1

CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
1
Infectious Pancreatic Necrosis and
Associated Aquatic Birnaviruses
P.W. Reno
Coastal Oregon Marine Experiment Station, Department of Microbiology,
Laboratory for Fish Disease Research, Mark O. Hatfield Marine Science
Center, Newport, Oregon 97365-5296, USA.
INTRODUCTION
Infectious pancreatic necrosis (IPN) is a disease which causes high mortalities in
young salmonid fishes; the aetiological agent of the disease (IPNV) was the first
virus to be isolated from teleosts (Wolf et al., 1960). In the three decades since
that work, agents biochemically and serologically identical to IPNV the
aquatic birnaviruses have been detected in a wide variety of both diseased and
non-diseased fishes and other aquatic animals.
The early work on IPNV was mainly responsible for the development of fish
virology. Techniques for the isolation of viruses from teleosts were modified
from methods used for the isolation of mammalian viruses. However, we now
know that IPNV and its interactions with the host are unique in several respects.
1. The virus itself has sufficiently unusual biophysical characteristics to warrant
its place as the archetype of a new family of viruses, the birnaviruses (Dobos et
al., 1979).
2. The carrier state associated with IPNV infection is unique among animal
diseases. The infection is lethal only to young animals, although virus can be
readily isolated in high concentrations from the viscera of infected fish during
the lifespan of the host, and there is no significant disease recrudescence, as in
herpetic infections, etc.
3. Serologically and biochemically related viruses induce a variety of disease
syndromes in taxonomically diverse host groups, which also include aquatic
invertebrates. The worldwide distribution of these viruses in cultured as well as
wild fishes and shellfish provides significant potential for economic damage.
Infectious pancreatic necrosis and other birnavirus-induced diseases can
cause a significant economic impact on cultured fishes, especially salmonids.
The cost can be as a consequence of lethal disease in young-of-the-year fish, or
the destruction of infected stocks even in the absence of disease. The virus is
vertically transmitted; therefore, the detection of virus in broodstock, even in the
2
P.W. Reno
absence of disease, often means the destruction of these valuable animals. Even
as recently as the late 1980s, there were instances in the USA where 7 million
young salmonids were destroyed to eliminate IPNV from a single hatchery (P.
Walker, Colorado, 1988, personal communication) and others where detection of
the virus in returning coho salmon resulted in the destruction of 2 million eggs
(Olson et al., 1994); similar occurrences were also reported in Canada (B.
Larson, Alberta, 1988, personal communication). In addition, regulations
currently in place to reduce the dissemination of diseases of salmonids often
result in restriction of the movement of IPNV carrier fish between local, regional
and/or national boundaries. These restrictions have attendant adverse economic
consequences to aquaculturists. In many instances, the slaughter of infected fish
and/or the restriction of movement of fish may be more economically
detrimental than the loss associated with direct mortalities.
The mandated destruction of salmonid stocks and restriction of movement
due to IPNV are controversial. From one perspective, the disease itself is not
highly destructive, although it has been in the past, and therefore need not be
regulated. Since only swim-up fry and fingerlings are generally affected, the
impact of the disease can be ameliorated by spawning additional brood fish to
compensate for the potential loss caused by the disease. Another perspective is
the dissemination into watersheds of a virus unresponsive to therapeutic agents
which can readily infect susceptible native or cultured stocks in adjacent
drainages. Given the myriad variables which come into play in the generation of
IPN and the potential for extensive, irreparable damage to stocks, it would seem
prudent to adhere to more stringent rather than lax requirements. Resolution of
the problem of IPNV management will be difficult and achieved only through a
thorough knowledge of the epizootiology of the disease and an adequate
database on both the disease and the virus. Many of these are unknown at
present.
Infectious pancreatic necrosis virus may also have an economic impact on
aquaculture because of fish health inspections on certain stocks of fish. Due to
the occult nature of the infection, these inspections are frequently mandated
prior to the movement or sale of fish across local, regional and national
boundaries. Tests required for the detection of IPNV and other teleost viruses
often represent a large out-of-pocket expense for aquaculturists. Again, this can
be a contentious issue because the regulations are not often applied uniformly,
even within regions regulated by one agency.
With the advent of molecular biological techniques, researchers are close to
the development of a vaccine for IPN, but the intricate lifestyle of the virus may
provide a considerable stumbling-block to disease prophylaxis. Future methods
designed to control the disease worldwide will also be difficult, given the ubiqui-
tous nature of the agent. Only the most stringent controls on the movement of
cultured fish, based on detection of the virus by the most sensitive and specific
diagnostic techniques, will lead to the successful elimination of this pathogen.
A number of reviews have been published on IPN disease and IPNV. The
extensive publication base includes more than 150 printed articles in the last
decade. In this review, I shall include more recent data and integrate information
into a logical summary.
3 Infectious Pancreatic Necrosis
THE DISEASES AND AGENTS
Aquatic birnaviruses are the most pervasive pathogens of aquatic animals. They
have been isolated from teleosts as well as aquatic invertebrates of freshwater,
brackish and sea-water environments. The ubiquitous nature of these agents and
the lack of association with disease have led to difficulty in nomenclature. In
order to maintain a consistency in this review, the term IPNV virus will be used
strictly to describe those isolates which have been shown to produce disease in
salmonids and aquatic birnaviruses will be used for those which are either
associated with other disease states or have not been shown to be the causal
agents of naturally or experimentally induced disease in aquatic animals.
HOST RANGE
Disease
Early reports of IPNV were limited to epizootics in cultured brook trout,
Salvelinus fontinalis (Wood et al., 1955; Snieszko et al., 1957, 1959; Wolf et al.
1960). With the development of several continuous cell lines from teleost fishes
(Wolf and Quimby, 1962; Wolf and Mann, 1980) and as more laboratories
became more proficient in the use of cell lines, it was found that IPNV was
responsible for disease in a variety of salmonid species, including members of
the genera Salmo, Salvelinus and Oncorhynchus. During the early 1970s, IPN
caused high mortalities in European and Japanese rainbow and brown trout (Ball
et al., 1971; Sano, 1971; Vestergrd-Jrgensen and Kehlet, 1971). More
recently, epizootics of diseases not characteristic of salmonid IPN have been
associated with birnavirus infections in epizootic proportions (Table 1.1).
The first report of birnavirus-induced disease in non-salmonids was in
Japanese eels (Anguilla japonica), first sampled in 1969 (Sano et al., 1981). This
disease of cultured eels was characterized as branchionephritis. Experimental
infection with the virus (eel virus European (EVE)) indicated that the branchial
lesions were probably due to supervening bacterial infections and that the
disease should be referred to as eel nephritis. Neutralization studies indicated
that the aetiological agent was a birnavirus which is serologically related to the
Sp (from Spjarup, Denmark) serotype of IPNV. Another birnavirus-induced
disease which caused mortalities in Japanese fish caused ascites in yellowtail,
Seriola quinqueradiata (Sorimachi and Hara, 1985). The condition was in feral
fry used as seed stock for mariculture. Because clinical signs occurred in fry, the
disease is similar to IPN. In addition, mortalities occurred in another flat-fish
species from Japan, the Japanese flounder (Paralichthys olivaceus). Fish held in
culture facilities died from ascites and cranial haemorrhage. An aquatic
birnavirus was isolated from moribund fish and an ascitic disease was
experimentally induced by intraperitoneal injection of the virus from moribund
fish showing either disease syndrome (Kusuda et al., 1989).
Stephens et al. (1983) isolated an aquatic birnavirus from the brain and other
tissues of menhaden (Brevoortia tyrranus) with spinning disease, a perennial
4
P.W. Reno
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6
P.W. Reno
high-mortality disease in the Chesapeake Bay region of the eastern USA. The
virus was isolated in a cell line from the same species (Stephens, 1981). The
disease was experimentally induced in susceptible menhaden after
intraperitoneal injection of the virus. Subsequent attempts to isolate the same
virus from diseased menhaden in other teleost continuous cell lines were
unsuccessful. Since the original menhaden cell line was lost, there have been no
further isolations of the virus from diseased or non-diseased fish.
In each of these cases, Rivers postulates have been fulfilled, indicating that
the aetiological agents of the diseases were aquatic birnaviruses which
cross-reacted serologically with IPNV. In several other instances, birnaviruses
have been isolated from aquatic animals undergoing epizootic disease, but as yet
there has not been irrefutable evidence that the isolated birnaviruses were causal
agents (Table 1.1).
A birnavirus infection was associated with haematopoietic necrosis and
caused high mortalities in turbot (Scophthalmus maximus) held at 18C in sea
water on the coast of France (Castric et al., 1987). Within 15 days after transfer
from fresh water to sea water, cumulative mortalities reach 25%, but no
mortalities occurred in the same population of fish held at the freshwater site. No
definitive studies have been done to confirm Rivers postulates for this disease
and a birnavirus is suspected.
In addition to a birnavirus isolated from Japanese eels with nephritis (Sano
et al., 1981), a birnavirus was also isolated in Japan from eels exhibiting
stomatopapillomas (Nagabayashi and Wolf, 1979). Although the virus was
isolated from moribund fish, there was no indication that the virus was capable
of producing the papillomatous lesions in homologous animals.
Transmitting papillomas in homoeotherms in often difficult, and the strong
association of the papilloma group of viruses with this disease in other animals
makes it unlikely that a birnavirus was the causal agent in eels. In retrospect, it is
possible that the eels affected with the stomatopapillomas were adventitious
carriers of the virus (Sano et al., 1981) and the isolated aquatic birnavirus had no
involvement in the development of papillomas.
An aquatic birnavirus belonging to the Sp (European) serotype was isolated
from visceral organs of snakehead fish (Ophicephalus striatus) and eyespot barb
(Hampala dispar) undergoing episodes of ulcerative disease in Thailand and
Laos (Wattanavijarn et al., 1988). Again, there is no further information to
indicate that this agent was responsible for the syndrome.
Another birnavirus was isolated from sea bass (Dicentrarchus labrax) off
the coast of France (Bonami et al., 1983) during a severe epizootic, in which
mortalities reached 95%. There was no evidence of bacterial infection or
parasites in the moribund fish, but again it is uncertain whether the virus is
merely associated with the mortality or was the aetiological agent.
With the rather large exception of IPN disease, the disease syndromes that
have been strongly associated with aquatic birnaviruses (yellowtail ascites,
Japanese flounder disease syndromes, eel nephritis, spinning disease of
menhaden and turbot renal necrosis) occur in marine animals or in those that
spend the bulk of their life in sea water. The IPN which affects the anadromous
Salmo and Oncorhynchus also fits into this category, although the susceptible
7 Infectious Pancreatic Necrosis
stage occurs in fresh water rather than in the marine environment. Recently,
postsmolt salmon died from an IPN-like disease in marine cage culture;
however, there was a confounding infection with the agent of pancreas disease
of an IPNV (Smail et al., 1992). Although the titres of virus from moribund fish
were not reported, titres were approximately 10
4
g
1
tissue 4 weeks after the
epizootic. This is low compared with the titres in trout succumbing to IPN
disease (Wolf et al., 1969; Reno, 1976).
It is likely that diseases caused by aquatic birnaviruses are more extensive in
nature because reports of this family of viruses are primarily in cultured animals,
which have economic importance. Relatively little information is available
regarding birnavirus infections in wild populations of aquatic animals that are
not of commercial importance and this is unlikely to change unless some of these
animals develop commercial importance.
Virus
Birnaviruses are the most ubiquitous pathogenic microorganisms in aquatic
species. During the 1960s, following on the heels of Wolfs early work on the
isolation and characterization of IPNV, many investigators detected the virus in
salmonids, especially those undergoing epizootics of frank disease. During the
1970s and 1980s, with closer scrutiny of cultured and wild non-salmonids, many
isolations of aquatic birnaviruses were made from a large number of aquatic
animals.
In the first report on birnaviruses in non-salmonids, Sonstegrd et al. (1972)
detected IPNV in common suckers (Catostomus commersoni) with no clinical
disease. They were resident in a tailway of a trout hatchery with perennial IPN
epizootics. It is a highly probable that the viruses in the suckers were from the
fish in the hatchery. Since that time, birnaviruses have been detected in nearly 80
species of aquatic animals (Table 1.2). Although these viruses belong to the same
serotype as IPN, most isolations were from apparently healthy animals.
Infectivity experiments have rarely been conducted to determine if the isolates
were capable of replicating in the homologous host. Most often only biophysical
and serological characterizations of the isolate were performed to confirm that
the agent was a birnavirus. There was little information on the virulence of these
viruses.
Several authors have attempted to determine if the isolates from non-
salmonid species are capable of inducing IPN disease in trout. It is difficult to
evaluate these experiments since a variety of exposure protocols were used.
Several investigators have standardized techniques for inducing IPN disease in
aquatic birnaviruses (Bootland et al., 1986; McAllister and Owens, 1986). In
some cases, aquatic birnaviruses from non-salmonids could produce classic
IPN disease in salmonids (Table 1.3). In 1965, Yasutake et al. demonstrated that
IPNV isolated from rainbow trout caused overt disease in chinook salmon
(Oncorhynchus tshawytscha). However, the birnaviruses from eels were
incapable of inducing disease in trout, although they were virulent in the
homologous species, A. japonica (Sano et al., 1981). Similar results were
8
P.W. Reno
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9 Infectious Pancreatic Necrosis
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11 Infectious Pancreatic Necrosis
reported in isolates from striped bass (Wechsler et al., 1986b), flounder
(McAllister and McAllister, 1988) and oysters (Hill et al., 1982).
GEOGRAPHICAL RANGE
Disease
While the disease was evidently first described from the Maritime Provinces,
Canada (MGonigle, 1940), and soon thereafter in the eastern USA (Wood et al.,
1955), it was later noted in many areas of the western USA (Yasutake et al.,
1965), especially in rainbow trout and brook trout transferred from the eastern
USA. Epizootics in rainbow trout with signs of IPN disease reported in France
(Besse and de Kinkelin, 1965) were caused by an aetiological agent which was
serologically distinct from the North American serotype (Wolf and Quimby,
1971). The detection of IPN in France was followed by diagnosis of
disease in rainbow trout in Denmark (Vestergrd-Jrgensen and Bregnballe,
1969), Norway (Hstein and Krogsrud, 1976), Sweden (Ljungberg and
Vestergrd-Jrgensen, 1973), the UK (Ball et al., 1971), Germany (Schlotfeldt
et al., 1975) and Italy (Ghittino, 1972). Outside Europe, the disease has been
documented in the Far East, usually in fish imported from enzootic areas or in
fish cohabiting the same water source as infected imported fish. The virus has
been isolated from Japan (Sans, 1971), Korea (Hah et al., 1984), Taiwan
(Hedrick et al., 1983a China (Jiang et al., 1989) and Thailand and Laos
(Wattanavijarn et al., 1988).
Virus
The isolation of aquatic birnaviruses from various geographical areas has
increased markedly since the isolation of IPNV from the eastern USA (Wolf et
al., 1960). In a few instances, there has been sufficient epizootiological evidence
to support the orderly spread of the virus from region to region. The ubiquitous
nature of aquatic birnaviruses on a worldwide scale is indicated in Figs 1.1, 1.2
and 1.3. When the viruses were serotyped, they proved to be serologically
identical to serotypes of IPN from epizootics (Caswell-Reno et al., 1986; Hill
and Way, 1995).
These agents have been isolated from regions where salmonids are reared
and from areas without salmonids. The data are often difficult to access and
summarize easily because of the inconsistent nature of the published literature
and the variety of sources from which samples have been taken. The presence of
these viruses in feral animals is a confounding factor. Since little current
information about the distribution of aquatic birnaviruses is available in the
literature, an attempt has been made in this review to update the information
(Figs 1.11.3).
Prior to the late 1980s, the virus was present in most areas of North America.
In many areas, the prevalence levels were low, but in some areas (Maritime
12
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13 Infectious Pancreatic Necrosis
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14
P.W. Reno
Canada, Pennsylvania, West Virginia and some north-eastern US states) a large
number of hatcheries were contaminated. However, because of increased
awareness of vertical transmission of the virus and regulations on movement of
fish eggs, the majority of states and provinces have significantly reduced or
eliminated the virus from hatcheries (Figs 1.1 and 1.2). There has been a marked
reduction in reports of the virus, especially in the western USA and eastern
Canada. This may be due to the reduction in rearing of brook trout, which serve
as reservoirs for the virus in the western states.
Unfortunately, the situation in Europe and Asia is not as positive (Fig. 1.3).
The virus has been found in virtually every European country (de Kinkelin,
1989, personal communication), with almost total contamination of facilities in
some countries (Melby et al., 1991). The pervasive dispersal of the virus in
Europe may be due to the transfer of contaminated stocks and an inability to start
with virus-free stocks without destroying the industry. The presence of IPNV in
trout is often considered an impediment to the industry, not because of the
consequences of disease but rather because of the restriction of fish movements
(Ghittino, 1982).
The virus and epizootic outbreaks of the disease are widespread in
salmonids in Asia, where importations from other countries, especially the USA
and Europe, have been extensive. Most probably, the increased detection of
IPNV in this region reflects an increased dispersal of the virus, an increased
ability of fish health workers to isolate the viruses, and the examination of a
wider variety of aquatic animals for birnavirus infection.
Iceland and Australia are the only countries which raise salmonids on a large
scale and where extensive fish health inspections are routinely performed that
have no reports of aquatic birnaviruses. Both countries have very strict
importation policies for fin-fish. It is interesting to note that, although Australia
is free of IPNV, chinook salmon returning to rivers in New Zealand were found
to harbour IPNV of the Sp serotype (Tisdall and Phipps, 1987). This chinook
salmon stock was imported into New Zealand from Japan, a country with
enzootic IPN.
The serotype A1 (WB archetype from brook trout in West Buxton, Maine,
USA) is found extensively throughout the USA and on many occasions in
Canada. However, the Canadian serotypes have rarely been isolated in the USA
(Caswell-Reno et al., 1990). The isolations of A1 viruses from other countries
have almost always been traced to the importation of specific batches of
contaminated eggs or fish from the USA (Sano, 1971; McAllister and Reyes,
1984; Bragg and Combrink, 1987a). Interestingly, the A1 serotype has only been
isolated from Europe in a single instance from turbot in Spain (Novoa et al.,
1993) although trout have been extensively transferred to Europe from the
USA. Serotypes A2 (Sp) and A3 (Ab from Abild, Denmark) have been
disseminated widely not only throughout Europe, but also to many countries in
Asia. Serotype A2 has been detected only once in feral fish in marine waters off
the coast of the USA (McAllister et al., 1984). The four serotypes Canada 1, 2
and 3 and Jasper, Alberta, are restricted to Canada, except for a few recent
isolations from facilities in watersheds that are contiguous in the USA and
Canada (Caswell-Reno et al., 1990). Although fish have been exported from
15 Infectious Pancreatic Necrosis
Fig. 1.1. Frequency of isolation of aquatic birnaviruses in the USA.
16
P.W. Reno
Fig. 1.2. Frequency of isolation of aquatic birnaviruses in Canadian Provinces.
Fig. 1.3. Frequency of isolation of aquatic birnaviruses in other countries.
17 Infectious Pancreatic Necrosis
Canada to many other countries, none of the Canadian 1, 2 or 3 serotypes have
been found outside of Canada or its borders with the USA. Thus, the distribution
of serotypes does not seem to support the hypothesis that the aquatic
birnaviruses are spread from continent to continent with the importation of fish
or fish eggs. It is likely that the virus had a global distribution prior to the
widespread dissemination of salmonids in the nineteenth and the early twentieth
century.
GENERAL SIGNS OF THE DISEASE
The clinical signs of diseases caused by different aquatic birnaviruses are
markedly different (see Table 1.1).
Infectious pancreatic necrosis virus causes mortality of fry and fingerling
salmonids and is characterized by behavioural changes and gross external,
internal and histopathological lesions. There are no specific pathognomonic
signs of IPN disease. Behavioural changes (Wood et al., 1955) include anorexia
and an agonal corkscrew swimming motion interspersed with ataxia. Non-
specific external signs include hyperpigmentation, exophthalmia and petechial
haemorrhage on the ventral surfaces. Internal gross lesions are visceral petechia
and an empty gut containing a yellow exudate. The disease may be manifested
with only a few, or even none, of these signs. Microscopically, there is focal
coagulative necrosis of the acinar and islet cells of the pancreas and of the
haemopoietic cells of the kidney. There are typical icosohedral virus particles
in the cytoplasm of pancreatic acinar cells (Lightner and Post, 1969; Hedrick
et al., 1985). Viral titres in the tissues of infected fish are usually quite high,
usually in the range of 10
7
10
10
(TCID
50
) g
1
(Wolf et al., 1969; Castric et al.,
1987).
Survivors of epizootics or those fish with no disease have few effects of
residual infection, although high titres of virus can be isolated from their viscera.
In carrier Atlantic salmon there are interstitial cells in the lamina propria of the
gut which appear necrotic, the so-called McKnight cells (McKnight and
Roberts, 1976). Studies examining the effect of IPNV infection on Atlantic
salmon which evince no clinical signs of IPN indicate that there is no adverse
effect on growth rate or susceptibility to other diseases (Smail et al., 1986).
Several studies have demonstrated that there are markedly variable virus
titres in carrier fish. Yamamoto (1975b) found that rainbow trout had IPNV titres
in the viscera ranging from 10
0.85
to 10
4.2
TCID
50
g
1
, while they ranged from
10
0.85
to 10
6.5
TCID
50
g
1
in brook char (Reno, 1976).
The virulence of IPNV isolates is quite variable. Most have been isolated
from aquatic animals with no evidence of disease. Only a few isolates from non-
diseased animals have been tested for virulence (Table 1.3). Wolf et al. (1969)
demonstrated differences in virulence with 15 serotype A1 (WB) isolates by
infecting brook trout fry. Mortalities ranged from 7 to 98%. The viral titre in the
mortalities was approximately 10
9
g
1
; there was no correlation between the titre
and mortality levels (regression analysis of data of Wolf et al. 1969).
Unfortunately, the titre of virus in the more resistant or in surviving fish was not
18
P.W. Reno
reported in rainbow trout infected with three European strains: Sp, Ab and Hecht
(He), the isolate from pike. Hecht was avirulent, while the two trout isolates
were causing significant mortality, albeit at different levels; virulence was
directly correlated with plaque size (Kohlmeyer et al., 1986). Interestingly, after
11 passages in vitro, the more virulent Sp strain lost much of its virulence. This
variation in virulence is a reflection of the complex nature of the disease, which
is not well understood. The two European serotypes are generally classified as
virulent for trout (Sp) or avirulent for trout (Ab) (Vestergrd-Jrgensen and
Kehlet, 1971).
The disease IPN primarily affects fry and fingerlings and rarely affects
yearling or older fish (Wolf et al., 1960; Elazhary et al., 1976). Predisposing
factors include temperature, age and stress (Frantsi and Savan, 1971a;
LaPierre et al., 1988). Six-month-old brook trout at 3 g are resistant to clinical
IPN disease; however, it is not known whether the host had cleared the virus
entirely or whether residual virus was present, as in typical IPN carriers.
Temperatures at which maximum mortalities occur vary, but temperature
effects may be obscured by the strain of virus and host species used: e.g. brook
trout infected with a Canadian isolate had maximal mortality at 10C, while
those infected with IPNV VR-299 succumbed most readily at 15.5C (Frantsi
and Savan, 1971a). In the epizootic affecting turbot, mortalities were markedly
higher in juvenile fish held in sea water at 18C than at 11C (Castric et al.,
1987). Similar results were obtained by Okamoto et al. (1987c) in rainbow
trout fry.
Nephritis in eels, caused by a birnavirus termed eel virus European (EVE),
occurs primarily during the winter, when water temperatures are lowest (Sano et
al., 1981). The compromised physiological condition of the fish during this
period may be a factor in the disease process (Egusa et al., 1971; Oka et al.,
1976).
The severity of the disease is dependent not only on the species of fish, but
also on the strain of fish within a species. Silim et al. (1982) demonstrated that
strains of brook trout from four genetic pools varied in their susceptibility to
IPN, with mortalities ranging from 40 to 85%. Similarly, Okamoto et al. (1987b)
found that rainbow trout from one hatchery (strain RT-101) were consistently
more susceptible than fish from two other hatcheries to lethal IPNV infection
over a course of 6 years. Additionally, these authors demonstrated that the
quantity of virus to which fish were exposed influenced mortalites. No
mortalities occurred in 0.26 g fish exposed to less than 10
2
TCID
50
ml
1
of
IPNV-Buhl, whereas 61% of fish died when exposed to 1000-fold higher virus
concentrations. The relationship between infection of salmonids and severity of
clinical disease is complicated and the IPNV carrier state has been difficult to
elucidate.
The diseases known as yellowtail ascites, Japanese flounder ascites and eel
nephritis are caused by birnaviruses. The clinical signs associated with these
diseases are listed in Table 1.1. All are severe and cause high mortalities in
cultured flat-fish and eels in Japan. In addition, three more diseases have been
found to be associated with birnaviruses, although unequivocal proof of their
involvement in the promulgation of the diseases is lacking. These diseases cause
19 Infectious Pancreatic Necrosis
sea-bass mortalities (Bonami et al., 1983), turbot haemopoietic necrosis (Castric
et al., 1987) and spinning disease of menhaden (Stephens et al., 1983).
THE AGENT
There are several excellent reviews on the biophysical and biochemical
characteristics, especially the molecular biology, of the birnavirus (McAllister,
1979; Dobos and Roberts, 1982; Wolf, 1988; Dobos, 1995). Consequently, the
emphasis in the present discussion will be placed on characteristics that may
play a role in the disease process and epizootiology.
Physicochemical characteristics
The virus is stable and it retains more than 90% of its infectivity after treatment
with chloroform or ethyl ether, at pH 3.0 for 60 min or 60C for 30 min (Roberts,
1975). It survives for nearly a year at 4C, for nearly 2 months at 15C in buffer
(Dorson, 1982) and for long periods in sea water, brackish water and unsterilized
fresh water (Toranzo and Hetrick, 1982). These characteristics are common for
icosohedral, unenveloped ribonucleic acid (RNA) viruses and early workers
suggested that IPNV most closely resembles members of the reovirus group.
After closer analysis of the genome (Dobos, 1976), it became apparent that
IPNV differs significantly from the reoviruses in its genomic constitution and
that it possibly warranted a new taxonomic niche.
The establishment of a new group of viruses, the birnaviruses (Dobos et al.,
1979), was based on the salient characteristics of IPNV: unenveloped
icosohedral viruses approximately 60 nm in size, containing a double-stranded,
bisegmented RNA genome, with a sedimentation coefficient of 435 S and a
buoyant density of 1.33 g cm
3
(Cohen et al., 1973; Dobos et al., 1977). The
group also includes the infectious bursal disease virus of gallinaceous birds and
the Drosophila X virus (Dobos et al., 1979).
The structural polypeptides associated with the virion fall into three size
classes: large approximately 100 kDa which is the putative RNA polymerase
(VP1); medium approximately 50 kDa which is the predominant capsid
protein (VP2); and small approximately 30 kDa also capsid proteins (VP3
and VP4) (Cohen et al., 1973; Dobos et al., 1977). Found in serotype A1 (WB)
viruses, VP4 is a post-translational cleavage product of VP3 and is structurally
similar to it (Dobos and Rowe, 1977; Dobos, 1995). The stoichiometric ratios of
the V1V4 polypeptide molecules in the virion are 4 : 24 : 17 : 14, respectively.
There is some variation in the apparent molecular weights of virion polypeptides
in viruses isolated from different species of aquatic animal and from different
geographical areas, this indicates some strain differences (Table 1.4). The
polypeptide VP2 is the major protein on the surface of the virus, as detected by
polyacrylamide gel electrophoresis (PAGE) and fluorescein isothiocyanate
labelling. It may contain carbohydrates (Estay et al., 1990), although there are
conflicting reports on this subject. There are sites on VP2 which could be
20
P.W. Reno
potential glycosylation sites (Mason, 1992), but the evidence for actual
glycosylation is not convincing (Estay et al., 1990; Kellerag et al., 1994).
Portions of VP3 are also at the virion surface, since many monoclonal antibodies
react with epitopes on this virion protein (Caswell-Reno et al., 1986; Wolski et
al., 1986; Lipipun, 1988; Chi et al., 1991; Shankar, 1991). In addition, infected
cells are known to contain an virion-encoded protease, which performs the
cleavages necessary for the maturation of the virion (Duncan et al., 1987;
Dobos, 1995).
Likewise, genomic analysis indicates that the molecular weights of the two
genome segments are similar (approximately 2.2 and 2.3 mDa), although they
also vary somewhat from isolate to isolate (Hedrick et al., 1983a) (Table 1.5).
The smaller genome segment (segment A) is polycistronic and codes for the two
capsid proteins; the larger segment (segment B) codes for the RNA-dependent
RNA polymerase (MacDonald and Dobos, 1981; Duncan et al., 1987; Lawrence
et al., 1989).
The bisegmented, double stranded RNA (dsRNA) genomes of different
aquatic birnaviruses can be discriminated from one another by acrylamide gel
electrophoresis since there are small differences in the molecular weights of the
segments (Table 1.5). When performed by one investigator, analysis of RNA
from several isolates of birnaviruses shows strong internal consistency;
however, there is less consistency when the results of RNA molecular weight
analysis of different authors are compared. For this reason, the comparison of
absolute molecular weights is inappropriate for classification, but comparisons
can be made for relative relationships among isolates.
Serological characteristics
While there are only minor differences in the biochemical composition of
aquatic birnaviruses, the serological relationships among them are complicated
and the problems of serological classification are compounded by the lack of
uniformity in analytical methods. Serological characterization of this group of
viruses is important for epizootiological reasons as well as for the development
of vaccines.
The first indication of the existence of distinct serotypes of aquatic
birnaviruses was that antibodies produced against viruses isolated from the USA
(American Type Culture Collection No. VR-299) incompletely neutralized two
new isolates from France (Wolf and Quimby, 1971). The French isolates are now
known to belong to the Sp (A2) serotype (Hill and Way, 1983, 1995). The
increasing number of isolations of aquatic birnaviruses from a broad variety of
host species and differing locations generated a number of studies which
serotyped the new isolates, but these studies usually involved a comparison with
VR-299 and perhaps a few other isolates (Leintz and Springer, 1973; Macdonald
and Gower, 1981; McMichael et al., 1975; Okamoto et al., 1983a). These viruses
are divided into three distinct serotypes: VR-299 from the USA and Sp and Ab
from Europe. While a number of serological tests have been used to classify
aquatic birnaviruses (see below for methods used), the reciprocal
21 Infectious Pancreatic Necrosis
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22
P.W. Reno
Table 1.5. Molecular weights ( 10
6
Da) of genome segments A and B of
aquatic birnaviruses and a non-aquatic animal birnavirus, infectious bursal
disease virus.
Segment Segment
Isolate Species Country Serotype A B Reference
VR-299 Rainbow trout USA A1 2.4 2.2 1
VR-299 Rainbow trout USA A1 Not detected 2.4 2
VR-299 Rainbow trout USA A1 2.4 2.1 3
VR-299 Rainbow trout USA A1 2.4 2.0 4
MH Menhaden USA A1 2.5 2.3 5
KRBT Rainbow trout Taiwan A1 2.4 2.0 4
Sp Rainbow trout Denmark A2 2.2 2.0 6
Sp Rainbow trout Denmark A2 2.3 2.0 3
Sp Rainbow trout Denmark A2 2.2 2.0 4
EVE J apanese eel J apan A2 2.2 1.9 2
EVE J apanese eel J apan A2 2.2 1.9 3
EVE European eel Germany A2 2.6 2.4 7
LKE J apanese eel Taiwan A2 2.1 1.9 4
LKT Tilapia Taiwan A2 2.1 1.9 4
DSE J apanese eel Taiwan A2 2.1 2.0 4
SGV Sand goby Thailand A3 2.4 2.0 6
Ab Rainbow trout Denmark A3 2.1 2.0 6
Ab Rainbow trout Denmark A3 2.3 1.9 3
Ab Rainbow trout Denmark A3 2.1 1.9 4
OV-3 Oyster UK A6 2.2 2.6 1
LTV Lake trout Canada A9 2.1 1.9 8
TV-1 Tellina UK B1 2.2 2.4 1
IBDV Chicken USA None 2.5 2.2 1
1, Dobos et al., 1979; 2, Hsu et al., 1989a; 3, Hedrick et al., 1983b; 4, Hedrick et
al., 1983a; 5, Stephens et al., 1983; 6, Hedrick et al., 1986a; 7, Schwanz-
Pfitzner et al., 1984; 8, Shankar, 1991.
cross-neutralization test has been a standard method for typing the viruses. Hill
and Way (1983) used this method to serotype nearly 200 isolates. They
suggested a standard serotyping scheme (Hill and Way, 1995) that has been
generally accepted by fish health workers. The serotypes and the species from
which they were isolated are listed in Table 1.6.
The aquatic birnaviruses have been divided by Hill and Way (1983, 1995)
into serogroups A and B, which have no cross reactivity by neutralization tests
but show some cross-reaction by immunofluorescence.
Serogroup B consists of a single serotype with fewer than ten strains, which
were isolated from fishes and marine invertebrates in Europe (Hill and Way,
1988b). Within this small serotype, some strains are virulent for trout and cause
typical IPN disease in brook trout (Ahne et al., 1989b); others are completely
avirulent in salmonids and non-salmonids (Hill and Dixon, 1977; Olesen et al.,
1988).
Serogroup A contains more than 200 isolates. They have been divided into
nine serotypes A
1
A
9
, based upon the reciprocal cross-neutralization test scheme
(Archetti and Horsfall, 1951), as carried out by Hill and Way (1983). All of the
archetype strains were originally isolated from North America or Europe and
23 Infectious Pancreatic Necrosis
Table 1.6. Proposed serotyping scheme for the aquatic birnaviruses
(modified from Hill and Way, 1988a).
Number of
Serotype Archetype isolates Class/family Species
Serogroup A
A1 WB 44 Salmonidae Salvelinus fontinalis
Salmonidae S. alpinus
Salmonidae S. naymacush
Salmonidae Oncorhynchus
gorbuscha
Salmonidae O. tshawytscha
Salmonidae O. nerka
Salmonidae O. kisutch
Salmonidae O. keta
Salmonidae O. rhodurus
Salmonidae O. clarki
Salmonidae O. mykiss
Salmonidae Salmo salar
Salmonidae S. trutta
Salmonidae Thymallus thymallus
Salmonidae Hucho hucho
4 Clupeidae Brevoortia tyrannus
1 Gadidae Gadus morhua
1 Percidae Morone saxatilis
1 Carangidae Seriola
quinqueradiata
1 Veneridae Illex illecebrosus
1 Cyprinidae Carassius auratus
A2 Sp 39 Salmonidae O. mykiss
Salmonidae O. gorbuscha
Salmonidae S. salar
Salmonidae S. trutta
Salmonidae T. thymallus
2 Anguillidae Anguilla anguilla
7 Cyprinidae Carassius auratus
Cyprinidae C. ceratisinis
Cyprinidae Carpio carpio
2 Pleuronectidae Pleuronectes
fluviatis
Pleuronectidae Phoxinus phoxinus
1 Cyprinidae Rutilus rutilus
A3 Ab 15 Salmonidae O. mykiss
Salmonidae O. tshawytscha
4 Anguillidae A. anguilla
Anguillidae A. japonica
2 Cobitidae Misgurnus
anguillicaudatus
1 Cichlidae Tilapia mossambica
2 Ostreidae Crassostrea gigas
1 Veneridae Mercenaria
mercenaria
A4 He 3 Salmonidae O. mykiss
10 Salmonidae H. hucho
24
P.W. Reno
most were associated with IPN disease in salmonids. The exceptions are the He
strain (serotype A4) from pike (Esox lucius) and serotype A5, isolated from the
marine bivalve mollusc, Tellina tenuis. However, there are isolates in both of
these serotypes which have been isolated from salmonids and more than 80% of
the isolates have been detected in salmonid fishes.
Several investigators have developed monoclonal antibodies (MAbs) to
aquatic birnaviruses (Caswell-Reno et al., 1986; Wolski et al., 1986; Christie
et al., 1988; Lipipun, 1988; Chi et al., 1991; Shankar, 1991). These MAbs were
used to classify aquatic birnaviruses. The patterns of reactivity in binding tests
with MAbs fairly closely follow the neutralization-based serotyping scheme as
outlined by Hill and Way (1988a, 1995). One of the MAbs (AS-1), generated
against a Canadian A1 isolate, reacted with all of the more than 200 aquatic
birnaviruses isolates tested, while others were isolate specific (Caswell-Reno et
al., 1986, 1989; Reno et al., 1994). None of the MAbs reacted with TV-2, an
isolate from serogroup B (P. Caswell-Reno, P.W. Reno and B.L. Nicholson,
unpublished results).
These studies indicate agreement among serotypes established with
Cyprinidae Blicca bjoerkna
Cyprinidae B. barbus
Cyprinidae C. carpio
Cyprinidae Chondrostoma
nasus
Cyprinidae Gobio gobio
Cyprinidae Scardinius
erythrophthalmus
Esocidae Esox lucius
A5 Te 16 Salmonidae S. salar
Salmonidae O. mykiss
Salmonidae S. trutta
Salmonidae S. fontinalis
1 Pleuronectidae Limanda limanda
1 Ostreidae Ostrea edulis
Tellinidae Tellina tenuis
A6 C1 2 Salmonidae S. salar
A7 C2 2 Salmonidae O. mykiss
A8 C3 1 Salmonidae S. alpinus
A9 J A 1 Salmonidae O. mykiss
Serogroup B
B1 TV-1 1 Tellinidae T. tenuis
1 Salmonidae S. trutta
1 Cyprinidae C. carpio
1 Pleuronectidae L. limanda
3 Ostreidae Crassostrea virginica
Ostreidae O. edulis
Table 1.6. Continued.
Number of
Serotype Archetype isolates Class family Species
25 Infectious Pancreatic Necrosis
polyclonal antibodies and reactions in MAb tests. There are studies which
suggest epitope variation within serotypes. In a study which included the nine
serotypes of serogroup A as well as other isolates, Caswell-Reno et al. (1986;
unpublished results) found that most strains within a serotype were constant in
their reaction pattern using MAbs directed against the WB (serotype A1) and
EVE (serotype A2) isolates. Others varied only slightly (one or two differences
in 11 MAb reactions) from the archetype (Table 1.7). Twenty isolates from the
A1 serotype were distributed into three subtypes: WB, Buhl and VR-299
(Caswell-Reno et al., 1989; unpublished results). In contrast, wide epitope
variation within the A3 (Ab) serotype exists, with seven different reaction
patterns among 20 isolates. Viruses belonging to the various serotypes reacted
with a wide range of the MAbs. For example, the He archetype (from the
northern pike in Germany) reacted with only two of the most universally reactive
MAbs, while the WB subtype reacted with nine of the 11.
Although there is variation in the reactivity patterns of aquatic birnaviruses,
there is epitope stability. Using the same 11 MAbs, there was no variation in the
epitope composition of viruses isolated from a single facility or trout population
during a period of 2712-14 years (P. Caswell-Reno, P.W. Reno and B.L.
Nicholson, unpublished results). Similar results were obtained when isolates of
either the WB or Buhl subtypes were tested. This indicates that these viruses
remain antigenically stable for long periods in vivo. This antigenic stability is
encouraging, as a vaccine developed against one serotype will probably remain
effective against that serotype over time. Caution, however, must be exercised in
interpreting these data, since 11 epitopes do not a virion make!
Christie et al. (1988) suggested the recognition of a new A serotype, based
on the reaction of isolates from Norwegian salmon hatcheries based on MAbs
against a Norwegian isolate. However, it is important when proposing a new
viral serotype to utilize polyclonal antiserum and heterologous pairings in
reciprocal cross-neutralization tests, since the high specificity of MAbs may not
give an overall picture of the antigenic composition of the virus isolate in
question. At this time, it is accepted that the Norwegian isolates belong to the A2
serotype.
Table 1.7. Variability of 69 aquatic birnavirus isolates in an immunodot test
using a panel of 11 monoclonal antibodies. For the sake of simplicity,
subtypes are defined as any reaction pattern variation from the archetype.
(From Caswell-Reno et al., 1989, and unpublished results.)
Serotype No. of isolates tested No. of subtypes
A1 (West Buxton) 22 3
A2 (Sp) 7 2
A3 (Ab) 20 8
A4 (He) 1 1
A5 (Tellina) 4 1
A6 (Canada-1) 7 1
A7 (Canada-2) 2 1
A8 (Canada-3) 1 1
A9 (J asper) 4 2
26
P.W. Reno
DIAGNOSTIC METHODS
The diagnosis of IPN has historically been predicated on clinical signs of the
disease, isolation and identification of the aetiological agent by cell culture
methods and confirmation using serological methods. Recently, there has been
an effort to utilize techniques developed in human and animal medicine for the
rapid identification of virus and increased sensitivity of detection. Fortunately
for diagnosticians, consistent detection of aquatic birnaviruses has proved to be
simple using cell culture systems. There are at least five reasons for this.
1. The virus is usually present in relatively high titres in tissues.
2. Unlike other fish viruses, such as infectious haemopoietic necrosis (IHN) and
channel catfish virus (CCV), isolations of aquatic birnaviruses are made most
frequently from non-diseased animals .
3. There is no indication of an occult stage, in which the virus cannot be
detected in cell culture.
4. The time required for isolation and identification of the agent, usually 23
weeks, is not often critical to the outcome of an epizootic.
5. Lastly, easily available continuous teleost cell lines are generally highly
sensitive to the virus and undergo a characteristic, readily observable cytopathic
effects (CPE) when infected.
Thus, isolation of virus from visceral samples in standardized teleost cell lines
remains the method of choice.
Cell culture methods: primary isolation
Many teleost cell lines are susceptible to IPNV infection, with the consequent
production of characteristic CPE, usually manifested as a stringy appearance
of the cells. Continuous cell lines proved to be susceptible to aquatic
birnavirus infection are listed in Table 1.8. The standard cell lines for aquatic
birnavirus isolation are: RTG-2 (rainbow trout gonad; Wolf and Quimby,
1962), CHSE-214 (chinook salmon embryo; Lannan et al., 1984), FHM
(fathead minnow; Gravell and Marlsberger, 1965), BF-2 (bluegill fry; Wolf
and Quimby, 1966) and EPC (epithelioma papulosum cyprini; Fijan et al.,
1983). For diagnostic purposes, variations in the susceptibility of the teleost
cell lines to infection become important and, for this reason, attempts at the
primary isolation of virus are usually made on at least two different, well-
characterized continuous cell lines (Hill, 1976; Gillespie et al., 1977; Amos,
1985). Hill suggested that the BF-2 cell line, derived from tissues of bluegill
(Lepomis macrochirus), is the most sensitive for the isolation of aquatic
birnaviruses in molluscs and invertebrates (Hill, 1982). Kelly et al. (1978)
compared the sensitivities of three cell lines to infection with IPNV from fish
tissues and found that EPC was the most sensitive. In many instances, if the
fish or shellfish to be examined is a non-salmonid, it is prudent to include a cell
line from the homologous or closely related species. For example, in
attempting primary isolation of the aetiological agent of spinning disease of
27 Infectious Pancreatic Necrosis
Table 1.8. Continuous teleost cell lines in which aquatic birnaviruses have
been propagated.
Cell line Species Virus yield Reference
RTG-2 Oncorhynchus mykiss 10
8.5
1
RF O. mykiss Not reported 2
RTH-149 O. mykiss 10
9.3
3
RTF-1 O. mykiss Not reported 4
STE-137 O. mykiss 10
9.5
3
CHSE-214 O. tshawytscha 10
9.7
3
CHSE-114 O. tshawytscha 10
9.2
3
CSE-119 O. kisutch 10
8.8
3
CHH-1 O. keta 10
10.0
3
SSE-5 O. nerka 10
8.8
3
SSE-30 O. nerka 10
8.8
3
KO-6 O. nerka 10
8.8
3
BB Ictalurus nebulosus Not reported 4
AS Salmo salar 10
8.0
5
CCT Cyprinus carpio 10
9.0
6
LF Misgurnus anguillicaudatus 10
7.33
6
TO-2 Tilapia mossambica 10
10.33
6
INEM-1 Stenodus leucichthys 10
9.9
7
SWT Xiphophorus helleri 10
8.0
8
GF-1 Haemulon sciurus 10
7.0
9
FHM Pimephales promelas 010
7.7
1
OMAKA Caranx mate 10
2
10
BF-2 Lepomis macrochirus Not reported 11
PG Esox lucius Not reported 12
SJ U-1 Carassius auratus 10
9.5
13
CL Ophicephalis lucius Not reported 14
WC-1 Stizostedion vitreum Not reported 15
MK Brevoortia tyrannus Not reported 16
EPC Cyprinus carpio 10
8.5
17
EK-1 Anguilla japonica 10
9.5
18
1, Kelly et al., 1978; 2, Wolf and Mann, 1980; 3, Lannan et al., 1984; 4, Wolf
and Quimby, 1969; 5, Piper et al., 1973; 6, Chen et al., 1990; 7, Follett and
Schmitt, 1990; 8, Kelly and Loh, 1972; 9, Moewus-Kobb, 1965; 10, Lee and
Loh, 1973; 11, Wolf and Quimby, 1966; 12, Ahne, 1978; 13, Lee and Loh, 1975;
14, Wattanavijarn et al., 1988; 15, Kelly et al., 1980; 16, Stephens et al., 1981;
17, Fijan et al., 1983; 18, Kusuda et al., 1989.
menhaden (B. tyrranus), the birnavirus could only be isolated in cells from this
fish species (Stevens, 1981).
While the in vitro host range of aquatic birnaviruses is wide, it is also erratic.
In one instance, only one of four cell lines derived from carp was susceptible to
a birnavirus from milkfish (Chanos chanos) (Chen et al., 1990). In vitro host
range variants of IPNV also occur: some strains do not replicate in FHM or EPC
cells, while others produce a high yield (Scherrer and Cohen, 1975; Kelly et al.,
1978; Nicholson et al., 1979; Castric et al., 1987). Furthermore, Kelly et al.,
(1978) found that the RTG-2 cell line was more sensitive than the FHM cell line
in detecting IPNV from tissues of carrier fish (50% vs. 12.5% of carriers
detected). The use of multiple cell lines is recommended and adopted by national
28
P.W. Reno
and international regulatory agencies for the detection of IPNV (Hill, 1976;
Gillespie et al., 1977; Amos, 1985).
Other methods which have been developed to enhance the sensitivity of
birnavirus detection in cell cultures are based on increasing the efficiency of the
sample preparation rather than on increasing the susceptibility of cells. Two
main methods of sample preparation are utilized. In both, homgenization of the
sample is followed by low speed centrifugation. In one method, the supernatant
was filtered through a bacteria-retaining filter, while in the others antibiotics
were added to inhibit bacterial growth (Gillespie et al., 1977; Okamoto and
Sano, 1984; Amos, 1985; Hedrick et al., 1986b); the two methods yield
comparable results. Cocultivation is another method; the separated host cells
(usually kidney or spleen) are allowed to settle on a preformed monolayer of
continuous cells, thereby infecting them by direct contact. This method has been
reported to provide greater sensitivity than homogenization techniques (Agius et
al., 1982, 1983).
No method other than cell culture isolation has been routinely employed,
since this remains the most sensitive and entrenched technique. For example,
one or two infectious virions in 1 ml of ovarian fluid can be expected (Amos,
1985) and, with concentration techniques currently available, it is feasible to
detect one virion per 10 l of hatchery water (Grinnell and Leong, 1979;
Watanabe et al., 1988; Maheshkumar et al., 1991).
Serological and biochemical methods: identification
Currently, a significant research effort is directed at superseding the use of cell
cultures, since the routine use of cell cultures for the isolation of viruses involves
considerable effort, expense and expertise. It would be advantageous to have a
technique that can be performed in the field or at less sophisticated laboratories.
Although these techniques have not yet achieved the sensitivities needed to
detect aquatic birnaviruses in carrier animals to the satisfaction of regulatory
agencies, they decrease the time and complex testing necessary to identify
isolated aquatic birnaviruses.
Two primary methods of identifying aquatic birnaviruses obviate the
requirement for cell cultures. The first is a group of serological techniques which
employ conjugated antibodies binding to viral antigen as the detector system; the
second utilizes nucleic acid-based probes.
Serological techniques have been used extensively in the identification and
classification of aquatic birnaviruses. The serological identification methods
have increased in sensitivity over the years, but the neutralization test remains
the benchmark for other tests due to its inherent sensitivity and its use for
classifying viruses. Neutralization tests utilizing polyvalent antisera are used to
identify serogroup A aquatic birnaviruses (Lientz and Springer, 1973; Amos,
1985). A cautionary note, however, was raised by Vestergrd-Jrgensen and
Grauballe (1971), who found that the method used to immunize rabbits in the
production of anti-IPNV antiserum could affect the specificity of the reaction.
The most frequently employed neutralization test uses a constant viral titre
29 Infectious Pancreatic Necrosis
(approximately 100 TCID
50
) and varies the antibody concentration. The test is
confirmatory for the identification of the virus and requires up to 1 week to
complete. Other serological techniques used include complement fixation
(Finlay and Hill, 1975), fluorescent antibody (Nicholson and Dunn, 1974; Tu et
al., 1974; Vestergrd-Jrgensen, 1974), immunoperoxidase tests (Reno, 1976;
Nicholson and Henchal, 1978), neutralization kinetics (Nicholson and Pochebit,
1981), Staphylococcus coagglutination (Kimura et al., 1984; Bragg and
Combrick, 1987b), counterimmunoelectrophoresis (Dea and Elazhary, 1983),
enzyme-linked immunosorbent assay (ELISA) (Nicholson and Caswell, 1982;
Dixon and Hill, 1983b; Hattori et al., 1984), immunodot (McAllister and Schill,
1986; Ramsey et al., 1986; Caswell-Reno et al., 1989; Babin et al., 1991; Ross et
al., 1991), and immunoprecipitation (Lipipun, 1988) tests. The tests vary in
sensitivity, as well as in specificity and in the complexity and efficiency of the
performance of the test (Table 1.9). The most commonly used tests, ELISA and
immunodot, have sensitivity levels that are sufficient to detect viral antigen in
the 10100 ng range (Nicholson and Caswell, 1982; Dixon and Hill, 1983b;
Caswell-Reno et al., 1986), which corresponds to approximately 10
6
infectious
doses of virus ml
1
of cell culture fluid or organ homogenate (Hattori et al., 1984;
Babin et al., 1991). However, viral titres in carrier animals are frequently more
than 10,000-fold lower than this (Wolf et al., 1968b; Yamamoto, 1975a; Reno,
1976). The sensitivity of these tests is, therefore, inadequate for confidently
detecting virus in carrier animals; hence cell cultures are still used for the
primary isolation of the virus. However, it has been demonstrated that viral
antigen is widespread in the tissues of carrier fish, even in the absence of high
viral titres (Reno, 1976, 1988; Hedrick and Fryer, 1982).
Serological techniques are used to confirm the identity of virus following
isolation in cell culture. Recently, Rodriguez Saint-Jean et al. (1991) reported
the use of flow cytometry to detect IPNV in the blood leucocytes in an IPN
epizootic. It was determined that at least 70% of the leucocytes must be infected
before a positive could be confirmed, although only small numbers of cells
(approximately 10,000) are needed for the test. The test is rapid and can be
performed more rapidly than enzyme-linked assays. A cocultivation method was
used to increase the percentage of leucocytes that were infected, but this
increased the time required for the assay.
Vestergrd-Jrgenson and Grauballe (1971) reported that the adjuvant used
for the immunization of rabbits with IPNV affected the specificity of the
response to different strains of the virus: Freunds complete adjuvant promoted
the generation of more specific antibodies than did Freunds incomplete
adjuvant. As with other viral systems, the assay that is used to determine
cross-reactivity of antibodies can play a role in the results obtained. For
example, Evensen and Rimstad (1990) determined that rabbit antisera against
strains Sp, Ab and VR-299 cross-reacted in neutralization and Western blotting
assays; however, in paraffin-embedded tissues from fish infected with strain Sp,
only the homologous antiserum reacted. Small differences in reaction patterns
were noted with neutralization reactions versus solid-phase immunoassays, even
when MAbs were used (Caswell-Reno et al., 1986).
Direct nucleic acid probes can be used for the detection of the viral genome
30
P.W. Reno
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31 Infectious Pancreatic Necrosis
and the amplification of genomic material can be achieved by the use of the
polymerase chain reaction (PCR) technique.
Rimstad et al. (1990) have generated an oligonucleotide deoxyribonucleic
acid (DNA) probe specific to the viral protease, which reacted with cell culture
harvests and RNA extracted from strains Sp, Ab and VR-299. The PCR
technique was developed and used for the detection of aquatic birnaviruses. At
present, it has a detection limit of approximately 1 ng of genomic RNA (Cepica
et al., 1991; McAllister et al., 1992; Shankar and Yamamoto, 1994). By using
the known molecular weight of the IPNV genome, 2.4 10
6
Da, it was
determined that 1 ng of genomic RNA would represent approximately 2 10
7
molecules of the A segment of the genome containing the primer. Since many
virions are known to be incomplete and non-infectious because of incomplete
genomes, the level of detection may be even higher. Rimstad et al. (1990), using
a double-nested PCR assay, followed by separation of the PCR-synthesized
DNA products on superparamagnetic beads, were able to detect 0.8 pg of
template ds-RNA, which is approximately 1.5 10
4
molecules of genomic
material. The sensitivity of these tests still does not exceed that of cell culture
isolation of virus. Thus, cell culture isolation and serological confirmation of the
identity of viruses isolated in cell culture remain the process of choice for the
detection and identification of aquatic birnaviruses.
CONTROL AND TREATMENT
Transmission of the disease and epidemiology
In the classic Culture and Diseases of Game Fishes, Davis (1953) makes the
following reference to MGonigles information (1940) on alleviating acute
catarrhal enteritis, now assumed to be IPN: MGonigle recommends that
affected fish be planted in small streams where they can get natural food such as
insect larvae. Since the disease is not due to infection by animal parasites or
bacteria, liberation of the fish in natural waters can do no harm. It is unknown
how many fish farmers adhered to this practice, which was promulgated before
the discovery of virus infections in teleosts. It is possible that the panzootic
nature of aquatic birnaviruses may be due in part to this type of remedy for a
disease of unknown aetiology.
The epizootiology of IPN is complex and is not thoroughly understood. The
disease affects young fish, is retained in the body with no clinical signs in older
fish and is transmitted vertically.
The prevalence of IPNV and IPN disease is not easily apppraised, although
it appears to be present at rather high levels in some instances (Table 1.10).
While there is considerable information in the literature on prevalence levels,
substantial variations exist within even small geographical locales and,
temporally, even at the same facility. This diversity precludes easy analysis or
compilation of information. It is generally accepted that, if the virus is present in
a facility, the infection will remain for long periods in the absence of active
measures to eradicate the agent. For example, in one hatchery, the disease was
32
P.W. Reno
present in the 1940s and mortalities were in excess of 90% in brook trout; by the
late 1970s, the disease was still present, but mortality had dropped to around
40% (L. McCullogh, personal communication). Conversely, a brook trout
hatchery known to have IPNV present at enzootic levels for many years has had
no virus-induced mortalities for more than 25 years (P.W. Reno, personal
observations). It is interesting to note that this hatchery has distributed
IPNV-infected brook trout to many other aquaculture facilities, but the virus has
not necessarily remained with all of the fish that were moved. For instance, one
fish farm received fish from the facility in 1975 and inspection of the stocks
indicated the presence of the virus. The only measures taken were to house the
fish at the tail-end of the impoundments. When these fish were tested annually,
starting in 1987, no virus was detected in them. This is one of only a few
instances in which IPNV has been eliminated from a population of carrier fish.
Bucke et al. (1979) report that in 1975 IPNV was detected in grayling at the
outfall of an IPNV-contaminated hatchery, but there was no evidence of virus
within 2 years in the same population of fish.
There is accumulating evidence that the mode of transmission within a
hatchery may be a combination of vertical and horizontal transmission. It is most
likely that the initial source of infection is from previously infected stocks or
from homoeothermic vertebrates or invertebrates. Horizontal transmission in
hatcheries is most probably due to contact with the virus in faeces and urine of
infected fish (Billi and Wolf, 1969; Frantsi and Savan, 1971b).
Table 1.10. Reported IPNV carrier rates in various aquatic animals.
Pooled Sample Prevalence
Year Species Disease? samples? tested (%) Reference
1976 BKT no no S, K, P, L, I, G. F 98.70 3
1975 BKT yes? no S. K. P. G. F 5090 1
1963 BKT yes yes O, F 50 2
1968 BKT yes? no F 17 4
1971 BKT yes? no S, K. P. L. F 1540 5
1982 BKT no yes S, K, P. L, I. G 60 6
1967 BKT yes? no FE, PW 92.7 7
1968 RBT no no F. PW 0.35 4
1975 RBT no? no S, K, P, G, F 23 1
1976 RBT no yes S, K, P 28 8
1975 RBT yes no S, K 20 9
1980 RBT yes no B, K, S 15 10
1981 RBT no no B, K, S 0 11
1979 RBT no 17 12
1976 BNT no no S, K, P 0.6 8
1976 ATS no no S, K, P 0.6 8
ATS, Atlantic salmon; BKT, brook trout; BNT, brown trout; RBT, rainbow trout.
S, spleen; K, kidney; P, pyloric caeca; G, gonads; I, intestine; L, liver; F, faeces;
PW, peritoneal wash; OF, ovarian fluid; B, blood.
1, Yamamoto, 1975b; 2, Wolf et al., 1961; 3, Reno, 1976; 4, Wolf et al., 1968b;
5, Frantsi and Savan, 1971b; 6, Hedrick and Fryer, 1982; 7, Billi and Wolf, 1969;
8, Munro et al., 1976; 9, Dorson, unpublished; 10, Dorson 1980, 1982; 11,
Dorson, 1981, 1982; 12, Bucke et al., 1979.
33 Infectious Pancreatic Necrosis
Little is known of the epizootiology of the other diseases caused by aquatic
birnaviruses (branchionephritis of eels, whirling disease of menhaden and
yamabe ascites of flat-fish). Much more work in this area is needed.
The aquatic birnaviruses are not restricted to hatchery environments or only
associated with fish released from contaminated facilities. The virus is in the
viscera of wild stocks in remote regions that have had no known plantings or
contacts with hatchery-reared fish. Souter et al. (1986) isolated IPNV from
nearly half of 229 anadromous Arctic char, Salvelinus alpinus, prespawners
collected from the Mackenzie river delta and rivers in the Yukon Territory, where
there was no known contact with hatchery-reared fish. The high level enzootic
nature of the virus in this location indicates that natural infections may be
widespread and that the maintenance of the carrier state in fish is efficient in the
absence of the high viral densities found in rearing facilities. In another remote
region of Canada (Hudson Bay), IPNV was enzootic in brook trout during the
late 1970s and early 1980s (A.J. Sippel, personal communication); likewise, the
virus was detected in wild Atlantic salmon in Loch Awe in Scotland (Munro et
al., 1976). In addition, aquatic birnaviruses have been isolated from wild brown
trout at the outfalls of IPNV-contaminated hatcheries in England and Wales
(Bucke et al., 1979). In Japan, yellowtail ascites virus was also detected in 15%
of wild yellowtail fingerlings some distance from mariculture sites; nearly half
of infected fish reared in the laboratory developed clinical disease (Isshiki et al.,
1989). However, it cannot be assured that these populations were isolated from
potential virus carriers in the sea or from fish upstream of the mouth of the bay.
Routine culture of yellowtail in Japan employs feral fingerlings as mariculture
stocks and these fish are probably the source of virus which causes the epizootics
in cultured yamabe. Thus, it appears that the aquatic birnaviruses are indigenous
to some fish populations, even wild stocks.
It appears that IPN disease is extremely contagious in hatchery facilities.
Fomites and humans are apparently capable of transmitting the virus within and
between fish-rearing facilities. The virus is transmitted both vertically and
horizontally. In most instances, it appears that if no recourse is taken to eliminate
IPNV from a trout culture facility, the virus will remain at the facility for years.
Many of the facilities from which IPNV was originally isolated still have virus
present (P.W. Reno, unpublished observations). The perennial pattern of
infection and disease in facilities that harbour IPNV led to early suspicion that
the virus was transmitted vertically. Wolf et al. (1968a) provided evidence that this
was the case with brook trout. This was later confirmed (Sano, 1971; Yamamoto,
1975a; Reno, 1976; Hedrick and Fryer, 1982; Luqi and Zhizhuang, 1988). The
exact location of the virus during embryonation is unknown, but it is extremely
difficult to isolate virus from the egg. Fijan and Georgetti (1978) detected the virus
at low levels in the chorion of the fertilized ovum. However, the virus is readily
demonstrable after hatching (Reno, 1976; Hedrick and Fryer, 1982).
Other animals may also be involved in transmission of the virus within and
between hatcheries. Sonstegrd et al. (1972) infected several mammalian and
avian species with IPN and found infectious virus in the faeces of mink (Mustela
sp.), great horned owl (Bubo virginianus) and chickens (Gallus domesticus) after
1 week. The virus was not detected in the faeces of great blue herons, common
34
P.W. Reno
eider, or common merganser. Similarly, Eskildsen and Vestergrd-Jrgensen
(1973) isolated IPNV from gulls (Larus ridibundus) administered virus per os.
More recently, Peters and Neukirch (1986) demonstrated that herons (Ardea
cinerea) which were fed virus either by direct intubation or by ingestion of
IPNV-injected trout harboured the virus at fairly high levels for about 30 days
after the last feeding. While these findings lend credence to the possibility of
transferring the virus by other animals, the consensus is that transfer most often
occurs either horizontally within the same facility or watershed or through
dissemination of contaminated eggs. It was also demonstrated that freshwater
crayfish (Astacus astacus) were susceptible to IPNV, retained the virus in their
tissues and haemolymph and shed virus constantly into the water (Halder and
Ahne, 1988). Transmission from crayfish to trout eggs occurred, as did the
infection of the crayfish from ingestion of IPNV-infected trout. There appeared
to be no diminution of virus titre in the crayfish haemolymph or tissues for at
least 6 months, but there was no indication of viral replication in the crayfish.
These findings indicate that crustaceans and other aquatic invertebrates may be
involved in the transmission of aquatic birnaviruses. Trout-rearing facilities
whose water-supply is from open sources or sources that are not treated with
ultraviolet irradiation or ozonation could have aquatic birnaviruses disseminated
by crustacean vehicles, such as crayfish or copepods.
The movement of fish and especially eggs, in the international market is
probably responsible for the dissemination of the virus worldwide, although no
thorough retrospective analysis has been done. The supposition is that, in many
instances, the advent of infection and disease can be traced directly to the
importation of fish. It is presumed that the viruses were originally indigenous to
salmonids and, as a consequence of widespread movement of infected fish, the
virus then infected non-salmonid animals. However, it must be pointed out that
non-salmonids, especially invertebrates, show little or no evidence of disease
when infected with aquatic birnaviruses and it is possible that the virus group
originated in these animals and were transferred to trout, which under artificial
rearing conditions were immunocompromised and susceptible to disease. In this
context, it has been reported by Hill (1982) that at least eight of the aquatic
birnaviruses isolated from marine shellfish species were capable of producing
characteristic signs of clinical IPN in experimentally infected rainbow trout.
Retrospective epizootiological studies of the diseases and infections caused
by aquatic birnaviruses are extremely difficult, due to a lack of consistency and
accuracy in record keeping. Future studies may be more fruitful since there is
considerable pressure within the aquaculture community to be cognizant of the
health status of fish prior to movement or sale and a much stricter accounting of
fish transfers than was previously kept.
Viruses belonging to the He and Tellina serotypes (types A
4
and A
5
of Hill
and Way) have been detected exclusively in Europe and Canada 1, 2, and 3 and
Jasper (types A
6
A
9
) have been confined to North America (Hill and Way,
1988b). This is interesting because it is known that trout infected with the WB
serotype (A
1
) were often transferred to Europe from North America in the 1960s,
but there is a single no report of a WB serotype virus isolated in Europe (Novoa
et al., 1993). There are, however, some instances where the presence of disease
35 Infectious Pancreatic Necrosis
in areas previously free from virus could be traced directly to the importation of
infected eggs. Such importations occurred in South Africa (Bragg and
Combrick, 1987a) and in Japan (Sano, 1971). In both instances, virus
accompanied eggs imported from the USA. In other instances, there was
circumstantial temporal evidence that the disease arrived with eggs imported
into France from the USA (Besse and de Kinkelin, 1965), but subsequent
serological evidence indicated that it was probably an indigenous strain (Wolf
and Quimby, 1971). A heightened awareness of the disease in France by fish
health workers, rather than the importation of virus in fish, was probably
responsible for the detection of these strains. In other instances, there have also
been indications that aquatic birnaviruses were transmitted to indigenous and
cultured species from imported fish or eggs: to Chile from the USA (McAllister
and Reyes, 1984; Espinoza et al., 1985), to Japan from Europe with European
eels (Sano et al., 1981) and to Taiwan from trout imported from Japan (Hedrick
et al., 1983a). It is very difficult, however, to accurately differentiate between
indiginous aquatic birnaviruses and those that were brought into a country or
region. The lack of efficient record-keeping at facilities, the lack of universally
applied inspection regimes for viruses and the complex serological composition
of the birnavirus group combine to make epidemiological studies less than
optimal. Given the geographical distribution of the serotypes, however, it may
well be that the aquatic birnaviruses detected in Asia were imported from the
USA and Europe.
Except for classic IPN disease in freshwater trout, all of the diseases
associated with aquatic birnaviruses affect either stenohaline or euryhaline
animals: yellowtail and Japanese flounder ascites, sea bass, menhaden, eels and
possibly clams.
The virus is quite stable, even under conditions that inactivate most other
fish pathogens. The virus survives for nearly a year at 4C and nearly 2 months at
15C in buffer (Dorson, 1982) and also for long periods in sea water, brackish
water and unsterilized fresh water (Moewus-Kobb, 1965; Toranzo and Hetrick,
1982). This stability increases transmission of the virus from carrier or clinically
ill fish in rearing facilities to susceptible fish upstream or downstream in the
watershed, or even to susceptible hosts in sea water. The spread of the virus
among freshwater, brackish and sea-water environments was facilitated by the
stability of the virus, the migration of anadromous and catadromous fishes and
the predatorprey relationships. The latter is supported by the recent finding of a
birnavirus-like agent in mass cultures of rotifers, Branchionus plicatilis (Comps
et al., 1991), which could potentially be transmitted to larval sea bass (D.
labrax), since rotifers are the primary food source for this species in culture. It
was suggested that rotifers could be responsible for the epizootics of the disease
in maricultured sea bass in France (Bonami et al., 1983).
Vertical transmission and the carrier state
Vertical transmission of the virus via the reproductive products of salmonids has
been substantiated (Wolf et al., 1968a; Sano, 1971; Yamamoto, 1975a; Reno,
36
P.W. Reno
1976; Hedrick and Fryer, 1982; Luqi and Zhizhuang, 1988); however, the actual
method by which this occurs remains obscure. The perennial nature of the
disease in salmonid-rearing facilities strongly suggests that this is the primary
transmission mechanism. This makes sense with trout which are reared in
freshwater facilities, often with a well-water source, which would preclude
infection via a water-borne route, although the possibility of contamination by
birds or transfer by aquatic mammals cannot be ruled out. This can be compared
to infectious haemopoietic necrosis, which was originally postulated to be
vertically transmitted but now appears to be horizontally transmitted (Leong and
Munn, 1991).
Wolf et al. (1968a) demonstrated that the virus was transmitted via
reproductive products. Eggs and milt were collected from IPNV-positive fish
and three pairings were made. The virus was isolated from two matings and
frank disease was noted in one batch of the offspring. Bullock et al. (1976)
found that the virus was transmitted to an uninfected facility via fertilized
brook trout eggs transferred from a contaminated hatchery. However, the
presence of the virus in the parents reproductive products or progeny does not
invariably lead to the development of clinical disease. Reno (1976) found low
levels (10
1.4
TCID
50
g
1
) of IPNV in all eight groups of progeny from matings
of naturally infected brook trout which harboured fewer than 10
2.4
TCID
50
ml
1
of IPNV in milt or ovarian fluid. Virus was not detectable during the
embryonation period or until feeding commenced, but IPNV was detected in
postfeeding fish for only 1 month. Similarly, in other species, vertical infection
has been transient and sporadic. In Atlantic salmon, Smail and Munro (1989)
detected virus for less than 1 h in eggs from brood fish exposed to IPNV at
titres of 10
6
10
9
plaque-forming units (pfu) kg
1
(injection) or 10
7
pfu kg
1
(immersion), and the resultant fry were negative for virus. However, when fry
were infected experimentally, the virus was isolated even after 2 years.
Dorson and Torchy (1985) experimentally infected rainbow trout 4.5
months prior to spawning. In addition, at spawning, they infected milt and ova
with high doses of IPNV Sp. Three of six infected females had detectable IPNV
in ovarian fluid at spawning, whereas none of the males had virus in the milt.
There was no evidence of IPN disease in the progeny. When milt was infected,
however, IPN disease developed in the fry. Similarly, Ahne and Negele (1985a)
demonstrated that experimental infection of rainbow trout and Arctic char
resulted in consistent isolation of virus from the chorion of the eggs during
embryonation and posthatch. In fry, virus was isolated after only 1 month
posthatch, but there was no evidence of disease in these fish. The variations are
most probably due to the differences in the species of animals used;
brook trout are more susceptible to disease than other salmonids. In addition, the
later works were experimental infections while the brook trout were natural
infections.
Survivors of IPN epizootics remain carriers for up to 6 years (Wolf, 1966;
Yamamoto,1975a; Reno, 1976). In a given population, the proportion of fish
from which virus can be isolated varies drastically. In naturally infected brook
trout, the prevalence ranged from 99% to less than 5% (Reno, 1976; unpublished
observations). The latter is the prevalence used in fish health inspections for the
37 Infectious Pancreatic Necrosis
detection of viruses in salmonids (Amos, 1985). The potential for the
dissemination of the virus through the faeces has been demonstrated (Billi and
Wolf, 1969; Frantsi and Savan, 1971b; Reno, 1976) and this shedding of virus,
albeit at low levels, may be responsible for the maintenance of the virus in
susceptible fish. Carrier states have also been reported in survivors of epizootics
associated with other aquatic birnaviruses: striped bass, turbot, yellowtail, eels
and menhaden (Sano et al., 1981; Stephens et al., 1983; Sorimachi and Hara,
1985; Wechsler et al., 1987a).
There are no reported instances of zoonotic infections contracted from any
aquatic birnaviruses.
Treatment and control
As with other virus diseases of fishes, there is no effective therapeutant for
aquatic birnavirus infections.
As yet, there is no effective prophylactic vaccine against infection with
aquatic birnaviruses, although recently several important advances have been
made in this area. An excellent, concise review of the history and prospects of
immunoprophylaxis against IPN has been written by Dorson (1988). The author
is pessimistic about achieving an efficacious vaccine against IPN disease in
salmonids for two reasons. First, the most effective method of administration of
inactivated IPN vaccines is by intraperitoneal injection (Dorson, 1977; Hill and
Dixon, 1977; Sano et al., 1981). This is not a viable option for the immunization
of the highly susceptible fry. Although this method could be used on brood stock,
it has been shown that natural carrier fish often have detectable circulating
antibodies while retaining the virus in the gonads and reproductive products
(Wolf et al., 1968a; Yamamoto, 1975b; Reno, 1976; Etchberger, 1987). Thus, it
is unknown whether vaccination will confer protection against vertical
transmission. Secondly, the use of attenuated vaccines could result in protection
if there is cross-protection against other viral serotypes and if the vaccine does
not revert to being virulent.
Recently, investigators have been working on using genetically engineered
IPNV immunogens produced in prokaryotic cells. Huang et al. (1986) have
cloned portions of the A segment of the IPNV Sp genome, which codes to VP2,
the virus protein which is the target of neutralizing antibodies, and have
synthesized the polypeptide in Escherichia coli for use as a subunit vaccine.
Injection of this immunogen into trout resulted in protection against challenge
with virulent IPNV Sp and IPNV Buhl (Manning et al., 1990).
Attempts have been made to break the transmission cycle by treatment of
fish or eggs with disinfectants. The virus is susceptible to iodophors (Economon,
1963; Amend and Pietsch, 1972), chlorine (Roberts, 1975; Dorson, 1982),
ethanol and methanol (Inouye et al., 1990), halocyamine A (Azumi et al., 1990),
amantidine (Hudson et al., 1988), ribavirin (Migus and Dobos, 1979) and
ammonium chloride (Farias et al., 1988) under in vitro conditions. However, it is
resistant to quaternary ammonium compounds at 12,500 mg l
1
(Dorson and
Michel, 1987), propanol and phenol (Inouye et al., 1990), invert soaps (JDK),
38
P.W. Reno
extracts from marine pseudomonads (Okamoto et al., 1988; Kimura et al., 1990)
and ultraviolet irradiation (Maisse et al., 1980).
In hatcheries, the most commonly used methods against transmission of
viral diseases are ultraviolet irradiation and iodophor treatment. Ultraviolet
irradiation is expensive to install and operate and IPNV requires 330,000
mW cm
2
to inactivate 80% of the virus, a significantly higher level than that
required to inactivate Egtved virus (Maisse et al., 1980). The disinfectants can
be used to sanitize implements contaminated with aquatic birnaviruses but are
toxic to fish and eggs. The iodophors (2050 p.p.m.) are most commonly used
to disinfect eggs prior to shipment or rearing (Amend and Pietsch, 1972;
Desautels and MacKelvie, 1975). Iodophors have limited ability to protect
embryonated eggs, since outbreaks of IPN disease have occurred even after
iodophor disinfection and rearing under quarantine conditions (Bullock et al.,
1976).
The best practice for the elimination of IPNV is prevention by the use of
virus-free stocks coupled with the construction of hatching and rearing facilities
that avoid opportunities for external contamination. This type of vigilance is
difficult to achieve, for economic or technical reasons. Nevertheless, it is the
method of choice for minimizing the impact of not only aquatic birnavirus
infection, but also many other infectious disease agents.
PATHOGENESIS AND IMMUNITY
Pathogenesis
Little has been done with natural IPN disease to determine the portal of entry,
dissemination of virus to target organs or effects on the target cells, except
histopathologically.
In experimental infections, the disease is manifest by 1 week postinfection
and generally has run its course within another week or so (Wood et al., 1955;
Reno, 1976; Reno et al., 1978). The acute nature of the disease is characteristic
of all aquatic birnavirus-induced diseases, including Japanese flounder and
yellowtail ascites, turbot disease, nephritis of eels and spinning disease of
menhaden (Sano et al., 1981; Bonami et al., 1983; Stephens et al., 1983;
Sorimachi and Hara, 1985).
Swanson and Gillespie (1981, 1982) experimentally infected 6-month-old
or yearling brook trout with IPN (strain VR-299) by intraperitoneal injection
and followed the distribution of virus in the gastrointestinal tract, kidney and
blood. While no clinical disease was produced because the fish were older than
the known age of susceptibility to disease, high viral titres were found in the
gut and kidney, and focal necrotic lesions were detected in the pyloric caeca
within 3 days postinfection and in the kidney within 5 days postinfection.
Indirect fluorescent antibody tests indicated the presence of foci of IPNV
antigen in pyloric caeca, kidney and liver and the virus was detected in both
the serum and the mononuclear cell fractions of the blood. Hornich et al.
(1986) indicated that fry undergoing acute IPN had the most intense
39 Infectious Pancreatic Necrosis
fluorescence in the gut epithelial cells. In contrast, in natural carrier brook
trout no virus was detected in the plasma, although virus was detected in the
leucocytic fraction (Yu et al., 1982). In addition, McAllister et al. (1987)
found that the cellular phase of the ovarian fluid of carriers contained up to 400
times more virus than the fluid portion. The distribution of virus within the
viscera is not uniform, with distinct tissue tropisms.
Immunity
The levels of immunity in salmonids during frank IPN disease are not known,
due in part to the small size of the fish. It has been found that the susceptible fry
for IPN disease are immunocompetent (Ellis, 1977; Manning and Mughal, 1985)
but it may be too little, too late.
A humoral immune response to IPNV infection is present in natural and
experimentally induced carrier fish (Wolf and Quimby, 1969b; Yamamoto,
1975b; Reno, 1976; Hedrick and Fryer, 1982) and that experimental infection in
adult salmonids induces antiviral activity (Wolf and Quimby, 1969b; Reno et al.,
1978; Kohlmeyer et al., 1986). Neutralizing antibodies have been demonstrated
in serum and in cutaneous, branchial and intestinal mucus (Dorson et al., 1989).
The serum antiviral activity in carrier brook trout was demonstrated to be an
antibody, since it was a polypeptide (> 200 kDa) which had the electrophoretic
mobility, and an immunoelectrophoretic pattern and reactivity with rabbit
antitrout antisera characteristic of trout immunoglobulin (Reno, 1976; Dorson et
al., 1989). The neutralizing antibody was evident at 30 days postinfection and
remained for several years (de Kinkelin et al., 1984). Hill and Way (1983) found
that IPNV vaccine inactivated by -propriolactone was not as effective as
formalized virus in protecting trout. It has been suggested that there is an
inverse correlation between IPNV titres in tissues and serum antibody titres
(Wolf and Quimby, 1969; Yamamoto, 1975a), but there is no statistical
correlation (Reno, 1976; analysis of data from Hedrick and Fryer, 1982).
Unfortunately, nothing is known of the levels of neutralizing antibody which
are necessary to prevent disease and/or infection, or whether animals with high
serum antibody titres are susceptible to superinfection with the same or
different serotypes.
The situation becomes more complicated as a non-induced, non-
immunoglobulin anti-IPNV molecule with a 6S sedimentation coefficient was
found in the serum of normal trout (Scherrer et al., 1974; Hill and Dixon, 1977;
Kelly and Nielson, 1985). The 6S-sensitive virus strains generated in vitro are
less virulent than 6S-resistant strains (Kelly and Nielson, 1985). The nature
and significance of this substance is unknown, but it may have a natural
protective effect. Gonzalez et al. (1989) determined that anti-trinitrophenol
antibodies in rainbow trout inhibit the replication of IPNV in vitro. In addition,
immunosuppression of IPNV-infected carrier trout with corticosteroids did not
promote viral replication or the induction of frank disease, although humoral
immunity was eliminated (Bucke et al., 1979; Bootland et al., 1986, Etchberger,
1987).
40
P.W. Reno
Another component of resistance to IPN is interferon, a broad-ranging
protective molecule generated by lymphocytes and other cells. In vitro
interferon production has been demonstrated and there is a correlation between
the optimal temperature for cell growth, maximal interferon production and
virus titre (Scherrer et al., 1974). Interferon has been demonstrated in serum of
trout infected with the virus (de Kinkelin et al., 1982), although its in vivo
significance is not known. In vitro, it has been shown that the cell density has an
effect on the virus yield, with cultures plated at higher densities producing more
interferon and concomitantly less virus (Okamoto et al., 1983b).
Little research has been done to examine the role of cell-mediated immunity
in protection against clinical IPN disease. There is some information that
suggests that there is a cellular response to the virus in carrier fish. Knott and
Munro (1986) demonstrated that Atlantic salmon inoculated with IPNV had a
depressed level of leucocytic mitogenesis to phytohaemagglutinin and
decreased number of natural cytotoxic cells (NCC). Similarly, Tate et al. (1990)
determined that, in addition to the alterations of mitogenesis and NCC,
experimentally-infected rainbow trout had higher titres of virus in adherent,
surface immunoglobulin-positive (Sig+) cells than in non-adherant Sig cells.
This indicates a greater affinity of the virus for B-cell lineages than T-cell
lineages. Etchberger (1987) indicated that cytokinins are elicited from
leucocytes of natural IPNV carrier fish as well as fish experimentally infected
with IPNV. Macrophage migratory inhibition factor was also produced in the
presence of IPNV. Natural cytotoxic cells may also play a role in the non-
specific response to IPNV infection, since Moody et al. (1985) found that
continuous teleost cell lines infected with IPNV were more sensitive to the
cytolytic action of trout leucocytes than were uninfected cells.
TOPICS FOR FURTHER STUDY
There has been a distinct emphasis on the characterization of the virus. Future
studies should include the following.
1. Vaccine development. Prophylaxis holds the best hope for elimination of
birnavirus diseases from aquaculture. Despite considerable research efforts in
the past 30 years, no efficacious vaccine against IPN or any other fish viral
disease has been produced commercially, although some successes have been
noted in the laboratory. However, with the advent of new techniques in
biotechnology, it may be possible to develop a subunit vaccine which is
enviromentally innocuous and effective in protecting fish from aquatic
birnaviruses.
2. A better understanding of the nature of the carrier state. The mechanism(s)
responsible for the generation and maintenence of the IPN carrier state is
unknown. Vaccines which simply reduce or eliminate mortalities will do little to
solve the problem of IPN on a worldwide scale. They must not only alleviate
disease, but also prevent infection and the development of carriers.
3. Mortalities due to IPNV in adult salmon. The emergence in European
41 Infectious Pancreatic Necrosis
Atlantic salmon culture of high mortality in postsmolt salmon may indicate a
different virulence mechanism(s) or new strains of virus that are responsible for
the mortalities. It is important to try to discern differences between the strains of
virus causing postsmolt mortalities and those of the same serotype which only
instigate mortalities in juvenile fish.
4. Improved diagnostic methods. Although the methods currently used for the
detection and identification of the aquatic birnaviruses are sensitive and specific,
they are based on cell culture isolation followed by identification, both of which
invariably consume much time and technical effort. Simpler, more rapid
techniques would improve the lot of fish health specialists desperately seeking
viruses.
5. Update information on aquatic birnavirus diseases other than IPN. Several
diseases caused by aquatic birnaviruses can have a significant impact on
aquaculture of non-salmonid species. Virtually nothing is known about these
diseases except that they are associated with aquatic birnaviruses. In those
countries where these diseases occur, it behoves investigators to expand their
information base about these diseases. In addition, this would aid in
understandiing the processes by which aquatic birnaviruses infect fish and cause
disease.
6. Assess the effects of management techniques in controlling the disease. Given
the expansive geographical and host range of aquatic birnaviruses, management
through direct action at facilities or restrictions on fish movement can have a
marked impact on promulgation of the disease. In Europe, especially, where IPN
is enzootic, this type of approach would be helpful. This can be done locally with
attention to the use of uncontaminated water sources and disinfectant methods
and especially the use of virus-free stocks. Even in areas where IPN is rampant,
perhaps some initial small scale efforts are required to establish stocks of
virus-free fish in hatcheries fish.
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