four amino acid residues of the capsid protein
VP2, as well as the size of the non-structural
protein VP5, appeared to be important. These
motifs have since been subject to further studies
to attain detailed knowledge of their individual
influence on the pathogenicity of the virus.
The reverse genetics technique has been used to
study the effect of VP5 on IPNV virulence. Recombinant viruses coding for different size
variants of the VP5 protein have been made, as
well as a VP5 knock-out mutant. Challenge
studies of both Atlantic salmon fry and postsmolt did not reveal any differences in the virulence of the VP5 variant strains. Additional
studies have demonstrated that VP5 is not essential for viral persistence. A cell protective
function (anti-apoptotic) has been recorded for
the VP5 protein of an Asian IPNV strain. This
anti-apoptotic effect was not found in cell culture or in fish infected with the recombinant
strains studied. Taken together, these results oppose a role of VP5 as a virulence factor of Norwegian IPNV strains.
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The significance of the individual VP2 amino
acids was also investigated using the reverse
genetics technique. These studies pinpoint VP2
residues 217 and 221 as the major virulence determinants of Norwegian IPNV isolates. Highly
virulent strains encode Thr217 and Ala221,
while moderate to low virulent strains have
Pro217 and Ala221. Strains with Thr at residue
221 are almost avirulent, irrespective of the
amino acid in position 217.
The molecular basis for cell culture attenuation
was realised by detection of an Ala-Thr substitution at residue 221 after a few passages of the
virus in cell culture. The cell culture adapted
strains are strongly attenuated and almost avirulent in experimental challenge of Atlantic
salmon fry. A single mutation at VP2 residue
221 can be detected after 10 passages in cell
culture. The resulting mutated strain has significantly reduced virulence, causing very low
mortality as compared to the original strain.
The three-dimensional structure of VP2 of
IBDV was recently determined. IBDV is a
birnavirus causing infectious bursitis in chicken. From the IBDV VP2 crystal structure it can
be predicted that the amino acids 217 and 221
of IPNV are exposed at the viral surface. Their
exposed orientation combined with the marked
effect on virulence may suggest that residues
217 and 221 are engaged directly with receptors
of the host cell. Further investigations are
necessary to decide if this is the case.
Field studies
In a field study of IPN including 20 different
fish farms with post-smolt vaccinated against
IPN, an accumulated mortality varying from
one to 45 per cent was registered three to nine
weeks after transfer to seawater. There might be
different reasons for the large variation in mor-
tality. One possibility was that salmon from different localities was infected with IPN viruses
of varying virulence, and IPN virus isolates
from these outbreaks were therefore sequenced.
Most of them showed the motif of the four
amino acids in VP2 consistent with a highly
virulent strain, while only a few of the field
strains were equal to the variants of moderate
virulence.
One field strain was interesting because it contained only two of the four “markers of high virulence”, Thr217 and Ala221, but nevertheless
was characterised as highly virulent in a challenge test. Again this indicated that the two
amino acids in position 217 and 221 are the
most important ones for virulence in related
IPN virus strains. All genetic characterisations
of the field isolates as highly or moderately
virulent were confirmed when the strains were
tested in challenge experiments under equal
conditions. Several trials have shown that nonvaccinated post-smolt challenged with strains
of high virulence usually results in 40–70 per
cent accumulated mortality and zero to 35 per
cent using strains of moderate to low virulence.
The mortality also seems to vary with the
susceptibility to IPN in the families of Atlantic
salmon used.
There is obviously a lack of correspondence between mortality registered out in the field and in
controlled challenge experiments. This reflects
that many factors, besides the quality of the IPN
virus itself, have an impact on the result of an
infection. It should be mentioned, though, that
no low virulent virus strain was isolated from
any of the IPN outbreaks. Results from all the
studies together show that the same virulence
factors are important for development of IPN
both in Atlantic salmon fry and post-smolt.
Whether this applies to other fish species susceptible to IPN is still to be investigated.
Carrier conditions, reactivation of the IPN
virus and virulence
Because the IPN virus is very common in Norwegian fish farms, primary infections may
occur after transfer of the salmon to the sea.
Nevertheless, there is reason to believe that
reactivation of a persistent IPN virus infection
could be responsible for a great part of the outbreaks. Increased resistance to IPN in new
generations of farmed Atlantic salmon may also
represent a genetic pressure allowing more
virulent IPN viruses to be carried asymptomatically in the fish. For instance, the N1 strain of
IPNV previously isolated from most of the
outbreaks of IPN in salmon fry and from virus
carriers is now characterised as a low virulent
strain.
Experimental challenge and knowledge about
the virulence of the IPN virus strains are also
used to ascertain the significance of virulence in
the establishment of a carrier condition, and in
reactivation of IPN as well. Experimental carriers of the IPN virus were established under
equal conditions by bath challenge in freshwater, using both a highly virulent and a low
virulent strain. The highly virulent strain was
reactivated after smoltification and transfer to
seawater, which resulted in an IPN outbreak.
The accumulated mortality was comparable to
that observed after challenge of post-smolt
salmon with the same strain in seawater. This
occurred even if the virus at some time points in
the freshwater phase was not detectable in cell
culture using a traditional carrier test. This type
of IPN virus was isolated from more than ¾ of
all collected field strains, but it is not known
whether the salmon was infected in seawater or
Thematic area: Health
177
already carried the virus in the early freshwater
phase.
The mechanisms behind an IPN virus carrier
condition are not known. Studies have shown
that the mutations that happen during passages
in cell cultures and result in a change from Alanine to Threonine in position 221 in VP2 may
explain a reduced virulence, which seems to coincide with the establishment of a persistent infection.
Virulence mechanisms for the
ISA virus
ISA virus
The ISA virus is an aquatic orthomyxovirus
(genus: Isavirus), belonging to the same family
as the influenza A virus causing disease in birds
and mammals. The virus is enveloped, 80–120
nm in size (Figure 3), with surface projections
performing hemagglutination (receptor-binding), receptor-destroying (viral release) and
fusion (virus entry into the cell) activity. The
genome consists of eight single-stranded RNA
molecules with negative polarity, encoding at
least 10 proteins, where nine are known to be
present in the mature virus particle. The largest
genomic segments (segments 1, 2 and 4) encode
the viral polymerases, while segment 3 encodes
the viral nucleoprotein. Segments 5 and 6 encode the two surface glycoproteins, i.e. the
fusion protein (F) and the hemagglutininesterase (HE). The HE is responsible for receptorbinding and -release, while the F protein
enables the entrance of viral nucleic acids into
the cell. The two smallest genomic segments
encode two proteins each. The largest open
reading frame (ORF1) of segment 7 encodes a
non-structural protein (NS), a putative interferon antagonist, while the function of the structural ORF2 protein remains to be characterised.
The matrix (M) protein is encoded by the ORF1
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Aquaculture Research: From Cage to Consumption
of segment 8, while the larger ORF2 encodes an
RNA-binding structural protein with putative
interferon antagonistic activity.
The ISA disease picture is complex, as observed by large differences in disease development and clinical signs. Typically, an ISA
outbreak may initially appear as diffuse, unspecific disease problems and variable mortality
over long time periods, before finally cumulating into an outbreak, while acute disease outbreaks occur more seldom. Virulence is
influenced by a) the genetic constitution of the
fish, b) the genetic constitution of the virus isolate, and c) environmental circumstances.
Possible virulence mechanisms
The study of ISA virus genes and gene product
is still in its infancy, and there are at present major gaps in our knowledge around the mechanisms behind ISA virus virulence. It was not
until 2002, for instance, that all the genes had
been sequenced, and not until 2005 that the total
sequence of one isolate (Glesvaer 2/90), including the non-coding 3’- and 5’-terminal viral
RNA sequences, was completed. This last work
was initiated as the first step in the establishment of a reverse genetics system on ISAV (see
below). Current knowledge of virulence is
based on results from experimental trials, sequence analyses of viral genes, and functional
studies on cloned recombinant viral proteins.
In orthomyxoviruses, reassortment of gene segments occurs frequently and is a major contributor to the evolution of these viruses and the
emergence of new virulent strains. Extensive
molecular and phylogenetic sequence analyses
of 14 full-length sequences of Norwegian ISA
virus isolates together with a parallel study on
the two surface glycoproteins provide strong
evidence for the occurrence of genetic reassortment involving several ISAV gene segments.
Moreover, properties like receptor-binding and
-release, fusion and interferon antagonism,
which are all important factors of virulence for
influenza virus, have also been identified for the
ISA virus. However, it is still not known to what
extent variations in these activities influence the
outcome of an ISA virus infection.
ISA virus HE protein
Cloning and early characterisation of the hemagglutinin-esterase (HE) gene was published
in 2001. It was soon realised that HE is the ISA
virus protein with the overall highest sequence
variation, concentrated in a small highly polymorphic region (HPR) near the transmembrane
region. This region is characterised by the presence of gaps rather than single-nucleotide substitutions. Alignments and analyses of a large
number of HE gene sequences suggested that
the polymorphism in this region could arise
from differential deletions of a theoretical full-
145x100//Kap10-fig01.eps
Figure 3. Electron micrograph of ISA virus particles (Ellen
length precursor gene (HPR0) as a result of
strong functional selection pressure, possibly,due to a newly or ongoing crossing of species barrier, or as a results of changes in
ecological conditions related to fish farming. So
far, the number of different HPR groups is 25 in
Europe and four in North America. The pattern
of variation is constrained to the 35 amino acids
defined as the HPR.
The presence of a long HPR0 gene has been
confirmed by RT-PCR of tissue samples from
healthy wild and farmed Atlantic salmon.
HPR0 is therefore assumed to represent a lowpathogenic /avirulent virus. The main reservoir
for the ISA virus is today believed to be wild
salmonids (brown trout and Atlantic salmon)
and the farmed salmon itself.
HPR/HPR0
All viruses isolated from ISA-diseased fish so
far contain deletions in the HPR region. It is
therefore assumed that deletions in the HPR are
a prerequisite for disease development, and that
the HPR most likely represents an important
virulence marker. In fact, this is so far the only
genetic marker associated more or less directly
with virulence in the ISA virus. The virulence
of the ISA virus cannot, however, be attributed
to the HE-HPR alone, as isolates with identical
HPR vary in virulence in experimental infections using standardised experimental fish.
Virus-host interactions
Experimental trials have been designed to study
the interactions between the ISA virus and the
salmon host in detail, with emphasis on disease
susceptibility and immune responses. To reduce
the effect of host genes, half-siblings identical
in MHC class I and II genes were used as
experimental fish. The ISA virus isolates were
selected according to a) variation in their HE-
Namork).
Thematic area: Health
179
145x100//Kap10-fig01.eps
Figure 4. Reverse genetics on the ISA virus.
A. The bidirectional transcription system. Viral cDNA is oriented in a positive direction to the RNA polymerase II promoter
(pII) and poly adenylation site (aII), and a negative orientation to the RNA polymerase I promoter (pI) and termination
sequence (tI). In this way both viral mRNA (positive-stranded) and viral vRNA (negative-stranded) may be produced from
the same construct following transfection into susceptible cells.
B. Total viral RNA sequence of each of the eight ISA virus genomic segments is converted to cDNA and placed in separate
plasmid vectors. The eight plasmids are co-transfected into fish cells (ASK or TO) and recombinant ISA virus synthesised.
C. Detailed description of plasmid-based replication of recombinant ISA virus.
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Aquaculture Research: From Cage to Consumption
HPR region and b) the isolates’ differences in
virulence in field outbreaks. The mortality
induced after cohabitant challenge ranged from
zero to 47 per cent in the test-group fish, and
three to 75 per cent in the injected cohabitants.
The use of MHC compatible experimental fish
made it possible – for the first time – to estimate
the relative importance of humoral versus cellular responses in protection against the ISA
virus. Most interestingly, the ability to induce a
strong proliferative response correlated with
survival, while induction of a humoral response
in itself was less protective. The differences in
induced immune responses could not be ascribed to the variation in the HE-HPR alone, as
two isolates with identical HE-HPR induced
opposite immune responses and hence survival.
ISA virus biobank material
Due to the lack of a reverse genetics system for
this virus at the time, detailed sequence- and
phylogenetic analyses were the methods of
choice in the search for additional virulence
traits. The 14 ISA virus isolates described
above were therefore cloned and full-length
sequenced. Following extended amino acid
sequence analyses coupled to data on virulence,
candidate genes and areas of genes of importance in ISAV virulence have been suggested
(see below). For final proof, however, a reverse
genetics system is needed.
ISA virus F protein
The fusion activity is associated with uptake of
a virus into the cell during infection, and changes in this protein may thus have important implications for the virulence of an isolate. Most
interestingly, when aligning all available fusion
gene sequences (segment 5), a 30-nucleotidelong insertion was found in several virus isolates, immediately upstream of the protein’s pu-
tative cleavage site. More detailed analyses
revealed extensive sequence homology in this
region. This, together with the fact that exactly
the same recombination occurred in two unrelated ISA virus isolates, suggests the presence
of a recombinational hot spot. Hypothetically,
this could lead to alterations in the cleavage
specificity of the fusion protein, with potential
changes in tissue or organ tropism, analogous to
avian influenza A virus HA of the subtypes H5
and H7, where sequential insertion of several
basic amino acid residues at the HA1/HA2 proteolytic cleavage site leads to systemic infection
and multi-organ failure in poultry.
ISA virus interferon antagonists
Induction of type I interferon responses represents an important first-line defence against
viral infections. Many viruses have developed
strategies to counteract this response. For the
influenza viruses, this is accomplished by a
non-structural protein with RNA-binding properties. The ISA virus, on the other hand, produces two proteins potentially interfering with
the cells’ interferon response: a non-structural
protein without RNA-binding properties, and a
small RNA-binding structural protein. The two
related virus groups may therefore seem to use
different strategies to achieve the same goal: to
inhibit the cells’ defence against viral infection.
The detailed mechanisms behind the ISA virus
interferon antagonism have not yet been characterised.
Reverse genetics on the ISA virus
Reverse genetics is the optimal tool for studying
virulence mechanisms and virus-host interactions. The method also has great potential in
future productions of vaccines against the ISA
virus. For several positive-stranded RNA viruses (such as the IPN virus) this technology has
already been established and been in use for
Thematic area: Health
181
several years. However, in contrast to positivestranded RNA molecules, negative-stranded
RNA molecules (vRNA; as the ISA virus) are
not infectious. By including expression of functional viral RNA polymerases and ribonucleoprotein complexes, synthetic vRNA segments
can be converted to an infectious virus from
full-length cDNA. A plasmid-based expression
system is used, similar to the one developed for
the influenza A virus, where viral cDNA from
each of the genomic segments are placed between a RNA polymerase I promoter and terminator sequence, flanked by a RNA polymerase
II promoter and polyadenylation site. The orientation of the two transcription units allows
synthesis of negative-stranded vRNA and posi-
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Aquaculture Research: From Cage to Consumption
tive-stranded mRNA from each cDNA template
(Figure 4).
The total vRNA sequences from all eight of the
ISA virus genomic segments have been mapped
and cDNA cloned into the pol I/II vector
pHW2000, and the work towards producing
viable artificial viral particles is promising.
After successful isolation of recombinant viral
particles, mutations will be introduced into candidate genes. The molecular analyses of the
above mentioned biobank material will help to
define relevant genes and areas of genes. The
effects of the mutations will be tested in vitro
(infection of cell cultures) and in vivo (experimental trials).
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Melby, H.P., Christie, K.E., 1994. Antigenic analysis of
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Børre Robertsen1), Ivar Hordvik2) and Trond Jørgensen1)
1) Norwegian College of Fishery Science, University of Tromsø 2) University of Bergen
The Fish Immune System
Why do Atlantic salmon smolts develop infectious pancreatic necrosis (IPN) when
released into the sea? Why are viral vaccines less efficient than bacterial vaccines
in salmon? Why does infectious salmon anaemia virus (ISA-virus) kill Atlantic
salmon, but not trout? These are some of the questions that occupy fish immunology researchers. It is the immune status of the fish, which determines whether
the fish develops disease after contact with an infectious microorganism. Molecular and functional studies of the fish immune system are thus very important
to understand why fish get sick and how fish vaccines can be developed to work
optimally. The defence against microorganisms of vertebrates consists of the innate and the adaptive immune system. The innate immune system is comprised
of cells and mechanisms that defend the host from infection by microorganisms,
in a non-specific manner. This means that the cells of the innate system recognize
and respond to molecules that are structurally conserved in viruses, bacteria or
fungi. The innate immune defence is active only for a short period of time and
may also be deceived by many pathogens. In contrast, the adaptive immune system is activated later during the infection and is directed specifically against the
invader. Moreover, the adaptive immune defence is more effective, possesses
memory and thus has a longer duration than the innate immune defence. The
main executing cells of the adaptive immune system are B and T lymphocytes. B
cells are the origin of antibodies whereas T cells kill virus infected cells and stimulate macrophages to kill intracellular bacteria. The goal of vaccination is to activate the adaptive immune system. Increased knowledge about the fish immune
systems may be used to improve fish vaccines and to develop better methods for
measuring the immune/health status of the fish. Such methods may be used to
reveal conditions that predispose the fish for infections.
Thematic area: Health
185
Atlantic salmon is one of the most important
model species for obtaining new knowledge
about the fish immune system and many of the
key genes involved have now been cloned and
sequenced. Research on the cod immune system has also made good progress. The Aquaculture Program of the Research Council of
Norway has contributed with significant funding in this field, which has resulted in increased
knowledge and competence in fish immunology. During the Program period several
projects have been devoted to studies of the
antibody repertoire of salmon and cod and on
the innate immunity against viruses of Atlantic
salmon. These topics are thus the main focus of
this chapter.
Innate immunity against virus
in fish
Although viral diseases cause major losses of
farmed Atlantic salmon, we tend to forget that
fish like mammals, are normally quite resistant
to viral infections. The fact is that vertebrates
possess a powerful innate immune defence,
which stops viral infections in an early phase.
The reason why fish or humans sometimes develop disease due to virus infection may be that
the innate defence is weakened by stress, poor
nutrition, poor environmental conditions or because some very aggressive viruses are able to
avoid or inhibit the defence mechanisms of the
animal. Atlantic salmon is for example quite resistant to IPN except for the early stages after
hatching and after release of the smolt into the
sea. Different salmon strains also display a
large variation in susceptibility to IPN-virus.
Atlantic salmon thus has an innate immunity
against IPN-virus that varies with inherited
traits, ontogeny/age and living conditions. As
described below Atlantic salmon appears to
have poor innate immunity against ISA-virus.
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Aquaculture Research: From Cage to Consumption
The interferon system
The interferon system plays a major role in the
innate defence against viruses and recent research has shown that teleost fish have an interferon system, which is very similar to that of
mammals [1]. Interferons (IFNs) are defined as
proteins that induce an antiviral state in host
cells. Virus infection activates the IFN system
of the host cell because the cell recognizes viral
nucleic acids [1, 2]. Henceforth, the infected
cell produces IFN, which is an alarm protein
that circulates in the body and tells other cells to
start production of antiviral proteins (Figure 1).
As a result the IFN system stops further invasion of the virus. The importance of the IFN
system in the innate immunity against viruses in
mammals is well documented. Mice that have
defective genes in the IFN system are for example very susceptible to viral infection. The IFN
system is likely to be equally important in the
antiviral innate immunity of fish. The mechanisms used by host cells for recognizing viruses
have recently been uncovered. This occurs by
binding of viral single-stranded or doublestranded RNA to intracellular receptor proteins
(RIG-I, MDA5, TLR3, TLR7, TLR8 or PKR).
The synthetic dsRNA poly I:C is in fact a powerful inducer of the IFN system because it imitates a virus infection in vertebrate cells.
Whereas the first IFN-genes from humans were
cloned in 1980, the first IFN-genes from fish including Atlantic salmon were cloned in 2003 [1,
3]. The reason why it has taken such a long time
to clone the first fish IFNs is that the sequence
identity between IFNs from fish and mammals
is very low. Similar to mammals, Atlantic salmon was shown to possess two types of IFN, type
I IFN, which is involved in innate immunity and
type II IFN, which is mainly involved in adaptive immunity. Type II IFN is identical to IFN-γ
and will not be further discussed here. Humans
Virus
IFN
IFN-receptor
dsRNA
receptor
Antiviral
proteins
IFN
IRE
NF-kB
IFN mRNA
IFN gene
Virus-infected cell
ISRE
mRNA
IFN stimulated
genes
IFN-stimulated cell
145x100//Kap11-fig01.eps
Figure 1. Role of interferon (IFN) in the defence against viruses. Upon infection by a virus, cytoplasmic receptor proteins
recognize double-stranded RNA (dsRNA) produced during viral replication.. Binding of dsRNA to receptor protein produces
signals that tell the nucleus to produce IFN mRNA. Accordingly the cell starts production of the alarm protein IFN, which is
secreted and circulates in the blood stream. IFN binds to a receptor protein present on the surface of most cells and this
starts a signalling process, which tells the cells to produce Mx, ISG15 and other antiviral proteins. As a result IFN protects
cells against virus infection. The arrows indicate signals that are produced in the cell as a response to dsRNA and IFN.
have many different subtypes of type I IFN
(IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFNτ,
IFN-ω and IFN-λ). Only three type I IFN subtypes have as yet been discovered in salmonids
[3, and unpubl. results]. These are most similar
to IFN-α from mammals.
Salmon interferon protects host cells
from IPN-virus infection
Recombinant IFN-α1 from Atlantic salmon
was produced by insertion of the cloned salmon
IFN gene into cultured human cells [3]. The recombinant IFN was demonstrated to induce
strong antiviral activity against IPNV in cultured Atlantic salmon cells (Figure 2). The test
was performed by incubating salmon cells with
serial dilutions of IFN for 24 hours after which
the cells were infected with IPN-virus. When
the control cells were killed by the virus after
about 72 h, the cells were fixed and stained with
crystal violet. Surviving cells become strongly
stained by crystal violet whereas dead cells remain colourless.
Thematic area: Health
187
Antiviral proteins are produced in
salmon cells in response to dsRNA and
IFN
In human and mouse, IFN has been shown to induce transcription of several hundred genes
some of which are encoding antiviral proteins
[2]. Several IFN-induced genes have recently
been identified in fish [2]. Mx protein is one of
the most studied antiviral proteins and is known
to inhibit replication of influenza virus and several other virus types. ISG15 is one of the proteins that is induced earliest and in the largest
amounts in cells by IFN. It has the ability to
conjugate to other cellular proteins and appears
to be involved in antiviral mechanisms. Both
Mx and ISG15 have been cloned from Atlantic
salmon and were shown to be induced by IFN
and dsRNA in live fish and/or cultured cells
[4–7].
Mx protein of Atlantic salmon inhibits
replication of IPN-virus
Genetic engineering methods have enabled
studies of the antiviral activity of Atlantic salmon Mx protein [8]. The gene encoding salmon
Mx protein was inserted into a plasmid, which
allows continuous expression of the protein in
animal cells. The plasmid was introduced into
salmon cells some of which incorporated the
Mx gene in their chromosomes. Cells producing Mx protein continuously were subsequently
selected and grown (Figure 3).
These Mx producing salmon cells were infected
with IPN-virus. Salmon cells, which had been
gene manipulated/engineered to produce green
fluorescent protein (GFP) were used as a negative controls. Three days after infection surviving cells in the different cultures were estimated
by crystal violet staining. Figure 4 illustrates the
145x100//Kap11-fig02.eps
Figure 2. Antiviral assay, which illustrates that recombinant Atlantic salmon IFN protects salmon TO cells against IPN virus
infection. The picture shows wells with TO cells in a 96 well culture plate after staining with crystal violet. Non-treated
cells or cells treated with different dilutions of recombinant salmon IFN were first incubated for 24 hours. Cells in wells
labelled IPNV or IFN+IPNV were next infected with IPNV whereas wells labelled Uinf or IFN were not infected. Three days
later the cells were fixed and stained with crystal violet. The colour intensity correlates with the number of living cells in
the wells.
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Aquaculture Research: From Cage to Consumption
Figure 3. Expression of Mx protein in Chinook salmon embryo cells after staining with a specific antibody. ASMx1: Cells
genetically manipulated to produce Atlantic salmon Mx protein continuously. IFN: Cells stimulated for 48 hours with IFN.
Control: Non-treated cells.
145x100//Kap11-fig03.eps
protection against IPN-virus obtained in the different cell types. Similar to IFN-treated cells,
almost 100 % of the Mx-producing cells survived the virus infection whereas the control
cells died. This demonstrates that Atlantic
salmon Mx protein protects the cells from the
damaging effects of IPN-virus and suggests that
Mx protein is very important for the ability of
salmon to resist IPN-virus infections.
ISA-virus is not inhibited by the IFN
system of salmon
ISA-virus kills Atlantic salmon very efficiently
in infection experiments. The aggressive nature
of this virus may in part be explained by its ability to trick the IFN system of the fish. Neither
IFN nor dsRNA was able to protect salmon cells
against ISA-virus in spite of the fact that the
cells contained large amounts of Mx protein [5,
9]. Moreover, the virus itself stimulates production of both Mx protein and ISG15 during infection of live salmon and salmon cells in vitro.
These results indicate that ISA-virus either
avoids or inhibits key antiviral proteins of the
IFN system in salmon.
The adaptive immune system in
fish
145x100//Kap11-fig04.eps
Figure 4. Effect of IPN virus infection on survival of salmon
cells, which produce Mx protein continuously (ASMx1). IFN
treated cells (IFN) were included as a positive control and
non-treated cells (Control) were included as a negative
control. The assay was carried out as described in Figure 2.
Percent surviving cells three days after infection were
calculated by measuring the light absorption at 550 nm
Cells and central molecules
The most important cells in the adaptive immune system are T cells and B cells (with their
derived plasma cells responsible for antibody
production). Antibodies recognise non-self
structures on for example bacterial or viral surfaces while T cells recognise foreign structures
on the surface of other host cells. These foreign
structures might be parts of proteins that are digested by multi-protease complexes in a specific manner and bound to a family of molecules
known as major histocompatibility complex
after staining of cells with crystal violet.
Thematic area: Health
189
T cell
CD4+
T cell
CD8+
T cell receptorcomplex
CD8
T cell receptorcomplex
CD4
MHC II
MHC I
Antigen presenting
cell
145x100//Kap11-fig05.eps
Figure 5. T cells recognize foreign non-self peptides displayed on other cells. The T cell receptor complex consists of a group
of molecules indicated in blue. Nearly all cells in the body are capable of displaying internally derived peptides on their
surfaces bound to MHC class I antigen (dark green). MHC class II antigens are expressed by many cells of the immune system
(light green) and bind proteins endocytosed from their surroundings. Interactions between MHC class I and II antigens and
T cells are supported by CD8 and CD4 co-receptors (violet and brown respectively)
190
(MHC) antigens. All cells constantly digest
proteins, which may include non-self proteins if
the cell is infected or has taken up foreign molecules. The peptides are loaded onto MHC
antigens and presented on the cell surface.
mune response in order to eradicate the foreign
material. While MHC class I peptides are derived from intracellular proteins, MHC class II
antigens display peptides that have been endocytosed from the extra-cellular environment.
MHC antigens are extremely polymorphic,
which means that the corresponding genes
show higher individual variation than any other
gene families. This polymorphism accounts for
different capabilities displayed by individuals
for the presentation of certain proteins. MHC
antigens loaded with peptide are transported to
the cell membrane where they are constantly
monitored by circulating T cells. Detection of
non-self proteins causes activation of the im-
Genes that encode antibodies, T cell receptors
and MHC antigens represent some of the most
complex genes studied. In fish there is much to
be done before these genes are characterised in
detail, but already we can say there are molecular and functional similarities between teleosts
and higher vertebrates [10–13]. The enzymes
and mechanisms responsible for the generation
of the almost infinite number of different antibodies and T cell receptors are also present in
Aquaculture Research: From Cage to Consumption
fish together with the machinery surrounding
the MHC antigens. Thus many of the established characteristics of the mammalian immune system can be found in fish.
Fish have three classes of
immunoglobulin
Antibody molecules are immunoglobulins composed of light and heavy glycoprotein chains. A
typical monomer consists of two identical light
chains and two identical heavy chains, the latter
of which determines the class and characteristics of the immunoglobulin. The different classes can form monomers, dimers, trimers,
tetramers, pentamers and even, hexamers.
For a long time fish were considered to have a
relatively simple immune system with only one
class of immunoglobulin, IgM, whereas mammals have IgG, IgD, IgA and IgE in addition.
However, a new immunoglobulin was cloned
from channel catfish and named IgD in 1997.
The name IgD was proposed due to its sequence
similarity with mammalian IgD and to its gene
localization i.e. adjacent to IgM. In addition
IgD and IgM can be expressed simultaneously
in the same cell, a unique property of IgD in
mammals. Circulating IgD in the blood has only
been found in channel catfish who express this
soluble form of IgD from a gene remote from
that coding for the membrane form albeit in the
same gene complex. In other teleost fish only
the membrane form of IgD transcripts has been
reported [14, 15]. The function of IgD is still
unknown in fish. Interestingly, a third class of
immunoglobulin has recently been cloned from
bony fish, designated IgT since it is found only
in teleosts. IgT can be both membrane bound
and soluble and is most similar to IgM [16, 17].
The role of IgT is also unknown at the present.
Identification of immune genes in fish: a
rapidly developing field.
In addition to salmonids other species such as
fugu, carp, Japanese flounder and zebrafish
have been prominent in the study of fish immunology during recent years. Several large-scale
sequencing projects have been initiated, providing a better basis for identification of immunerelated genes. Mapping of gene positions has
also made it possible to find many previously
unidentified genes by their comparative locations in the genome (so-called synteny analysis).
Salmonids: a duplicate set of genes.
Due to a tetraploid ancestry, many genes in
salmonid fish have two very similar copies. In
Atlantic salmon, for example, there are A and B
sub-variants of IgM, IgD and (probably) IgT
[18, 19]. The pseudo-tetraploid state of the genome in salmonid fish has been known for a
long time, but the biologically consequences of
this phenomenon are not, so far, known. It has
been speculated that salmonid fish with their
anadromous life cycle have benefited from their
pseudo-tetraploidy to adapt more quickly to
various environmental conditions.
The cod immune system
Atlantic cod has a unique position within the
Norwegian fishing industry and the efforts in
establishing cod as an aquaculture species will
hopefully give this industry an extra dimension.
Many see great potential in cod farming, but the
road ahead is still long and several multi disciplinary research programs are needed before the
domestication can be fulfilled in a scale comparable to Atlantic salmon.
Thematic area: Health
191
Variable“domain”/sites IgM & IgD
Constant domains, IgM & IgD
IgD, cod
IgD,
salmon
L-chain
H-chain
Active site
IgM, cod and salmon
Fc part
B cell
B cell
B cell
145x100//Kap11-fig06.eps
Figure 6. Comparison of IgM and IgD in salmon and cod.
A research field that has received attention for
some years are studies of the immune system in
cod, or more precisely, the antibody dependant
or humoral part of it. These studies started almost 20 years ago, and a finding that surprised
the researchers was the relatively low amount of
antibodies produced after immunisation of cod
with various antigens. Compared to salmon,
cod was clearly a non- or low-responder against
both bacterial and model antigens, although serum concentrations of immunoglobulins in nonimmunised cod was approximately 10 times
higher than in the Atlantic salmon [20, 21]. The
reason for this “discrepancy” is not known in
any detail but both regulatory and genetic issues
have been discussed.
A main issue has been to compare the antibody
repertoire in cod with the one in salmon, hoping
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Aquaculture Research: From Cage to Consumption
that such studies could explain the species difference in antibody production [22]. The antibody genes are relatively complex, but have the
same organisation “pattern” in vertebrates as divergent as fish and mammals. In short, the antibody gene cluster contains several gene
segments (V, D, J and C) with individual genes
that are combined in a complete transcript that
is translated into the H- and L-chain of the antibody molecule. What makes these molecules
special is that they possess so-called active sites
(see figure 6) which bind to particular antigen
structures / epitopes on the pathogens. In addition, the antibody “tail” or Fc part determines
the functions or immune mechanisms involved
during infection and eventually humoral immune protection.
Immunoglobulin classes
mammals
IgM
birds
amphibians
Class shift
bony fish
cartilaginous fish
IgD
IgG
IgE
IgA
IgM
IgG
IgA
IgM
IgY
IgX
IgM
IgD, IgT
IgM
NAR, NARC
145x100//Kap11-fig07.eps
Figure 7. IgM is the predominant antibody class found in fish, amphibians, birds and mammals. In teleosts IgM is found
mainly as a tetramer whereas in higher vertebrates it is a pentamer. Previously it was thought that IgD occurred late in
evolution due to a duplication of IgM but it is now known that IgD was present more than 400 million years ago. Several
lines of evidence suggest that the modern Ig classes found in mammals are present as a result of the duplication of the IgD
gene. The advent of “class switching” has been an important development of the immune system of higher vertebrates.
This involves the switching of a specific IgM antibody production to IgG, IgA or IgE production with the same specificity in
progeny cells. Recently a new class of immunoglobulin has been identified in teleosts, called IgT, which most closely
resembles IgM.
The gene segments encoding the variable (V)
part of an antibody H and L chains contain
about 100–150 genes (V, D and J genes), but the
ultimate diversity is composed of random recombination of these genes (combinatorial diversity), in addition to various mutation
mechanisms normally giving a functional diversity of at least 109 antibody specificities.
The low antibody responses in cod might reflect
a limited number of functional V-genes encoding antibody sites compared to the repertoire
possessed by i.e. the salmon. However, scientists have now characterised and compared the
genetic variation of the antibody genes in cod
and salmon [23–25] and although differences
were shown, these variations cannot fully explain the low responses in cod.
Humans produce nine different antibody classes / subclasses, bony fish produce only two or
three (isotypes) characterised as IgM, IgD and
IgT / IgZ (see above).
The studies of IgD genes in cod demonstrated a
peculiar organisation of the IgD constant (Fc)
genes compared to the ones in salmon and other
fish species [26]. It was further shown that this
antibody class does not exist in a secreted/serum form, but only as membrane bound antibody receptors on B lymphocytes. This
function of IgD is probably the same in cod and
salmon. The peculiar organisation of the IgD
constant genes in cod does not fully explain
why cod B-lymphocytes do not express a proper
IgD receptor diversity, because membrane
bound IgM and IgD receptor molecules share
the same antibody V-gene repertoire. Altogeth-
Thematic area: Health
193
er, the antibody gene repertoire in cod and salmon B-lymphocytes seem similar and is probably
not responsible for the diverse antibody responses in these two species.
As already mentioned, cod express much higher
natural immunoglobulin concentrations in
blood than salmon [20, 21]. These immunoglobulins or antibodies do not, however, reflect a
response towards known antigens or pathogens
from natural infection or immunisation. It is not
known whether antigens such as bacterial components taken up through the intestines may
stimulate such production as is probably the
case in mammals. The immunoglobulin production happens either through a massive stimulation of the repertoire of specific (BCR)
receptors, or more likely, by the pan-B-cell or
mitogen receptors present on these cells. An interesting question is, whether these “unspecific” antibodies protect against pathogenic
infections and represent a “natural” immune
strategy in cod? As haddock and coalfish are
also low antibody responders, this may reflect a
common “strategy” of the gadoid species. Further investigations are needed to verify this “hypothesis”.
It should also be mentioned that the low responsiveness in cod is not absolute, as recent data
have shown that the bacterial fish pathogen
Aeromonas salmonicida expresses antigens that
to some degree stimulate antibody production
[27]. Since the antigen “nature” is unknown, it
is too early to speculate about tentative mechanisms of this stimulation.
Stimulation of B cells and antibody production
is only one of several immune reactions against
pathogens and belongs to the so-called “adaptive” arm of the immune system. These mechanisms include immunological education and
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Aquaculture Research: From Cage to Consumption
memory, which means that antigens / vaccines
“educate” the immune system to remember the
antigen structure and mount stronger reactions
when the antigen reappears i.e. during a secondary infection. Stimulation of B-lymphocytes
with protein antigens also includes stimulation
of T-lymphocytes providing helper molecules
(cytokines) important for B-cell proliferation
and differentiation into plasma cells. The Tcells express specific antigen receptors (TCR)
encoded by genes related to the antibody genes
and with a similar number of V-genes expressing the TCR repertoire (see above). As the Band T- lymphocytes cooperate during an antibody response, it could be that a limited antibody response is caused by inefficient
stimulation of T cells and a subsequent lack of
helper molecules important for B-cell stimulation and proliferation into antibody producing
cells. Although the mechanisms for T and B cell
cooperation in cod and other fish species have
not been studied in sufficient detail, the genes
encoding the cod TCR are well characterised.
Neither the diversity nor the organisation of the
cod TCR genes differ substantially from the
ones in other fish species, including salmon. As
also mentioned above, a third cell type, the antigen presenting cell (APC; macrophages, dendritic cells etc.) plays an important role in
immune stimulation by presenting antigen peptide fragments to the T–cell receptor. The APC
phagocytose (“eat”) antigens / pathogens and
process protein antigens into peptides which are
then bound to the MHC molecules, and present
this peptide-MHC complex to the TCR on Tlymphocytes. Although MHC molecules exist
as two classes (MHC I and MHC II), only the
class II is involved in stimulation of the T-lymphocytes and helping the B-cell in antibody
production. Thus, if cod lack or possess crippled MHC II molecules, stimulation of cells involved in antibody responses will be subopti-
mal and result in low levels of specific antibodies [28]. In support of this idea, several
research groups searching for MHC genes in
cod have so far discovered only the MHC I
class, but a final conclusion in this issue awaits
further gene screening, eventually genomic sequencing.
Over the years many vaccine experiments have
been performed against bacterial pathogens
causing cold-water vibriosis (Vibrio salmonicida), classical vibriosis (Vibrio anguillarum) and
atypical furunculosis (atypical Aeromonas salmonicida). The overall conclusion is that vaccinated cod is protected against disease to the
same degree as vaccinated salmon [29]. The
mechanisms involved in this protection are not
known, but commercial vaccines are now under
way and will hopefully be as valuable as vaccines developed for the salmon farming industry.
The lack of specific antibodies after immunization of cod has challenged scientists for almost
two decades, and has also led to studies of the
innate arm of the fish/cod immune apparatus.
The best studied molecule so far is the so-called
“Bacterial Permeability Increasing Protein”,
BPI [30] and later also the lysozyme C molecule. These molecules are well known from
other species to protect against bacterial infections, and presumably also in cod. The search
for “new” molecules and potential immune
mechanisms against bacterial infections in cod
continue, and hopefully, the future cod farming
industry will benefit from this research to the
same extent as for established aquaculture species.
Prospects and challenges
The molecular characterisation of the fish immune system is important for future applications not only for improvement of vaccination,
but also for description of various types of immune responses of fish to different stimuli. The
regulation of immune genes is both dependent
on the type of pathogen that the animal encounters and on the physiological and immunological state of the animal. In human medicine
blood tests have long been used to trace immune responses that might indicate early development of disease. One example is the test for
C-reactive protein, which is an indicator of bacterial infection. So-called “immune profiling”
may also be applied within aquamedicine in the
near future. Mx, IFN and other molecules may
be important as molecular markers for the
health status of the fish. Such markers may be
used to, reveal conditions that down-regulate
the immune system of the host and indicate how
to stimulate the defence against viruses during
critical stages of production. Information about
immune genes may also be useful for breeding
programs.
The research on the IFN system of Atlantic
salmon has revealed strengths and weaknesses
in the innate immune defence against IPN- and
ISA-virus, two of the most important viral
pathogens in the Norwegian aquaculture industry. Future experiments will study how these
viruses counteract the IFN-system of salmon.
From mammalian research it is well known that
the IFN-system collaborates with the adaptive
immune system to combat infection. IFN and
IFN-stimulating compounds can function as adjuvants by augmenting the protective effect of
vaccines. This area of research also deserves
more attention in aquamedicine. So-called
CpG-oligonucleotides represent one of the most
Thematic area: Health
195
promising vaccine adjuvants and may also be
beneficial in fish vaccines since recently they
have been shown to induce Mx protein and protection against IPN-virus infection in salmon
[30].
The research on fish antibodies has made good
progress during the last 30 years. Research has
also established that fish have a T cell mediated
adaptive immune system similar to that of
mammals. Although methods for measuring
antibody responses have been available for a
long time, methods for measuring T cell mediated immune responses in fish are still lacking.
The importance of continued research on the
adaptive immune system of salmon and cod is
underlined by the fact that protection obtained
by vaccination does not always correlate with
the concentration of circulating IgM antibodies.
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