Fish 5

177
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
5
Spring Viraemia of Carp and Other Viral
Diseases and Agents of Warm-water Fish
N. Fijan
Department of Biology and Pathology of Fish and Bees, College of Veterinary
Medicine, University of Zagreb, Heinzelova 55, PO Box 190, 10000 Zagreb,
Croatia.
INTRODUCTION
Warm-water fin-fish which spend their entire life cycle in fresh water are
considered in this chapter. They form the largest segment of the world
aquaculture production and a major part of the catch-fish industry in inland
waters. The harvest of farmed warm-water fish is increasing, due to investment,
refinement in technologies, rapid transfer of advances and a continuous
widening of the spectrum of cultivated species.
Wolf (1988) in his review on fish viruses and fish viral diseases described 59
viruses and virus-like particles, as well as related diseases. Subsequent to that
review, the number of known fish viruses has surpassed 100 and continues to
increase.
Viruses of cultivated cyprinid and other warm-water fishes of major
importance are listed in Tables 5.1 and 5.2. Infectious pancreatic necrosis (IPN)
virus and lymphocystis virus are omitted and are considered elsewhere in this
book.
Of the 27 viruses enumerated in Tables 5.1 and 5.2, two cause diseases
which constitute the major part of this chapter: spring viraemia of carp virus
(SVCV), which induces spring viraemia of carp (SVC), and channel catfish
virus (CCV), inducing channel catfish virus disease (CCVD). Cyprinid
herpesvirus I infection (CHI) or carp pox, grass carp haemorrhagic disease
(GCHD), caused by a reovirus, golden shiner virus disease (GSVD), pike fry
rhabdovirus disease (PFRD) and viraemic iridovirus diseases (including
epizootic haematopoietic necrosis (EHN) are also described. Viruses causing
unapparent infections in cultivated species, those of very low virulence or
causing hyperplastic lesions in feral fish and those reported only once are not
discussed.
Up to 1985, viruses and viral diseases of warm-water fish were accessibly
reported from Europe and North America. Sano et al. (1985a, b) and Langdon et
al. (1986b) were the first to publish data on such viruses in Japan and Australia.
178
N. Fijan
The review on the grass carp reovirus disease in the Peoples Republic of China
(Wolf, 1988) was the first widely accessible account of a viral disease of a warm-
water fish in mainland Asia. Expansion and progress in aquaculture and
awareness of the importance of fish mortalities in farming operations as well as
in feral populations are increasing and they stimulate research on viral fish
diseases.
Viruses of warm-water fishes are not known to replicate in higher animals or
humans. However, some of them can be propagated in reptilian, avian,
mammalian and human cell lines under certain conditions (see Spring viraemia
of carp).
Three of the diseases described in this chapter SVC, EHN and CCVD are
currently of international concern due to their socio-economic importance
within affected countries and their significance in the international trade of
Table 5.1. Viruses of cultivated cyprinid fishes.
Virus: Taxonomy;
Geographical range*;
Gross pathology
;
Status
Main host Reference

Cyprinid herpesvirus I; Common carp Sano et al. (1985a,b)
A, E, M, NA; hyp, hs; I (Cyprinus carpio)
Goldfish herpesvirus 1; Goldfish J ung and Miyazaki
A; ns; I (Carassius auratus) (1995)
Carp iridovirus; Common carp Linnik et al. (1972)
E; 0; I
Goldfish iridoviruses Goldfish Berry et al. (1983)
1 and 2; NA; 0; I
Grass carp picornavirus; Grass carp Mao et al. (1988)
A; hs; I (Ctenopharyngodon idella)
Carp coronavirus; Common carp Sano et al. (1988)
A; ns; I
Spring viraemia of carp Common carp Fijan et al. (1971)
virus; E, M; hs; I
Golden shiner reovirus; Golden shiner Plumb et al. (1979)
NA; l; I (Notemigonus crysoleucas)
Grass carp haemorrhagic Grass carp Chen and J iang
disease reovirus; (1984)
A; hs; I
Tench reovirus; Tench (Tinca tinca) Ahne and Klbl
E; 0; I Ide (Leuciscus cephalus) (1987)
Carp reovirus; A; 0; I Common carp J iang et al. (1991)
Unclassified grass carp Grass carp Ahne et al. (1987)
virus; E; 0; I
Unclassified golden Golden shiner Hedrick et al. (1989)
shiner virus; NA; 0; I
* A, Asia; E, Europe; M, Middle East; NA, North America.
hyp, hyperplastic or proliferative lesions; hs, haemorrhagic

septicaemia; ns, necrotic septicaemia; l, low pathogenicity; 0, unapparent
infection.
I, isolated in cell culture.
179 Spring Viraemia of Carp
aquatic animals and aquatic animal products. The Office International des
pizooties (OIE), with its Fish Diseases Commission, is an intergovernmental
organization that advises its worldwide member countries. Its primary role is to
advise on international trade in animals and animal products, including aquatic
animals and aquatic animal products, on the basis of health control and
preventive measures (Hstein, 1996). Spring viraemia of carp and EHN are
listed in the International Aquatic Animal Health Code among five diseases
notifiable to OIE (OIE, 1995a). These diseases are also enumerated among the
OIE list B diseases of animals (OIE, 1997). Channel catfish virus disease is in
Table 5.2. Viruses of catfishes and other fishes of major importance for
warm water aquaculture and fisheries.
Virus: Taxonomy*;
Geographical range
;
Gross pathology
;
Status
Main host Reference

Channel catfish virus; Channel catfish Fijan et al. (1970)
NA, CA, E; nhs; I (Ictalurus punctatus)
Silurid herpesvirus; Sheatfish Bksi et al. (1984b)
E; hyp; EM (Silurus glanis)
Black bullhead Black bullhead Alborali et al. (1996)
herpesvirus; E; nhs; I (Ameiurus melas)
Esocid herpesvirus; Northern pike Yamamoto et al.
NA; hyp; EM (Esox lucius) (1984)
Epizootic haematopoietic Redfin perch Langdon et al. (1986b)
necrosis virus; A; hs; I (Perca fluviatilis)
Sheatfish iridovirus; Sheatfish Ahne et al. (1989)
E; hs; I
Black bullhead Black bullhead Pozet et al. (1992)
iridovirus; E; hs; I
Largemouth bass Largemouth bass
iridovirus; NA; l,?; I (Micropterus salmoides) Plumb et al. (1996)
Pike fry rhabdovirus; Northern pike de Kinkelin et al.
E; hs; I (1974)
Pike rhabdovirus; Northern pike J rgensen et al.
E; hs; I (1993)
Perch rhabdovirus; Redfin perch Dorson et al. (1984)
E; l; I
Channel catfish Channel catfish Amend et al. (1984)
reovirus; NA; l; I Hedrick et al. (1984)
Pike epithelial Northern pike Winquist et al. (1968)
proliferation retro-VLP;
E, NA; hyp; EM
Esocid lymphosarcoma Northern pike, Winquist et al. (1973)
retro-VLP; muskellunge
E, NA; hyp; EM (Esox masquinongy)
*VLP, virus-like particles
A, Australia; AS, Asia; CA, Central America; E, Europe; NA, North America.
hs, haemorrhagic septicaemia; hyp, hyperplastic or proliferative lesions; nhs,

necrohaemorrhagic septicaemia; l, low pathogenicity.
EM, demonstrated by electron microscopy, but not isolated in cell culture; I,

isolated in cell culture.
180
N. Fijan
the group of other significant diseases which are of current or potential
international significance in aquaculture (OIE, 1995a,b). The Fish Diseases
Commission has also prepared the Diagnostic Manual for Aquatic Animal
Diseases (OIE, 1995b), which proposes the basis for fish health surveillance,
sampling and diagnostic procedures for certain pathogens of aquatic animals.
The latter publication is cited in this chapter as OIE Manual (1995b).
SPRING VIRAEMIA OF CARP
Introduction
Spring viraemia of carp is an acute, systemic, contagious disease caused by a
rhabdovirus. The term SVC and the name Rhabdovirus carpio (RVC) for the
causal agent were introduced by Fijan et al. (1971). The virus is also known as
SVCV and as RVC. It belongs to the vesiculo group (vesiculovirus genus
(Wunner and Peters, 1991)) of rhabdoviruses (Lenoir and de Kinkelin, 1975) and
affects primarily the common carp (Cyprinus carpio). Several other cyprinids
and some other species are also susceptible. Spring viraemia of carp typically
occurs at water temperatures below 18C, and predominantly in the spring.
Prior to the description of SVC as a separate aetiological and clinical entity,
this disease was part of the condition known as infectious dropsy of carp (IDC)
or infectious ascites, haemorrhagic septicaemia, red contagious disease or
rubella. Infectious dropsy of carp was initially considered to be a bacterial
disease. Distinction of two forms of IDC an acute or ascitic and a chronic or
ulcerative one was also to some extent a matter of controversy. Mixed
infections are common in fish. The cytopathic agents from IDC described by
Toma sec et al. (1964) and by Osad caja (1964) might have been SVCV. Fijan
(1972) indicated that IDC and its synonyms cover several distinct aetiological
and pathological entities. It is clear that the acute or ascitic form of IDC
encompassed SVC, and some of the chronic IDC corresponded to carp
erythrodermatitis (Fijan, 1972; Bootsma et al., 1977). Swim-bladder inflamma-
tion (SBI), Pseudomonas fluorescens infection, motile aeromonad infection and
some cases of columnaris disease with skin lesions were also covered in IDC.
Review papers on SVC have been compiled by Bootsma and Ebregt (1983) and
Wolf (1988).
Spring viraemia of carp virus can induce serious mortality and economic
losses, which fluctuate from year to year. These irregularities in epizootical
patterns are poorly understood. Mortality is usually around 30%, but may exceed
70%. Most outbreaks occur after stocking of ponds in the spring. Ghittino et al.
(1980) estimated the loss of 1-year-old carp in Europe to be 4000 tons annually,
i.e. 1015% of this age-group. The loss is more disastrous when 2-year-old carp
are used for stocking. The need for restocking increases the cost of production
(cost of additional fingerlings, increased labour). Outbreaks of SVC have been
less frequent during the past 15 years (Lehmann et al., 1986; N. Fijan, personal
observation).
Species of fish affected and geographical distribution
Carp is the main species affected by SVC. All four scale-distribution varieties as
well as coloured koi carp can be infected. Hill (1977) found that feral carp were
more susceptible to SVC than farmed ones. Both young fish and adults are
susceptible to the virus. Due to the seasonality of the disease, the principal
victims are 912 and 2124 months old.
Other species of fish that are known to be susceptible are listed in Table 5.3.
Additional unspecified young pond fishes are also susceptible (OIE Manual,
1995b). The rhabdovirus isolated from bighead carp (Hypophthalmichthys
molitrix) by Rudikov et al. (1975) is serologically unrelated to SVCV (P.R.
Pichugina, cited in Shchelkunov and Shchelkunova, 1989). Experimentally
infected young pike kept at 9C did not develop disease, but SVCV persisted in
fish and was transmitted to pike by predation (Ahne, 1985b). The virus can
replicate in the fruit fly (Drosophila melanogaster) under experimental condi-
tions (Bussereau et al., 1975), but the involvement of this or other insects in
transmission of the disease is improbable.
Spring viraemia of carp has been diagnosed in the following countries with
low water temperatures during winter: Austria, Bosnia and Hercegovina,
Table 5.3. Spring viraemia of carp virus: known extent of susceptible and
resistant fishes.
Susceptibility
Species Natural Experimental Reference
Carp + + Fijan et al. (1971)
Cyprinus carpio
Goldfish Ahne (1973)
Carassius auratus
Crucian carp + + Klbl (1975)
Carassius carassius
Grass carp + + Roudikov (1980)
Ctenopharyngodon idella
Silver carp + + Shchelkunov and
Aristichthys nobilis Shchelkunova (1989)
Bighead + + Shchelkunov and
silver carp hybrid Shchelkunova
(1989)
Roach + Haenen and Davidse
Rutilis rutilus (1993)
Pike + de Kinkelin
Esox lucius et al. (1973)
Sheatfish + + Fijan et al. (1984)
Silurus glanis
Guppy + Ahne (1973)
Lebistes reticulatus
Pumpkinseed + Fijan (1976)
Lepomis gibbosus
Rainbow trout Haenen and Davidse
Oncorhynchus mykiss (1993)
182
N. Fijan
Bulgaria, Croatia, Czech Republic, France, Germany, Great Britain, Hungary,
Italy, Israel, Poland, Rumania, Slovakia, Spain, Yugoslavia and several countries
which were part of the former Soviet Union. Serological surveys demonstrated
virus neutralizing antibodies in 95% of carp farms in Austria (Klbl and Kainz,
1977), 86% of farms in Bavaria (Wizigmann et al., 1983) and in Schleswig-
Holstein (Hoppe and Werner, 1985). Spring viraemia of carp virus infection is
mostly clinically unapparent (Wizigmann et al., 1980); for instance, some farms
in Germany with serologically positive fish had no record of SVC outbreaks. In
Croatia, the percentage of serologically positive carp varies from one pond to
another and usually reaches a peak at the end of the summer (N. Fijan, D.
Sulimanovi c and Z. Petrinec, 19721976, unpublished).
Neither the disease nor the virus has so far been reported from North or
South America, Japan or China with climates similar to those in affected parts of
Europe.
The disease
Signs and pathological changes
Multiplication of virus in capillary endothelium, as well as in haemopoietic and
excretory kidney tissues, causes an impaired saltwater balance, which is often
lethal. Characteristic macroscopic lesions include oedema, haemorrhage,
anaemia, enteritis and peritonitis. Mixed and secondary infections can lead to
additional clinical signs and increased mortality. The disease signs outlined
below are based on uncomplicated natural SVC outbreaks and experimental
infections in the laboratory (Fijan et al., 1971; Bachmann and Ahne, 1973, 1974;
Fijan, 1972, 1973, 1975).
Fish in the initial stages of disease congregate where there is a slow flow of
water and near pond banks or lie on the bottom. The rate of respiration, reaction
to sensory stimulation and swimming speed are progressively reduced.
Terminally, the fish are sluggish, swim on their side, move slowly and aimlessly
or rest in a normal or abnormal position.
External signs of SVC include skin darkening, swollen belly, exophthalmia,
petechial haemorrhage and ecchymoses in the skin, gills and anterior eye
chamber, anaemia and pale gills, as well as protrusion and inflammation of the
vent (Figs 5.1 and 5.2). Shedding of long, white to yellowish and thick mucoid
casts from the vent is readily visible in fish kept in experimental facilities.
Internal signs are dominated by oedema in all organs, haemorrhage,
peritonitis and catarrhal enteritis. Small to large quantities of a transparent fluid
in the peritoneal cavity may be tinged by haemorrhage. Fibrinous peritonitis
causes adhesions of organs to each other and to the parietal peritoneum.
Petechial haemorrhage is evident in internal organs and muscles. Haemorrhage
is easily recognizable in the lamina epithelialis of the swim-bladder. The
intestine is void of food and appears stretched from the abundant mucus. The
spleen is enlarged and sometimes affected by haemorrhagic or ischaemic
infarcts.
Histopathological changes in some organs of experimentally infected carp
were described by Negele (1977). Blood vessels in the liver show a varying
degree of oedematous perivasculitis, which leads to necrosis. The liver
parenchyma is hyperaemic, with multiple focal necroses and degeneration. The
pancreas is inflamed and shows multifocal necrosis. The hyperaemic spleen
shows hyperplasia of the reticuloendothelium and enlarged melanomacrophage
centres. In the kidney, both the excretory and haemopoietic tissue is damaged.
Tubules are clogged with casts and the cells undergo hyaline degeneration and
vacuolation. The peritoneum is inflamed and the lymph vessels are filled with
detritus and macrophages. Changes in the intestine are dominated by
perivascular inflammation, desquamation of the epithelium and atrophy of the
Fig. 5.1. Spring viraemia of carp. (a) Lateral view of carp showing distended abdomen and
haemorrhage in skin and in anal and caudal fin. (b) Ventral view of gross abdominal distension in
experimentally infected carp (top) compared with control fish (bottom).
184
N. Fijan
villi. Figure 5.3A shows the intestine of a moribund carp. In the swim-bladder,
the epithelial lamina changes from a monolayer into a discontinuous multilayer.
Vessels in its submucosa are dilated, while lymphocytes infiltrate nearby areas.
The heart is affected by pericarditis and by infiltration of the myocardium, which
is followed by focal degeneration and necrosis. Osad caja and Rudenko (1981)
found similar changes in both natural and experimentally induced SVC. They
also noted encephalitis, haemorrhage and inflammation in the spleen, as well as
acute catarrhal enteritis, with necrosis and desquamation of the epithelium.
Sulimanovi c et al. (1986) encountered two types of severe changes in the
premortal stage of experimentally infected carp: acute, necrotic alterations with
weak inflammatory reactions (Fig. 5.3B,C) or prolonged host response with
hydrops and pronounced inflammation (Fig. 5.3D). Perivascular inflammation,
endocarditis and lesions in the myocardium are illustrated in Fig. 5.4.
Fig. 5.2. Spring viraemia of carp. Pale gills with haemorrhage in experimentally infected upper
carp compared with gills in control fish below.
Fig. 5.3. Histopathology of the moribund stage in experimentally induced SVC. Courtesy of M.
Sulimanovi c and T. Miyazaki. Haematoxylin and eosin (H & E). A. Intestine: necrosis and
sloughing off of the epithelial layer; infiltration, oedema and necrosis in submucosa; oedema in
the internal muscular layer; oedema and infiltration between internal and external muscular layer
and in visceral peritoneum. 80. B. Trunk kidney: diffuse necrosis of haematopoietic tissue. 100.
C. Detail from B: almost all cells of haematopoietic tissue are necrotic; a varying degree of cell
degeneration and some necrosis in tubuli. 250. D. Trunk kidney: focal infiltration in
haematopoietic tissue; peritubular oedema, partial degeneration and necrosis in tubuli. 120.
186
N. Fijan
Predisposing factors
As in almost all fish disease, temperature is the main factor determining the
course and outcome of SVCV infection. Under experimental conditions, high
mortality occurs within 1015 days at 1617C but later at 1115C (Fijan et al.,
1971). The disease does not develop in infected fish kept at 2022C or higher
(Fijan, 1976) or at a constant temperature of 18C (Baudouy et al., 1980a,b,c) or
20C (Ahne, 1980). In several long-term laboratory experiments, Baudouy et al.
(1980a,b,c) investigated the influence of increasing and decreasing temperature
on the infection. A gradual increase from 11 to 16C will lead to faster disease
Fig. 5.4. Histological sections of gills and heart of carp with signs of SVC 7 days after
experimental infection. A. Degeneration of a small blood-vessel (a) near the gill arch, surrounded
by inflammation in oedematous loose connective tissue; b =cartilage. B. Ventricle of the heart.
Endocarditis (c), focal degeneration and necrosis in myocardium (d). H & E. 180.
development than a decrease in temperature. The gradual decrease of
temperature from 11 to 5C for 60 days, followed by a slow increase to 20C
during the next 140 days, led to low mortality during the decreasing temperature
and massive mortality during increase from 7 to 14C (Baudouy et al., 1980c).
These findings are in agreement with field observations about the predominant
occurrence of SVC outbreaks in the spring. The disease is first registered from
March to June (Fijan et al., 1971), but it can also occur in the autumn (Pfeil-
Putzien and Baath, 1978). In Bavaria, most cases are diagnosed in April and May
and several in June, while single or low numbers occurred in March, July,
November and December (Wizigmann et al., 1983). In the Ukraine, the majority
of cases appeared in April and May; a single case in carp brood stock was
diagnosed in June and a few affected young fish in July (Osad caja and Rudenko,
1981). In summary, SVC can be expected from November to July, with a peak in
AprilJune (Fijan, 1988). Tropical and subtropical climates are unfavourable for
the development of SVC.
The immaturity of the defence system in up to 1-month-old carp and
sheatfish (Silurus glanis) permits the development of SVC under rearing and
laboratory conditions at 23C, although older fish resist the infection at this
temperature (Fijan et al., 1984; Pasco et al., 1987).
The age of fish influences the resistance to SVC. Shchelkunov and
Shchelkunova (1989) ascertained that young fish were susceptible and 1.5-year-
old fish were resistant. Sheatfish up to 25 days old are susceptible, but older fish
become resistant (Pasco et al., 1987). Farmed carp are more susceptible than
grass carp, which in turn are more susceptible than the bighead silver carp
hybrid (Shchelkunov and Shchelkunova, 1989).
The causes of differences in susceptibility of groups and individuals of the
same species, scale-distribution variety and age to SVC are not known. Field
observations indicate that the crowding of fingerlings in winter or in spring and
long transportation prior to stocking favour outbreaks and high mortalities.
Physiological status of the fish, water quality and stress caused by handling also
influence the course and outcome of infection. Dehydroabietic acid is
immunotoxic and it can increase the susceptibility of carp to SVC (Munoz et al.,
1994).
The virus
The aetiological role of RVC in SVC was confirmed by Baudouy (1975),
Rudikov et al. (1975), Ahne (1977), Hill (1977), Tesar ck et al. (1977), Bksi
and Szab (1979), Bucke and Finlay (1979), and Osad caja and Rudenko (1981).
Ahne (1973) and Bachmann and Ahne (1973, 1974) reported a rhabdovirus
from carp with clinical signs of SBI, established its pathogenicity and named it
SBI virus. They noted its serological similarity with rhabdovirus carpio
(Bachmann and Ahne, 1974). The virus induced haemorrhage and inflammation
of the swim-bladder, a typical clinical sign in the initial stages of SBI. However,
serological comparison by de Kinkelin and Le Berre (1974) and Hill et al. (1975)
showed the SBI virus to be indistinguishable from Rhabdovirus carpio.
188
N. Fijan
Furthermore, Kri zanac et al. (1981) could not isolate virus from typical
(Arshaniza and Bauer, 1973) cases of SBI.
Spring viraemia of carp virus is 6090 nm wide and 90180 nm long. It has
the typical bullet form of the family Rhabdoviridae. Virions bear a regular array
of spicules on the surface (Fig. 5.5). They pass through filters of 450 and 200 nm
porosity, but are retained by 100 nm membranes. The inner nucleocapsid has
helical symmetry, consists of a ribonucleic acid (RNA)protein (polymerase (L),
nucleocapsid (N) and non-structural (NS)) complex and measures about 50 nm
in diameter. The nucleocapsid is surrounded by a lipid-containing envelope
(metric (M) protein) with spikes (glycoprotein (G protein)).
Defective interfering particles of SVCV (de Kinkelin and Le Berre, 1974)
usually appear in cell cultures infected with a high virus input. Production of
normal virions in cell culture can be secured by dilution of inocula to ensure a
low multiplicity of infection. These particles measure about two-thirds of the
length of infective virions (Bishop and Smith, 1977).
The purified SVCV has a buoyant density of 1.1951.200 g ml
1
in caesium
chloride (Ahne, 1973; Bachmann and Ahne, 1973) and 1.16 g ml
1
in a linear
1560% sucrose gradient (Lenoir, 1973). The single-stranded (ss) linear RNA
molecule sediments at 3840 S in a 525% sucrose gradient (Hill et al., 1975).
Structural proteins of SVCV are similar to those of the vesicular stomatitis
virus (VSV) (Lenoir, 1973; Sokol et al., 1974; Roy and Clewley, 1978a; Deuter
et al., 1982). In these studies molecular weight ( 10
3
) for L protein varied from
90 to 190, for the G protein from 70 to 88, for the N protein from 40 to 52, for the
NS protein from 43 to 53 and for the M protein from 19 to 27 Da. The RNA
polymerase of the virus is RNA-dependent, with optimum in vitro enzymatic
activity at 22C; its 5 nucleotide is pppAP (Roy and Clewley, 1978b). The
Fig. 5.5. Bullet-shaped virion of SVCV showing an array of surface spicules. Courtesy of P. de
Kinkelin.
glycoprotein on the virions surface is responsible for its infectivity and
immunogenicity (Bishop and Smith, 1977). The NS protein contains two related
phosphoproteins, NS1 and NS2 (Roy, 1981). Studies on the 5-terminal structure
of RNA (Gupta et al., 1979), the mechanisms of messenger RNA (mRNA)
capping (Gupta and Roy, 1980, 1981), the base sequence at the 3 end of the
RNA (Roy et al., 1984) and the sequence analysis of mRNA coding for the M
protein (Kiuchi and Roy, 1984) have elucidated the specificities of SVCV and its
similarities with the VSV. They indicate a common ancestor of SVCV and VSV
(Kiuchi and Roy, 1984). Characteristics of L genes (Bjrklund et al., 1995),
glycoprotein genes and internal gene junctions place SVCV firmly in the
vesiculovirus genus of Rhabdoviridae (Bjrklund et al., 1996). The virus lacks
the non-virion (NV) gene which exists in three lyssa-type fish rhabdoviruses
(Kurath et al., 1994) belonging to the recently suggested new genus
aquarhabdovirus (Bjrklund et al., 1996).
Spring viraemia of carp virus is inactivated by lipid solvents, heating (60C
for 15 min), glycerol, ozone and diethylpyrocarbonate, as well as by pH below
4 and above 10. Formalin (3%), sodium hydroxide (NaOH) (2%), chlorine
(500 mg l
1
), actomar (0.01% I
2
), gamma irradiation (10
3
krads) and ultraviolet
(UV) irradiation (254 nm) inactivate the virus within 10 min (Ahne, 1982a,b).
Normal handling in the laboratory does not cause undue loss of infectivity. Serum
concentrations of 2 or 5% have a pronounced protective effect on the infectivity
during storage at room, 4C and freezing temperatures, freezethaw cycles and
lyophilization (Ahne, 1973; de Kinkelin and Le Berre, 1974). The infectivity is
retained in tap water at 10C, in mud (pH 7.4) at 4C for 42 days, in stream water
at 10C for 14 days and after drying at 421C for 21 days (Ahne, 1982a,b).
The SVCV adsorbs to the plasma membrane and enters the host cell by
receptor mediated endocytosis. The first sign of viral replication is the formation
of inclusion bodies in the cytoplasm. A budding process at the plasma membrane
and, later in infection, at membranes of dilated Golgi vesicles secure maturation
and release of virus (Granzow et al., 1997).
The virus replicates in a variety of fish and other vertebrate primary cell
cultures and cell lines, causing a clear cytopathic effect. The best systems for
viral replication are cell lines from cyprinid fishes, such as epithelioma
papulosum cyprini (EPC) (Fijan et al., 1983), fathead minnow (FHM) (Gravell
and Malsberger, 1965) and carp leucocyte culture (CLC) (Faisal and Ahne,
1990) cells. Each produces high virus yields (10
8
10
9
tissue culture infective-
dose at 50% end-point (TCID
50
ml
1
). Channel catfish ovary (CCO) cells
(Bowser and Plumb, 1980a,b) are also quite susceptible to SVCV (N. Fijan,
unpublished data). A number of other widely used fish cell lines, such as bluegill
fry (BF-2), brown bullhead (BB) and rainbow trout gonad (RTG-2), also support
replication of SVCV, but the yields are somewhat or much lower. The chicken
embryo, fetal calf kidney and pig kidney cells, mammalian cell lines baby
hamster kidney (BHK/21), Vero, MDCK, SK (Ahne, 1973; Bachmann and
Ahne, 1974), human diploid lung (WI-38) and several reptilian cell lines (Clark
and Soriano, 1974), are also susceptible if incubated at 2022C. Pesticides
(atrazine and lindane) in EPC cell cultures do not influence virus titres
(Cossarini-Dunier and Hattenberger, 1988).
190
N. Fijan
Replication in cell cultures takes place between 4 and 32C, with the
optimum at 2022C. The cytopathic effect is characterized by rounding,
detachment and lysis of cells (Fig. 5.6). Margination of nuclear chromatin and
cytoplasmic vacuolation precede rounding of cells and can be seen in fixed and
stained cell sheets. Fathead minnow cells incubated at 20C synthesize first
progeny virus 46 h after infection and peak titres of both cell-associated and
cell-free virus are reached between 10 and 22 h. One growth cycle lasts 810 h
(Bachmann and Ahne, 1974; de Kinkelin and Le Berre, 1974). Well-defined
plaques are formed after 3 days at 20C.
Antigenic studies with rabbit polyclonal neutralizing antibodies indicate
that all isolates examined belong to a single serotype. Spring viraemia of carp
virus is serologically unrelated to salmonid rhabdoviruses which cause viral
haemorrhagic septicaemia (VHS virus) and infectious haemopoietic necrosis
(IHN virus) and to other fish rhabdoviruses, except the pike fry rhabdovirus
(PFR). The possibility of a serological relationship between these two
rhabdoviruses of the vesiculo group was confirmed by Jrgensen et al. (1989).
Immunochemical and biological examination of 22 rhabdovirus isolates from
Cyprinidae, Esocidae and Siluridae shows that SVCV and PFR share common
antigenic determinants on the G, N and M proteins which prevents the two
viruses being distinguished by an indirect fluorescent antibody test (IFAT).
Spring viraemia of carp virus and PFR can be differentiated by an enzyme-
linked immunosorbent assay (ELISA) and by a virus neutralization test, if the
Fig. 5.6. Cytopathic effect of SVCV in EPC cells. Rounding up and detachment of cells in a focal
area. 230.
rabbit antiserum is heat-inactivated and no complement is added. Jrgensen
et al. (1989) suggest that SVCV and PFR are two serotypes of a single virus
species. Heat-inactivated hyperimmune carp anti-SVCV serum can also
distinguish SVCV from PFR (Petrinec, 1984).
The virulence of individual isolates may vary and can be specifically higher
for the species or age of fish from which the virus was isolated (Ahne, 1986;
Shchelkunov and Shchelkunova, 1989). It can be reduced by passage in
mammalian and fish cell lines (Fijan et al., 1977b; Klbl, 1980).
Diagnostic methods
Diseased fish
Mortality of fingerlings and older fish can be expected at temperatures below
1820C, i.e. in the spring and at the beginning of the summer, but less
frequently in the autumn and winter.
Clinical diagnosis is based on behavioural and external and internal signs.
Haemorrhage in the skin, pale gills, ascites and protruding vent, as well as
enteritis, peritonitis, oedema and varying degrees of petechial haemorrhage in
the swim-bladder, muscles and other organs, warrant sampling of fish for
laboratory examination. The wide variation of disease signs among individual
fish in a population or the simultaneous presence of fish with other disease signs
will require the selection of separate sample groups. At least ten diseased or
moribund fish from each species or group should be selected for laboratory
confirmation of SVCV in suspected outbreaks. Live or sacrificed specimens are
transported under refrigeration or on ice but never frozen. The OIE Manual
(1995b) suggests the immediate collection and dispatch of all internal organs
and the encephalon for fish of 46 cm or the kidney, spleen and encephalon for
specimens above 6 cm. Such material from up to five fish is combined to form a
tissue pool of 11.5 g. Each pool is placed into a sterile vial with a fivefold
volume of transport medium containing antibiotics. Vials are sent and stored at
4C. Virus extraction should be carried out within 24 h and not later than 48 h.
Diagnosis is based on direct methods, i.e. virus identification. The isolation
and identification of virus in cell culture are the most sensitive and widely used
approach. Spring viraemia of carp virus can also be demonstrated in fish tissue
or tissue extracts by immunofluorescence or ELISA.
Isolation of SVCV from diseased fish is readily accomplished by
inoculation of susceptible, young (24 h) and active cell cultures with
homogenized, decontaminated, clarified and diluted (three serial 10 dilutions)
organ samples. The OIE Manual (1995b) recommends EPC cells for virus
isolation. At least 2 cm
2
of cell monolayer should be inoculated with 100 l of
each dilution. After adsorption for 0.51 h at 1015C, the inoculum is not
withdrawn and the added medium with 2% fetal calf serum should be buffered at
pH 7.6 and to maintain pH at 7.37.6 for 7 days. Positive controls have to be
prepared simultaneously. Cultures should be incubated at 15 (OIE Manual,
1995b) or 20C and monitored daily for 7 days. The cytopathic effect develops
within 12 days but more time may be required. Initially, areas of cell rounding
192
N. Fijan
spread over the sheet and finally all cells are detached and lysed. Depending on
the stage of disease, the virus concentration can vary from 10
2
to over 10
8
TCID
50
g
1
tissue. If a sample remains negative for cytopathic effect for 7 days, the
inoculated cell culture must be subcultured.
Shchelkunov and Shchelkunova (1989) noted virus inhibitors in tissues with
high amounts of SVCV, and these may cause absence of cytopathic effect when
cultures are inoculated with low dilutions.
Identification of isolated virus is carried out using serological techniques,
i.e. virus neutralization (Petrinec, 1973; de Kinkelin and Le Berre, 1974; Fijan,
1976; OIE Manual, 1995b), IFAT (Faisal and Ahne, 1983, 1984; Rodak et al.,
1993; OIE Manual, 1995b), ELISA (Way, 1991; Rodak et al., 1993; OIE
Manual, 1995b) or immunoperoxidase (Faisal and Ahne, 1980, 1983, 1984;
Rodak et al., 1993). Virus neutralization is the preferred assay. The titre of
neutralizing antibody solution to SVCV for the virus neutralization must be at
least 2000 for 50% plaque reduction (OIE Manual, 1995b). When a suspected
SVCV isolate remains unaffected by neutralizing antibody to SVCV in a virus
neutralization test, it is mandatory to conduct an IFAT (OIE Manual, 1995b).
Kits for IFAT and ELISA to identify SVCV in cell cultures are commercially
available. Detection of SVCV by hybridization with biotinilated probes
(Oreshkova et al., 1995) is a promising approach which needs refinements for
increased specificity and sensitivity.
Virus isolation accompanied by simultaneous virus neutralization of SVCV
accelerates the obtaining of a diagnosis.
Spring viraemia of carp virus is serologically distinct from all other fish
rhabdoviruses except PFR. An IFAT cannot distinguish SVCV from PFR. Virus
neutralization with selected antisera and ELISA are specific. If complement is
used in virus neutralization, the distinction between SVCV and PFR is
impossible (Clerx et al., 1978; Jrgensen et al., 1989).
Identification of SVCV antigen in tissues from moribund and dead infected
carp is possible by IFAT (Faisal and Ahne, 1984) and by ELISA using tissue
extracts in 1 h (Way, 1991; Rodak et al., 1993). The sensitivity of ELISA for
detection of subclinical virus levels is lower than that of isolation in cell cultures,
but it is superior for recognizing SVCV antigen in fish carcasses which undergo
some degree of decomposition and loss of virus infectivity (Way, 1991; Rodak
et al., 1993).
Spring viraemia of carp virus is a poor antigen for rabbits and none of the
immunization programmes tested by Hill et al. (1981) gave entirely satisfactory
and predictable results. Serum with the highest neutralizing antibody titre of 1 :
6500 was obtained by a single intramuscular (i.m.) inoculation of concentrated
virus, combined with complete Freunds adjuvant, followed by two intravenous
booster injections. However, antisera from some rabbits had titres of less than
100. Similarly, Jrgensen et al. (1989) obtained relatively low (1 : 1280) but
highly specific neutralizing antibody titres from rabbits immunized with purified
SVCV after 22 weeks. Dixon and Hill (1984) could not develop a satisfactory
ELISA for SVCV due to the low titre (1 : 3500) of antiserum. Way (1991) had
good SVCV antibodies for ELISA by purification of gammaglobulin from rabbit
antiserum. Monoclonal antibodies have not been developed.
Non-clinical spring viraemia of carp virus carrier fish
Gill biopsy was recommended by Baudouy et al. (1980c) for screening carp for
SVCV at temperatures below 11C.
Attempts at virus isolation from survivors of SVC outbreaks and suspected
carrier fish have been mostly unsuccessful. Nevertheless, OIE Manual (1995b)
specifies sampling and laboratory procedures in surveillance programmes for
establishing the SVC-free status of carp production units. Encephalon of any
size fish and/or ovarian fluid from brood fish at the time of spawning have to be
sampled from a number of fish, specified by Ossiander and Wedemeyer (1973),
to detect a prevalence of infection in the examined population equal to or higher
than 2% at a 95% confidence level. Samples are processed for virus isolation and
identification, as specified above for overtly infected fish.
The only case of subclinical SVCV was reported by Bksi and Csontos
(1985). They examined reproductive products of carp brood stock that was
induced to spawn by hypophysation and recovered virus from three of 491
samples of ovarian fluid. All 211 samples of seminal fluid were virologically
negative. Fijan (1988) reported negative findings in 86 samples of seminal and
ovarian fluids. Ahne (1983) could not find virus in seminal fluid of
experimentally infected carp. Wolf (1988) recommended using immuno-
suppression or low-temperature stress on brood stock and subsequent
examination for the presence of virus in ovarian fluid and urine and in leucocytes
by cocultivation.
Previous contact of a fish with SVCV and the presence of virus in a fish
population can sometimes be detected indirectly by demonstration of virus-
specific neutralizing antibodies in the serum. This approach was not accepted as
a routine diagnostic method, due to insufficient knowledge of the serology of
virus infections in fishes (OIE Manual, 1995b). Circulating antibodies to SVCV
in clinically healthy and virologically negative carp survivors were first found in
Croatia by an indirect haemagglutination test (Sulimanovi c, 1973) and by virus
neutralization (Petrinec, 1973). As already mentioned, examination of carp sera
for neutralizing antibodies was also used in epizootiological studies on farms in
several other European countries (Klbl and Kainz, 1977; Wizigmann et al.,
1980, 1983). Kretschmar and Dresenkampf (1987) applied a solid-phase ELISA
for detecting anti-SVCV antibodies. The method was 100 times more sensitive
than virus neutralization in detecting antibodies in hyperimmune carp sera, but
detected a lower percentage of antibody-positive carp than virus neutralization
in surveys of suspected populations. The competitive immunoassay (Dixon et
al., 1994) appeared to be more sensitive than virus neutralization.
Control and treatment
Epizootiology
Spring viraemia of carp is generally transmitted horizontally. Overtly infected
fish shed virus in faecal casts and possibly in urine and gill mucus (Pfeil-Putzien,
1977; Ahne, 1978; Pfeil-Putzien and Baath, 1978; Baudouy et al., 1980a,b,c).
Demonstration of a persistent viraemic phase in experimentally infected carp
194
N. Fijan
kept at temperatures below 1314C (Ahne, 1977, 1978, 1980; Baudouy et al.,
1980b,c) before the appearance of visible symptoms (Baudouy et al., 1980b,c)
suggests that SVCV shedding by a few fish during winter is the most important
factor in transmission.
Virus shedding by survivors of infection has not been demonstrated but
appears to be important for survival of virus in fish populations. The virus prob-
ably circulates from farm to open water and back by small wild fish and escaping
cultivated fish, as well as by water draining and filling ponds (Fijan, 1984).
Bloodsucking parasites, such as the carp louse, Argulus foliaceus, and the
leech, Piscicola geometra, are vectors and can passively transfer the virus from
infected to healthy carp (Pfeil-Putzien, 1977; Ahne, 1985a). They do not support
replication or long-term survival of the virus (Ahne, 1985a). Fish-eating birds
can probably carry the virus. Regurgitated infected fish from heron (Ardea
cinerea) fed SVC contained virus up to 120 min after feeding (Peters and
Neukirch, 1986). The fate of virus in digestive systems of other fish-eating birds
and of turtles eating fish carcasses has not been investigated.
Since SVCV retains infectivity for a long time in water or mud or in a dry
state (Ahne, 1982a,b), the pond environment and the fishery equipment on
SVCV-positive farms should be regarded as infected with the virus.
Gills are the natural portal of virus entry and primary replication. Fish can be
experimentally infected using a virus bath suspension. Higher concentrations of
virus in the bath result in higher mortality (Baudouy et al., 1977). Entry by the
intestinal route seems to be of minor importance. The application of virus
directly into the anterior gut did not induce disease (Varovi c and Fijan, 1973) and
the bile rapidly inactivated virus in vitro (N. Fijan, unpublished data).
For experimental purposes, SVC can be readily induced by immersion and
intraperitoneal (i.p.) injection. Ahne (1977, 1978) found that i.p. infection causes
a higher mortality rate (90%) than immersion of carp in the virus (20%) at 13C.
Intracerebral and intrapneumatic (by the swim-bladder) injection of virus also
induces disease but less frequently than i.p. injection (Varovi c and Fijan, 1973).
Vertical transmission of SVCV seems possible. Bksi and Csontos (1985)
found virus in about 0.6% of the examined ovarian fluids. However, the scarcity
of natural SVC outbreaks among fry and fingerlings indicates that vertical
transmission is of minor importance.
Prevention and control
Avoidance of SVC seems feasible on small farms supplied by spring or well
water and with drainage which prevents the entrance of fish from open recipient
water.
Eradication by slaughter and disinfection has little chance of success on
most large European carp farms fed by surface waters from streams or rivers,
mainly due to the presence of SVCV in water supplies (Fijan, 1984).
Outbreaks of SVC can be prevented or stopped in mature fish by raising
water temperatures above 1920C. The application of this approach has not
been reported. The cost of maintaining such temperatures in a temperate climate
limits the applicability of this approach to indoor operations using suitable warm
underground water.
Prophylactic measures on farms should include disinfection of eggs by
iodophore treatment (Ahne and Held, 1980), regular physical and chemical
disinfection of ponds, chemical disinfection of equipment, careful handling of
fish to minimize stress and safe disposal of dead fish. Avoid crowding during
winter and early spring to reduce the spread of the virus.
The resistance of carp to SVC can probably be increased by genetic
selection. A long-term selection programme resulted in high resistance of the
Krasnodar strain of carp (Kirpi cnikov et al., 1987; Wolf, 1988). However,
observations on mortality rates in carp strains were not accompanied by
virological diagnosis, and laboratory challenges with virus were not carried
out.
Alikin et al. (1996) studied the potential of double-stranded (ds) and ss yeast
RNA preparations to protect carp against infection with SVCV. Intraperitoneal
injection of dsRNA shielded carp for over 3 weeks but the bath administration
was much less efficient. The authors also studied the capability of carriers for
dsRNA to increase the efficacy of preparation.
Sensitivity of SVCV to antiviral chemotherapeutic compounds has not been
tested and there are no prospects for their applicability in the near future.
Secondary bacterial infections in carp exposed to SVCV can be controlled at
temperatures above 15C by oral delivery of broad spectrum antibiotics,
providing fish are taking pelleted food.
An effective and safe vaccine for prevention of SVC is not available.
However, there are encouraging results in laboratory studies and the pilot field
trials with live attenuated and, to some extent, with inactivated preparations.
Information on vaccination is reviewed by Fijan (1984, 1988).
The protective immunity after vaccination at 1920C with an attenuated
live virus in autumn lasted for about 9 months both in fish receiving 10
6.2
TCID
50
of virus orally and in those receiving 10
5.8
TCID
50
by i.p. injection; only in the
latter group was protection still effective at about 11 months (Fijan et al.,
1977b). Similar results were obtained by Klbl (1980), who pointed out the
feasibility of vaccination with live attenuated vaccines in countries where the
majority of farms were serologically positive. Immersion in virus also provides
protection from challenge by i.p. injection (Hill, 1977; Ahne, 1980; Fijan and
Mata sin, 1980). Klbl (1990) orally vaccinated about 820,000, mostly 2-year-
old, carp with attenuated strains in field trials over 5 years. Vaccine was
delivered in the food, twice at 1-month intervals. No case of SVC developed in
immunized fish.
An inactivated vaccine was tested for several years in the former
Czechoslovakia (Tesar ck et al., 1984). A lyophilized preparation of two virus
strains inactivated with -propiolactone was injected i.p. in spring. The vaccine
contained approximately 10
4
TCID
50
ml
1
. The dose of vaccine in oil emulsion
was 0.5 ml for fish up to 0.5 kg and 1.0 ml for larger fish. Resistance to SVC was
obtained as early as 7 days after vaccination and was claimed to last up to 11
months, but the mechanism of protection was not explained. The vaccination
was economically efficient. The percentage of total losses at harvest in the
autumn was lower in vaccinated than in control populations. Virus neutralization
antibodies were present in small samples of vaccinated fish. The inactivated
196
N. Fijan
vaccine of Mata sin et al. (1980, cited in Fijan, 1988) also increased survival rates
in carp in field trials, but did not protect fish under controlled laboratory
conditions.
A possible strategy of vaccination adapted to carp culture was discussed by
Fijan (1988). The vaccine should preferably be delivered at water temperatures
above 1920C. Brood fish could be vaccinated immediately after spawning by
i.p. injection. The 4060-day-old carp could be immunized orally or by bath. In
autumn, only oral vaccination of fingerlings can suit the present technology. The
valuable 2-year-old carp for stocking could be vaccinated by i.p. injection in
spring. Immunization is inefficient in already infected, parasitized or stressed
carp (Klbl, 1980; N. Fijan, unpublished data).
Pathogenesis and immunity
Uptake and multiplication of SVCV in carp was studied by Ahne (1977, 1978) at
13C. After infection by immersion, the virus was detectable in gills up to 2 h
and then on the fourth day, when it was also present in kidneys, liver, spleen and
gut. Viraemia was evident on day 5 and reached 5 10
7
plaque-forming units
(pfu) ml
1
on day 6. Virus was detected in faeces on day 11. At 11C Baudouy et
al. (1980c) found the virus in gills and viraemia on day 2 and by day 3,
respectively. High viral titres in kidney, liver and spleen of naturally and
experimentally infected carp (Fijan et al., 1971; Ahne, 1977, 1978) indicate that
these organs are primary targets of virus replication. Twenty-four days after
infection kidneys have a much lower (or none) virus titre than liver and spleen
(Faisal and Ahne, 1984).
Reduced haematocrit and haemoglobin demonstrated in experimentally
infected sheatfish by Jeney et al. (1990) are a consequence of damage to the
haematopoietic tissue. Necrosis at this and other sites is accompanied by
increased blood transaminase activity.
The defence system has an important role in the pathogenesis of SVC and
encompasses neutralizing antibodies, resistance to reinfection (Fijan et al.,
1977a,b; Hill, 1977; Ahne, 1980; Baudouy et al., 1980b) and interferon
synthesis (Baudouy et al., 1977; de Kinkelin et al., 1982). Nothing is known
about the cellular immune response that is probably involved in protective
immunity in fish without neutralizing antibodies.
Temperature has a decisive influence on the carpSVCV interactions. The
virus replicates in vitro at a wide temperature range, with an optimum at
2022C, but the defence system of immunologically mature fish restricts this
activity in vivo to suboptimal temperature. Viraemia, detectable virus shedding
and mortality occur mainly below 15C, when carp are not capable of rapid
interferon and neutralizing antibody synthesis (Fijan, 1988).
After i.p. injection of the virus, the incubation period and survival times at
1617C are 37 days and 810 days, respectively (Fijan et al., 1971).
Incubation after infection by immersion lasts 715 days at 1617C and is much
longer below 10C (Baudouy et al., 1980c). Production of antibodies against
SVCV is influenced by the age and condition of the fish, route of infection and,
most importantly, temperature. Intraperitoneally injected adult carp produce
neutralizing antibodies slowly and unevenly at about 14C, but at 25C
production is more consistent, faster and with higher titres (Fijan et al., 1977a).
Most fish that survive infection at 13C have peak neutralizing antibody titres
after 22.5 months, which decline at 4 months (Baudouy, 1978). Ahne (1980)
found neutralizing antibodies in carp infected by immersion after 7 days at 20C
and after 7 weeks at 13C. However, Hill (1977) did not locate measurable
neutralizing antibodies in fish kept at 15C 10 weeks after exposure by
immersion, while Fijan et al. (1977b) detected neutralizing antibodies in only
7.5% of 118 sera collected 1 and 2 months after oral application of a live vaccine.
In both cases the serologically negative carp were refractory to i.p. challenge with
virus, indicating that specific resistance was mediated by other mechanisms.
Infection of carp by the Asian tapeworm Bothriocephalus gowkongensis or
blood flagellates and other parasites may hamper the development of protective
immunity against SVCV (Klbl, 1980; N. Fijan, unpublished data).
Topics for further study
Refinement of ELISA for greater sensitivity and development of other sensitive
methods for detection and identification of virus are needed. The polymerase
chain reaction (PCR) for an SVCV RNA component could offer the greatest
sensitivity. Such methods will have to be applied in studies on dormant
infection, virus shedding and other aspects of epizootiology.
Studies on the axonomic status of SVCV and PFR by molecular biology
methods could contribute to clarification of differences between these two
closely related viruses.
A better understanding of hostvirus interactions should be achieved by
studies on humoral and cellular immune response including clarification of the
point made by Wolf (1988) about the unclear meaning of a neutralizing antibody
presence (freedom from SVCV or a carrier state). Effects of stress and
husbandry practices on the outcome of infection and virus shedding should be
studied in order to reduce losses by application of improved husbandry and
production technology.
Since eradication of SVC does not seem to be possible, research on
vaccination and other preventive methods is a high priority. Information is needed
on vaccine formulation (live, inactivated or synthetic, mono- versus polyvalent,
adjuvants, etc.), delivery, fate and effectiveness under various conditions. Genetic
selection for resistance to SVC and other diseases should be continued.
PIKE FRY RHABDOVIRUS DISEASE
Pike fry rhabdovirus disease is an acute to subacute contagious disease
characterized by haemorrhage, hydrops and high mortality in fry and young
northern pike (Esox lucius) and in a few other species. The causal agent of PFRD
is closely related to SVCV.
198
N. Fijan
Mortality of pike fry and young fish has been a significant problem in
several hatcheries in the Netherlands since 1956. Two clinical entities,
hydrocephalus and red disease, were described (Bootsma, 1971). Their viral
aetiology was proved by fulfilment of Rivers postulates (de Kinkelin et al.,
1973) and electron microscopy (Bootsma and van Vorstenbosch, 1973). Pike-fry
rhabdovirus disease was inflicting severe economic losses in pike hatcheries in
the Netherlands and in Hungary (Bksi et al., 1984a), but since then mortality
has been infrequent.
Pike fry rhabdovirus and PFRD are geographically limited to Europe. In
addition to the northern pike, other hosts include the grass carp (Ahne, 1975),
tench (Tinca tinca), white bream (Blicca bjoerkna) (Ahne et al., 1982), gudgeon
(Pseudorasbora parva) (Ahne and Thomsen, 1986), brown trout (Salmo trutta)
(Adair and McLaughlin, 1986), roach (Rutilus rutilus), golden ide (Leuciscus
idus var. auratus) (Haenen and Davidse, 1989) and sheatfish (S. glanis)
(Jrgensen et al., 1989). Young roach, carp, grass carp and golden ide develop
PFRD after experimental infection, but not brown trout (Haenen and Davidse,
1989). The roach isolate was not pathogenic for pike (Haenen and Davidse,
1989).
Infected young pike lose their schooling behaviour, move slowly at the
surface or lie motionless on the bottom. In fry, infection follows a subacute
course and manifests itself in hydrocephalus, with a slight to very pronounced
unilateral or bilateral swelling on the dorsal part of the head behind the eyes,
accompanied by exophthalmos. Acute red disease in young fish is characterized
by haemorrhage, typically in the form of a red and swollen area around pelvic
fins. The abdomen is distended, gills are very pale and exophthalmos may be
present. Both groups of clinical signs may appear in the same fish. Ascites,
changes in the colour of the liver and blood accumulating in the pericardial
cavity may be present at necropsy.
Histopathological findings (Bootsma, 1971) include accumulation of fluid
in the ventricle of the mesencephalon and haemorrhage in the connective tissue.
Petechiae may be present in the spinal cord, optic tectum, spleen and
haemopoietic tissue in the kidney. Proximal tubules of the kidney show marked
degenerative changes.
Age is a predisposing factor to PFRD. Fish longer than 45 cm have
increased resistance to infection by immersion (Bootsma et al., 1975). Sexually
mature fish are completely resistant.
Pike fry rhabdovirus has the typical properties of a rhabdovirus. It measures
125 10 nm 80 8 nm (de Kinkelin et al., 1973, 1974; Hill et al., 1975). Its
protein composition is closely similar to that of SVCV. The RNA of PFR was
initially studied by Clerx et al. (1975). Clerx and Horzinek (1978) showed PFR
and SVCV to be antigenically related with regard to the N protein. Jrgensen et
al. (1989) demonstrated that relatedness also exists with regard to G and M
proteins. The latter authors recommended that PFR and SVCV should be
considered as two serotypes of one virus.
Methods for the aetiological diagnosis of PFRD do not differ from those for
SVC. The method of choice for virus identification is ELISA (Jrgensen et al.,
1989). Antibody titres in rabbit sera are low.
Reservoirs of PFR are unknown. Transmission is possible by immersion and
by injection. Pike fry rhabdovirus was not detected in spawning pike, but, if
added to fertilized eggs, it is transmitted to fry and causes 100% mortality
(Bootsma et al., 1975). Experimental egg transmission could be interrupted by
disinfection with an iodophore. Disinfection at 50 mg l
1
available iodine for
1015 min has largely diminished the frequency of PFRD at two hatcheries in
the Netherlands (Bootsma, 1976). Other specific measures for control of PFRD
are not available.
Northern pike can be the host of another specific rhabdovirus, probably
from the vesiculovirus family (Jrgensen et al., 1993). Although isolated from
healthy fry, this virus is strongly pathogenic for pike fry but not rainbow trout.
Disease signs resemble those caused by other systemic rhabdoviral infections.
This unnamed disease should be taken into account in differential diagnosis of
PFRD.
Topics for further study largely correspond to those for SVC.
CHANNEL CATFISH VIRUS DISEASE
Introduction
Channel catfish virus disease is an acute, communicable and highly species-
specific disease of young channel catfish (Ictalurus punctatus) in the USA. The
causal agent is known as channel catfish virus (CCV) or ictalurid herpesvirus 1.
Infection of susceptible, almost exclusively juvenile, fish results in viraemia and
mortality, which occasionally reach nearly 100%.
Historically, awareness of a viral disease affecting fry and fingerlings began
when the channel catfish industry expanded in the mid-1960s. Severe hatchery
mortality occurred in the southern USA. Evidence for a herpesvirus aetiology
and a proposed name of the disease were discussed by Fijan (1968) and Fijan et
al. (1970). The virus was described in more detail by Wolf and Darlington (1971).
Reviews are presented by Wolf (1988), Plumb (1989) and Davison (1994).
Channel catfish virus disease can be economically devastating and is
considered a serious problem. It has been partially responsible for the closing of
at least two catfish farms in the USA and causing decreased production in others
(Plumb, 1988). Precise data on economic damage inflicted by CCV are not
available.
Natural outbreaks of CCVD occur almost exclusively in cultivated channel
catfish. There is only one brief account of a natural outbreak in blue catfish
(Ictalurus furcatus) fingerlings (Plumb, 1989). The virus has not been reported
from wild channel or any other wild catfish.
Experimental induction of disease by i.p. injection of virus is possible in
fingerlings of blue catfish and of channel catfish blue catfish hybrids.
200
N. Fijan
However, CCVD could not be induced in these fish by oral administration of
virus or by cohabitation with infected channel catfish. White catfish (Ictalurus
catus) is susceptible to experimental infection, but disease incidence and
mortality are low (Plumb, 1971a; N. Fijan, unpublished data). Fingerlings of
brown bullhead (Ameiurus nebulosus) and yellow bullhead (Ameiurus natalis)
did not develop disease after injection of the virus (Plumb, 1989). Sheatfish, the
African catfish (Clarias gariepinus), the Asian catfish (Clarias batrachus) and
the bluegill (Lepomis macrochirus) are resistant to CCV (Plumb et al., 1985;
Boon et al., 1988; Chumnongsitathum et al., 1988); the virus was detected in
some specimens of these resistant species only within a few days of infection.
Channel catfish virus and CCVD are endemic in reared channel catfish in
most parts of the USA. Plumb (1994) listed 15 states as their range. Evidence
suggests that there are infected local populations with no history of CCVD
outbreaks. The CCVD status in other countries with channel catfish culture is
not known. It is (or was) present in Honduras (Plumb, 1994) after a shipment of
fry from the USA (J.A. Plumb, personal communication). After introduction of
channel catfish into the former USSR, a syncytium-forming virus was isolated in
BB cells from fish with the clinical signs of CCVD (A.E. Osad caja, 1976,
personal communication). Plumb (1989) reported similar information about
CCVD and virus isolation after the import of channel catfish into Russia.
Isolates from the two latter cases were not examined serologically, but they were
most probably CCV.
The disease
Channel catfish virus disease occurs during warm weather (Fijan et al., 1970),
from May to September (Plumb, 1971a). Outbreaks are more frequent in years
with high water temperatures (Plumb, 1989). The importance of high
temperatures for development of the disease is documented by experimental
data. Virus-injected susceptible fingerlings suffer no or a low mortality when
kept at or below 15C (N. Fijan, unpublished data) and the moving of infected
fish from 28 to 19C markedly reduces mortality (Plumb, 1973a).
Channel catfish virus disease affects up to 10 cm channel catfish in ponds,
raceways or holding tanks. Adult channel catfish raised in the laboratory without
contact with CCV as juveniles are susceptible to infection by bath and single fish
are killed (Hedrick et al., 1987). The first sign of a CCVD outbreak in ponds is
an increase in morbidity and mortality at temperatures above 25C. The course
of an outbreak without secondary infection is acute. Most fish die within 10 days
and mortality normally ceases within 23 weeks. Mortality can vary from low to
almost 100%, but 4060% is common.
Infected fish swim convulsively, often in spirals. In terminal stages, they lie
quietly on the bottom, respiring rapidly but superficially. Some or up to 50% of
moribund fish may hang head-up at the water surface, but this is not
pathognomonic for CCVD.
External signs of disease can vary and depend on the degree of kidney
dysfunction and capillary damage resulting from the replication of CCV. Some
or all of the following signs are present: distension of abdomen, exophthalmia,
swollen and protruding vent (Fig. 5.7); haemorrhage at the base of ventral and
caudal fins, in gills and skin (especially abdomen and peduncle); pale gills.
Secondary infections with Flexibacter columnaris and/or aeromonads often
occur at later stages of the disease and occasionally simultaneously, causing a
combination of CCVD and secondary lesions as well as prolonged mortality.
At necropsy, the peritoneal cavity is hyperaemic and contains a clear,
yellowish or slightly reddish fluid. Liver and kidney may be pale, with or
without haemorrhage or petechiae. The spleen is congested and dark. A
yellowish mucoid material, but no food, is found in the digestive tract.
The histopathology of CCVD is well documented (Wolf et al., 1972; Plumb
et al., 1974; Major et al., 1975; Plumb and Gaines, 1975). Fish with natural and
experimental infections show severe changes, consisting of oedema,
haemorrhage and necrosis. Kidneys are the first and the most severely affected
organ. The haematopoietic tissue shows an increase in lymphoid cells, oedema,
necrosis and accumulation of macrophages. Necrosis and occasional
haemorrhage develop in nephrons (Fig. 5.8). The liver shows oedema, necrosis,
Fig. 5.7. External signs of channel catfish virus disease in fingerlings. Top: Lateral view:
distended abdomen and urogenital vent, bleedings at the bases of fins. Bottom: Dorsal view:
bilateral exophthalmia, abdominal distension and haemorrhage in anal fin.
202
N. Fijan
haemorrhage and occasional eosinophilic cytoplasmic inclusions in the
hepatocytes. The oedematous submucosa of the gastrointestinal tract shows
multiple focal accumulations of macrophages and, in some specimens,
haemorrhage. Necrosis and sloughing of intestinal lining are infrequent. The
spleen is congested and its lymphoid tissue is reduced. Haemorrhage and mild
necrosis are occasionally noted. Cardiac tissues may be affected by focal
haemorrhage and/or necrosis.
The virus is most abundant in kidney, spleen, intestine and encephalon of
overtly infected fish. Kancharla and Hanson (1996) found high virus quantities
also in skin and gills of experimentally infected fingerlings.
Survivors of experimental CCV infection have a retarded growth rate
(McGlamery and Gratzek, 1974), but this is not obvious in natural CCVD
outbreaks in commercial operations (Plumb, 1989).
The virus
The aetiological agent of CCVD, CCV, is herpesvirus-like (Fijan et al., 1970)
and was named Herpesvirus ictaluri (Wolf and Darlington, 1971). Robin and
Rodrigue (1980b) suggested the provisional designation ictalurid herpesvirus 1.
Davison (1992) classified CCV as a sole member of a new, fourth, herpesvirus
subfamily. The characterization of immediateearly genes in the CCV genome
suggests that the virus is most closely related to the alpha subfamily of
Herpesviridae (Silverstein et al., 1995).
Fig. 5.8. Posterior kidney from channel catfish fingerling 3 days after injection with CCV. Large
number of lymphoid cells and necrotic tubules (T). 280. Courtesy of J .A. Plumb.
Dimensions of complete virions (Fig. 5.9) vary between 175 and 200 nm
and the average diameter of the icosahedral nucleocapsid is about 100 nm. Virus
synthesis takes place in the nucleus (Fig. 5.10).
The virions linear ds deoxyribonucleic acid (DNA) has a terminal repeat
sequence at both ends (Chousterman et al., 1979; Cebrian et al., 1983; Davison,
1992). The buoyant density is about 1.715 g ml
1
(Goodheart and Plummer,
1975; Robin and Rodrigue, 1980b) and corresponds to a base composition of
56%. This estimate is in agreement with DNA sequence analysis, which revealed
the total nucleotide composition to be 56.2% C+G (Davison, 1992). Several
authors (Chousterman et al., 1979; Robin and Rodrigue, 1980b; Cebrian et al.,
1983) found the molecular weight of DNA to be about 8.5 10
7
Da.
Chousterman et al. (1979) presented details on molecular structure and
restriction endonuclease sites. Dixon and Farber (1980) reported 32 virus-
specific polypeptides within the molecular weight range of 12,000300,000.
Lacasa (1990) detected and characterized a protein kinase-related gene in
terminal repeats of the genome. Davison (1992) sequenced the whole 134,226-
bp CCV genome in the Auburn 1 strain and predicted the presence of 77 genes.
Two of the immediateearly transcripts were characterized (Silverstein et al.,
1995). The CCV capsid proteins, which are products of specific genes (Davison
and Davison, 1995), showed no relationship to other herpesviruses in their
sequences (Booy et al., 1996). The thymidine kinase (TK) encoded by CCV is
unique among herpesviruses and differs from the TK in the CCO host cell line
Fig. 5.9. Negatively stained virions of channel catfish virus (CCV). The membrane-bound
envelope is very distinct. Nucleocapsid is located somewhat excentrically. 298,000. Courtesy of
R.C. Bird, K.E. Nusbaum and M. Toivio-Kinnucan.
204
N. Fijan
(Hanson and Thune, 1993). The TK gene mapping by marker rescue and marker
transfer genetic analyses showed the amenability of CCV to investigations used
in the molecular biology of other herpesviruses (Hanson et al., 1994). Assays for
PCR amplification of TK and protein kinase-related genes were recently
described by Tham and Moon (1996).
Polyclonal rabbit antibodies recognize CCV isolates as a homogeneous
group in virus neutralization, IFAT and ELISA tests. On the other hand, virus
neutralization patterns of four CCV isolates with four monoclonal antibodies
indicated possible antigenic differences between strains (Arkush et al., 1992).
Diversity in DNA restriction digestion patterns among 12 CCV isolates was
reported by Colyer et al. (1986). These differences may be useful in
Fig. 5.10. Intranuclear channel catfish virus in spleen cell of channel catfish. The virus has
migrated to the nuclear membrane, which in some places has been destroyed. Intranuclear
lamellar-like inclusions are also present. 22,000. Courtesy of J .A. Plumb.
epizootiological studies for instance, to identify the source of a particular virus
or epizootic.
The virus infectivity is inactivated by ether, chloroform and glycerol. It is
sensitive to acid pH, heat and UV light and is unstable in sea water (Robin and
Rodrigue, 1980a). Under simulated farm pond conditions, CCV survives less
than 24 h on dried concrete chips and less than 48 h on glass cover slips or dried
fishnets and is immediately inactivated by pond mud (Plumb, 1974). The finding
of Brady and Ellender (1982) that soil sediment rapidly absorbs the virus helps
explain the immediate inactivation by pond mud. In pond water the virus persists
for about 2 days at 25C and about 28 days at 4C, but somewhat longer in
dechlorinated tap water (Plumb et al., 1973).
The rate of virus degradation in decomposing fish is temperature-dependent
and relatively rapid. It is impossible to isolate virus from dead fish at 22C after
48 h. Icing or freezing at 20 or 80C preserved infectivity for 14, 162 and 210
days respectively (Plumb et al., 1973).
The in vitro replication of CCV occurs in catfish cell cultures. These include
primary cultures from channel catfish, BB cells (Fijan et al., 1970), CCO cells
(Bowser and Plumb, 1980a,b,c), continuous lines of channel catfish leucocytes
(Chinchar et al., 1993) and K1K cells from the more distantly related walking
catfish (C. batrachus) (Noga and Hartmann, 1981; Walczak et al., 1981). Three
cell lines from marine fish (Fernandez et al., 1993) are also susceptible. In
stationary cultures, maximum virus yields (about 10
8
pfu ml
1
) are obtained in
the CCO cells and the lowest ones in the K1K line and in some leucocyte lines.
CCO cells show approximately 1 log greater sensitivity than BB cells at
2533C (Bowser and Plumb, 1980c) and a more rapid development of
cytopathic effect. Channel catfish virus replicates in vitro in the temperature
range between about 10 and 35C, with an optimum at 2530C. The cytopathic
effect is characterized by formation of syncytia (Fig. 5.11), which are usually
interconnected with cytoplasmic strands, followed by pyknosis, lysis and total
destruction of the cell sheet.
Changes in infected BB cultures at 30C were monitored by Wolf and
Darlington (1971). The degenerative changes started at 2 h and the first
enveloped virions were completed by 10 h. These authors also described the
course of virus replication and growth in BB cells. Bowser and Plumb
(1980a,b,c) studied viral growth in CCO cells, while Walczak et al. (1981)
conducted studies in K1K cells. Somewhat more than 50% of virus remained
cell-associated in BB and CCO cells, while most of it was released by K1K cells.
Noga and Hartmann (1981) and Walczak et al. (1981) produced a distinct
attenuated strain in K1K cells, recognizable by the small plaque size, and
claimed that it is suitable for vaccination.
Diagnostic methods
The diagnosis of CCVD is based on the isolation and identification of CCV or on
immunological demonstration of CCV antigen in infected fish tissues. The
detection of the dormant CCV infection by these methods is recommended by
206
N. Fijan
the OIE Manual (1995b), but references on the success of the procedure are
unavailable.
Increased mortality among channel catfish fry or fingerlings during warm
weather, especially after stress, warrants examination for CCVD and sampling
for laboratory tests. Hydropic conditions in fish with external signs of
columnaris disease or of other bacterial infections may also be an indication of
primary involvement of CCVD. The OIE Manual (1995b) recommended the
selection of a sample of ten moribund or clinically affected fish. Living or
sacrificed specimens should be properly packed, labelled and transported under
adequate cooling. They should not be frozen. It is preferable and recommended
to collect viscera (including kidneys) from fish of 46 cm or kidney, spleen and
encephalon from larger fish. Up to 1.5 g of such material from up to five fish
should be pooled in a sterile vial with a fivefold volume of transport medium
with antibiotics for suppression of bacterial contaminants. Whole fish shorter
than 4 cm should be in such a transport medium. Material for examination
should reach the laboratory at a time which allows processing within 24 or a
maximum of 48 h after sampling.
Inocula for CCV isolation are prepared by homogenization of the sample,
Fig. 5.11. Formation and contraction of syncytia in CCO cells about 14 h after inoculation with
CCV and incubation at 30C. Wright stain. 230.
dilution 1 : 10, decontamination of supernatant with antibiotics or filtration and
by making two additional tenfold dilutions. Channel catfish ovary cells are
recommended for isolation (OIE Manual, 1995b). Aliquots of 100 l from each
of the three serial tenfold dilutions serve for inoculation of at least 2 cm
2
of 24-h-
old and drained CCO cultures. After adsorption at 2530C for 0.51 h and
addition of a medium buffered to maintain pH between 7.3 and 7.6, cultures are
incubated at 2530C for 7 days. Positive and negative controls are mandatory.
The cytopathic effect may become visible after 1012 h in the form of focal cell
granulation, which is soon followed by cell enlargement and formation of
syncytia. If cultures inoculated with the test material remain negative, they
should be subcultivated.
Formation of syncytia in CCO or BB cells is specific for CCV. Titres of
CCV in infected fish may reach 10
5
10
6
TCID
50
or pfu g
1
of tissue, but are
usually lower, both in early and in late stages of an epizootic. Virus identification
is carried out by virus neutralization, IFAT or ELISA, using positive and
negative controls. The titre of neutralizing antibody solution for virus
neutralization must be around 2000 in the 50% plaque reduction assay (OIE
Manual, 1995b). The first monoclonal antibodies against CCV (Arkush et al.,
1992) are opening better possibilities for identification, detection and
quantification of CCV.
Attempts at viral isolation from survivors of CCVD outbreaks and from
suspected brood-fish carriers were unsuccessful (Plumb and Jezek, 1983), until
Bowser et al. (1985) examined a population suffering sustained mortality during
the winter, using cocultivation of tissue extracts or leucocytes and blind passages
of inoculated CCO cultures. Immunosuppression of brood fish by i.m. injection
of dexamethasone increased the virus isolation rate by cocultivation of
leucocytes to 100%. Using normal procedure and cocultivation, Wise et al.
(1988) could not isolate virus from stressed, non-clinically infected fry. There
are no further reports on testing of suspect carrier populations using the method
described by Bowser et al. (1985).
The first indication of the CCV antigen in brood-fish ovaries was provided
by Plumb et al. (1981), using an IFAT. Focal areas of fluorescence were recorded
in spent ovaries from two immunosuppressed catfish and in the primary cell
cultures from this tissue, but there was no virus replication in cultures. The
authors believed the fluorescence to be a specific reaction, possibly with
incomplete virus. Detection of latent CCV in clinically healthy catfish was
successfully carried out by nucleic acid hybridization methods and by PCR.
Wise and Boyle (1985) cloned the terminal fragment of the CCV genome and
used it as a specific probe for detection of viral DNA. The probe demonstrated
CCV DNA in liver and other soft tissues and in erythrocytes of some channel
catfish with no history of CCVD (Wise et al., 1985). Bird et al. (1988) selected
probes for detection of viral DNA expressed late in infection and one of them
was highly sensitive for CCV-specific sequences. Boyle and Blackwell (1991)
applied the PCR for detection of CCV DNA in latent carrier fish. Kancharla and
Hanson (1996) developed a quantitative PCR which detected over 500 times
more virus DNA molecules than the plaque assay of Buck and Loh (1985).
Refinements of these techniques may lead to the selection of appropriate tests
208
N. Fijan
for diagnostic procedures. The OIE Manual (1995b) disclosed that ELISAs
using certain monoclonal antibodies to virus nucleocapsid antigen may become
the recommended procedure for the detection of virus carriers by screening fish
for viral antigen in the encephalon.
Epizootiology
Reservoirs of virus are the overtly and the dormantly infected fish. Transmission
is mostly horizontal but the claimed vertical or egg associated transmission
from carrier brood fish to eggs and fry seems to be important for virus survival
and for perpetuation of infection in catfish culture.
Horizontal transmission occurs by contact and through water, and is easily
demonstrable by cohabitation of virus-free catfish with infected fish. Channel
catfish virus is probably shed via faeces and urine. Fingerlings infected by bath
and kept at 28C shed the virus from the second to the sixth day in considerable
quantities (Kancharla and Hanson, 1996). Plumb (1988) assumed that
fingerlings contracted the virus by cannibalizing dead or moribund fish with
CCVD. The short CCV survival in pond water and mud and on tools indicates a
low significance of vectorial transmission over long time intervals. The role of
biological vectors has not been investigated. Nothing is known about potential
virus reservoirs in natural catfish populations.
Plumb and Jezek (1983) never isolated the virus from suspected brood fish.
Nusbaum and Grizzle (1987b) could not demonstrate the vertical transmission.
Yet there is circumstantial epizootiological and other evidence for vertical
transmission. Outbreaks of CCVD in progeny of brood stock with high titres of
CCV-neutralizing antibodies were regular (Amend and McDowell, 1984), and
the viral antigen was present in ovaries of spent females (Plumb et al., 1981).
The virus was isolated from brood fish in the winter (Bowser et al., 1985) and
the latent virus was detected in adult and in brood fish with no clinical signs and
in their apparently healthy offspring, using hybridization methods or PCR (Wise
and Boyle, 1985; Wise et al., 1985, 1988; Boyle and Blackwell, 1991). Egg
infection, if it occurs, is accomplished by different mechanisms from those
involved in the surface infection of salmonid eggs by IHN virus (Nusbaum and
Grizzle, 1987b).
The disease is easily induced in susceptible channel catfish fry and
fingerlings by exposure to virus in water, swabbing the gills or feeding with
virus, as well as by i.p. or i.m. injection (Fijan et al., 1970). However, some
attempts to induce CCVD by exposure to virus in water have failed (Wolf et al.,
1972; McConnell and Austen, 1978) for unknown reasons. Some adult fish can
replicate CCV after water-borne exposure (Hedrick et al., 1987).
Strain and age of fish and water temperature are the decisive predisposing
factors for outbreaks of CCVD.
Plumb et al. (1975) found striking differences among strains of channel
catfish fingerlings in their susceptibility to the virus. The interstrain hybrids can
be significantly more resistant to infection than pure strains.
Overt disease develops at an age between 2 weeks and 6 months. Natural
overt infections are generally confined to fish weighing less than 10 g. Virus
injection induces disease in fish of up to 50 g (Plumb, 1971a) and the virus has
been occasionally isolated from 1-year-old fish (Plumb, 1989). Experimental
water-borne infection of adult channel catfish with no prior viral contact resulted
in very low mortality. Survivors become asymptomatic carriers, with specific
neutralizing antibodies (Hedrick et al., 1987).
McConnell and Austen (1978) postulated that the high mortality rates in
commercial operations were not due solely to the virus but resulted from
simultaneous infection and water-quality problems. High stocking rates,
oscillating environmental conditions, low oxygen and poor water quality
probably increase the chances and severity of mortality. The relationship of these
factors to the course of CCV infection has not been explored experimentally.
Prevention and control
Approaches and methods for the control of CCVD need further refinement. Wolf
(1988) suggested avoidance and the use of resistant channel catfish strains and
possibly of resistant species hybrids. Plumb (1989) considered avoidance,
quarantine of infected fish populations, sanitation and optimization of hygienic
(environmental) conditions in rearing facilities to be practical. Vaccination is in
the experimental phase, while the manipulation of temperature is impractical.
Chemotherapy is unavailable.
Avoidance is the most desirable and practical approach and it can be carried
out only if virus-free stock is available. To control the spread of CCVD, Plumb
(1973b) recommended avoidance of serologically positive brood stock. Amend
and McDowell (1983, 1984) and Plumb (1989) warned about the limited value
of serological examinations of brood stock for absence of CCV. New methods,
especially PCR and ELISA, may become crucial for brood-stock screening and
the reliability of the avoidance approach.
Slaughter has been recommended for prevention of the spread and for
eradication of the disease. Plumb (1971c) advocated the destruction of both the
affected fish and the brood stock and disinfection of premises and equipment.
Wolf (1988) mentioned instances where affected fish were killed and ponds
disinfected with 2050 mg l
1
chlorine. After dissipation of chlorine and
biological testing for residual CCV, ponds were stocked and the susceptible fry
remained free of CCVD. Drying out of ponds should also effectively inactivate
the virus. It should be pointed out that the stamping-out policy can only be
effective if both the water-supply and the fish for restocking are free of virus.
Channel catfish virus is sensitive to UV irradiation (Yoshimizu et al., 1989) and
its application in hatcheries could reduce risks of virus dissemination among fry.
When quarantine and restriction of movement are applied to control the
spread of CCVD, survivors should be delivered exclusively to a processing
plant. Under no circumstances should such fish be used as brood stock.
The reduction of water temperature to 19C or lower decreases the mortality
rate if carried out soon after infection, but not much after the onset of disease
(Plumb, 1973a, 1989). Such temperature manipulation may be useful, but is
rarely practical in large-scale commercial operations.
210
N. Fijan
Two in vitro studies on a candidate for chemotherapy of CCVD (Koment
and Haines, 1978; Kimura et al., 1983) showed that phosphonoacetic acid was
not promising.
As the control of secondary bacterial infections may reduce mortality, fish
farmers provide medicated feed in CCVD-affected catfish ponds (Plumb, 1988).
The OIE Manual (1995b) recommends thorough disinfection of eggs and
complete protection of premises for further developmental stages from contact
with virus carriers and fomites. No literature on the success or failure of such an
approach is available.
Immunization has so far received only limited attention. The potential
vaccination against CCVD is therefore unknown. Economic obstacles are
considerable, perhaps even prohibitive (Clem et al., 1996). Progress is also
hindered by the need to convince the catfish industry and potential producers of
a commercial vaccine about the need for a vaccination programme (Plumb,
1989). An immersion or oral vaccination for fry, when fish are being moved
probably has the best chance of becoming acceptable (Clem et al., 1996).
The first report on experimental vaccination was presented by Noga and
Hartmann (1981). They attenuated CCV by 60 in vitro passages in K1K cells.
Intraperitoneal inoculation of attenuated virus protected the fish effectively
against challenge. Adequate protection using hyperosmotic infiltration of the
vaccine required two doses at a 3-week interval (Walczak et al., 1981). A
constructed recombinant TK gene-deletion mutant of CCV also induced
protective immunity against a lethal dose of wild-type CCV (Zhang and Hanson,
1995). This CCVTK can express foreign genes and induce antibodies against
their products, thus indicating the potential of recombinant CCV for vaccine and
vaccine vector development (Zhang and Hanson, 1996). Reduced shedding and
persistence of this recombinant virus is also an encouragement for further
research in this direction (Kancharla and Hanson, 1996).
A subunit vaccine from CCV envelope was prepared and tested in a
preliminary study by Awad et al. (1989). Eyed eggs and 7-day-old fry were
vaccinated by bath. Subgroups obtained booster dose 2 weeks after vaccination.
Vaccinated fry survived the challenge at significantly higher rates than controls.
Impressive protection obtained by application of antigen to the life stages which
are generally considered to be immunologically immature is surprising.
However, in the light of accessibility and ease of manipulation of this age of
channel catfish, such vaccination trials should at least be repeated.
Breeding for resistance and hybridization of channel catfish strains are a
promising approach. Plumb et al. (1975) showed differences in susceptibility of
six inbred strains to infection with CCV and a significantly higher resistance of
two interstrain hybrids compared with parental strains. Cultivation of blue
catfish or of its hybrids with the channel catfish on farms frequently affected
with CCVD could be a successful and relatively easily applied tactic.
Considering the important role of stress in CCVD, it seems that improved
management practices can reduce the incidence and severity of the disease.
Thune (1993) suggested proper water flow and circulation and daily removal of
eggshell debris and of wasted feed, as well as feeding with nutritionally
complete fry starter with at least 50% protein in hatcheries. On farms with
endemic CCV, stocking densities for production of fingerlings should be below
220,000 ha
1
and daily feeding rates below 75 kg ha
1
, especially during high
water temperatures. Fingerlings should be harvested and handled only at
temperatures below 20C. Supplementary aeration should secure adequate
levels of dissolved oxygen, especially during high water temperatures.
Radiolabelled CCV enters fish through gills and possibly also through the
intestine and is concentrated in the liver (Nusbaum and Grizzle, 1987a). The
kidney seems to be the primary site of virus replication, followed by other
parenchymatous organs (Plumb, 1971b). A special (antiviral) class of cytotoxic
cells from catfish peripheral blood leucocytes is capable of lysing virus-infected
cells in vitro (Hogan et al., 1996). Fast virus replication in the host causes a short
incubation time of about 3 days at 2530C and of about 10 days at 20C.
Destruction of infected cells leads to major damage of the circulatory system and
of excretion causing osmotic imbalance as well as haemorrhage. Data on
haematology and other clinical parameters are lacking.
The virus elicits a variable humoral immune response and resistance to
reinfection. Plumb (1973b) was the first to recognize anti-CCV neutralizing
antibodies in sera. Brood stock which had produced virus-infected fingerlings
for 2 consecutive years had fairly constant neutralization indices throughout 1
year of examination. Gratzek et al. (1973) described virus neutralization in
microcultures for quantitation of neutralizing antibodies. Heartwell (1975)
characterized neutralizing antibodies as immunoglobulins. Virus neutralization
activity can be found in juveniles 1 week after i.p. and i.m. infection (Plumb,
1973b; Heartwell, 1975) and at the same time after waterborne exposure of
adults (Hedrick et al., 1987). Activity increases up to 9 weeks before declining.
Survivors of experimental infection have anti-CCV activity in plasma 2 years
after the last known exposure to virus (Hedrick et al., 1987). Under natural
conditions, neutralizing antibodies can be found 4 weeks after the original
disease outbreak (Bowser and Munson, 1986). Neutralizing antibodies may be
absent in populations which suffer a low (0.1%) mortality (Amend and
McDowell, 1983). Neutralizing antibody titres vary with water temperature and
are highest during summer and lowest in winter (Bowser and Munson, 1986).
Findings on farms with and without a history of CCVD indicate that vertical
transmission with disease outbreaks in offspring occurs when brood stock have
higher antibody titres and a higher percentage of positive sera (Plumb et al.,
1981). Continuing virus neutralization activity in sera seems to be stimulated by
expression of certain viral antigens or periodic reactivation of virus (Hedrick et
al., 1987). Concerning cellular immunity, Hogan et al. (1996) described a
population of cytotoxic cells among peripheral blood leucocytes that killed
CCV-infected cells. Even the early virus gene products rendered cells
susceptible to lysis.
Juvenile catfish can be immunized passively by injection of antiserum from
adults (Hedrick and McDowell, 1987).
212
N. Fijan
Continued efforts to develop and test specific, sensitive, simple and practical
methods for detection of latent-state CCV in fish are the prerequisite for further
epizootiological studies and the control of CCVD by avoidance. The
development and testing of subunit and other vaccines and exploring the
potential of breeding for resistance to CCVD should be pursued.
CYPRINID HERPESVIRUS I INFECTION (CARP POX)
Introduction
The cyprinid herpesvirus I (CHV) of common carp manifests itself in three
clinical forms: chronic and benign epidermal proliferation or carp pox (CP),
acute septicaemia in very young carp and dormant infection.
Benign epithelial skin tumours (fish pox, CP, epithelioma papulosum
cyprini, epithelioma papulosum, papillomatous skin growth, epidermal
hyperplasia, epidermal proliferation) in carp were first reported in Europe in
1564 (Hofer, 1904). This disease and similar conditions in other fish were
covered by the name pox or fish pox by Hofer (1904), Schperclaus (1941),
Nigrelli (1952) and Wolf (1988). A minority of authors used the term CP but
extended the range of affected species (Ghittino, 1985; McAllister, 1993b).
The aetiology of CP was attributed to a herpesvirus by von Schubert (1964),
who visualized its particles in epidermal proliferation by electron microscopy.
Sano et al. (1985a,b) isolated CHV (or Herpesvirus cyprini) from common and
coloured carp with CP. Experimental infection of 24-week-old fry with this
virus induced an acute, generalized and species specific-infection with high
mortality. Survivors developed typical CP lesions after several months
(Sano et al., 1991). In accordance with the generally high host specificity of
Herpesviridae, the CHV was not pathogenic to fry of two other cyprinids (Sano
et al., 1991). Hedrick et al. (1990) confirmed the presence of herpesviruses in
epidermal hyperplasia of coloured carp and their consistency with CHV, but did
not report their isolation in cell culture.
Fish pox or CP is actually inappropriate for epidermal proliferation and,
according to Nigrelli (1952), Hines et al. (1974) and Wolf (1983), it is not a true
epithelioma or papilloma. All these terms should be used with caution or
abandoned. The three postulated disease forms caused by CHV are referred to
here as CHI. Herpesviral epidermal proliferation in carp (HEPC) is used instead
of pox and the systemic infection in fry is denoted as herpesvirus septicaemia
in carp (HSC). The name fish pox remains for similar, but aetiologically
unclear epithelial skin proliferations in cyprinids and other fish.
The HEPC causes economic damage to carp farmers by reducing fish
growth, rendering them unsightly and making them unmarketable. Although
frequent in the past, HEPC is now rare in European and Israeli pond farming
probably due to reduced inbreeding.
Herpesvirus epidermal proliferation in carp affects all scale-distribution and
colour varieties of carp in Europe, Israel, Japan, South Korea, China and North
America. Fry of goldfish (Carassius auratus), grass carp and willow shiner
(Gnathopogon elongatus caerulescens) are resistant to CHV infection under
controlled conditions and survivors remain free of skin neoplasms (Sano et al.,
1991). Herpesvirus septicaemia in carp has yet not been diagnosed in farmed or
feral populations.
The disease
Herpesvirus epidermal proliferation in carp is a chronic and benign hyperplastic
skin disease. Lesions may first develop on one or several locations, commonly
on the fins. Initially they are flat, firm, smooth and translucent, but they soon
grow thicker, giving the impression of paraffin drops on the skin. Multiple
hyperplasia of squamous cells can form islands over the entire body (Fig. 5.12).
Further proliferation results in irregularly shaped papillomatous formations,
several centimetres wide and 46 mm thick. These have a warty surface and a
milky to greyish white colour, which is sometimes tinged with pink by capillary
dilatation. Proliferations rarely coalesce to cover almost the whole skin. Some
cells have cytoplasmic and Cowdry type A inclusions in nuclei, with marginated
chromatin. The normal strata are lacking, the number of mucous cells is reduced
Fig. 5.12. Herpesviral epidermal proliferation in carp (HEPC) or carp pox lesions in mirror-type
common carp (Cyprinus carpio). Glistening patches of varying size, thickness and shape; some are
translucent, others milky.
214
N. Fijan
and serous glands are absent. Mitoses are frequent. Proliferating cells are not
invasive and they are not metastatic. The underlying structures appear normal in
thin proliferations, but the subepidermis of large papillomatous lesions form
finger-like projections to reach and nourish the thick and folded epithelium.
Retarded growth and emaciation were consistently observed in advanced
natural cases of fish pox. The tail of such a carp can be easily bent to touch its
head. Such flabby fish have a reduced muscular tone and osteomalacia with very
low levels of ash, calcium and phosphorus in the vertebrae (Mann, 1951). After
recovery, some may have spinal deformities. The causal relationship of this
disorder in metabolism of minerals and the CHV has not been established
experimentally.
In 2-week-old fry infected experimentally by bath, HSC appeared after 1
week of incubation at 20C (Sano et al., 1991). Mortality was over 80% in
2-week-old fry and about 30% in 4-week-old fish. In 8-week-old fingerlings, the
infection did not cause mortality. At 15C, mortality is highest and it decreases
with increased temperatures to 20 and 25C (Sano et al., 1993a). Sano et al.
(1993b) showed that 2-week-old fry kept at 20C did not develop HSC, and
HEPC appeared 20 weeks after bath immersion. Clinical signs of HSC include
loss of appetite, swimming in straight lines and occasional loss of movement.
Fish have dark pigmentation, a distended belly, exophthalmia and haemorrhage
on the operculum and abdomen. Infected 4-week-old fingerlings only exhibit
spiral swimming. Necrosis is the main histological finding; it affects the liver,
kidney and lamina propria of the intestinal mucosa. Papillomatous skin
neoplasms develop in over 50% of survivors infected as fryfingerlings and in
about 10% of infected 1- or 2-year-old carp. Spontaneous outbreaks of HSC
mortality have not been reported.
The virus
Several attempts to isolate the virus from fish pox or CP lesions either failed
(Sonstegard and Sonstegard, 1978; Fijan et al., 1983; McAllister et al., 1985) or
had a dubious outcome (Grtzner, 1956; Ghittino et al., 1984). Such an attempt
resulted in the establishment of the EPC cell line (Fijan et al., 1983). Sano et al.
(1985a,b) recovered CHV from the Asagi strain of coloured carp and from the
common carp. This finding has so far not been confirmed by another laboratory.
An aquareovirus isolated from CP in China is not related to the disease and is
innocuous to fish (Jiang et al., 1994).
The CHV has a diameter of 190 27 nm in FHM cells and its nucleoid
measures 113 9 nm (Sano et al., 1985a). In European carp with natural HEPC,
virions have a diameter of 140 nm (von Schubert, 1964). Sano et al. (1985a,b)
described the isolation and growth of the virus in FHM, MCT and EPC cell lines
at 1520C, which is the optimal temperature (Sano et al., 1993a). Cyprinid
herpesvirus I multiplied in FHM cells at a temperature range from 10 to 25C,
but not at 30C (Sano et al., 1993a). At primary isolation, a cytopathic effect
appears in 23 weeks. During in vitro passages, it becomes evident after about 5
days. The cytopathic effect is characterized by vacuolation, pyknosis, slow
detachment of cells, margination of chromatin and occasional Cowdry type A
inclusions. The highest titre of about 10
5.2
TCID
50
ml
1
is obtained in FHM cells.
Virus can be reisolated from moribund and dead carp fry, as well as from about
50% of induced tumours (Sano et al., 1991).
Diagnostic methods
Diagnosis of HEPC can be based on external signs. Histological findings will
strengthen its reliability. Isolation and identification of CHV in cell cultures are
possible (Sano et al., 1985a, b, 1991, 1992, 1993b) but have not been reported
from natural outbreaks of HSC in fry. Sano et al. (1991) demonstrated virus in
tissues of carp fry and in skin neoplasms, using IFAT. Detection of its genome
was carried out by in situ hybridization in infected cell cultures and in tissues of
experimental fry with acute infection and with neoplasms (Sano et al., 1992).
The virus genome can be detected with this probe at a time when CHV cannot be
isolated or viral antigen detected in injected carp, i.e. between the acute infection
and neoplasm formation (Sano et al., 1992).
The prevalence of HEPC on carp farms varies from less than 1 : 10,000 to over
70%. It appears during low to moderate temperatures from late winter to early
summer. Skin tumours are transient they usually desquamate after some time
and scar tissue may develop. Hines et al. (1974) noted the disappearance of these
lesions as the water warmed up during the summer. Under experimental
conditions (Sano et al., 1993a) regression occurred after shifting the water
temperature from 7.5C to 2030C but not after the raise from 7.5 to 15C.
Sano et al. (1991) noted HEPC recurrence in experimentally infected carp after
7.5 months. Reappearance has long been known from pond environment
(Schperclaus, 1941). Young carp develop these tumours more rapidly than
older ones (Sano et al., 1991). Herpesvirus epidermal proliferation in carp does
not generally cause mortality.
Experimental transmission of CP to common carp and common carp
goldfish hybrids by cohabitation and by rubbing lesion tissue against abraded
epithelium was reported by Sonstegard and Sonstegard (1978). After about 60
days at 10C, HEPC developed. This incubation time was shorter than the ones
reported by Sano et al. (1985a,b, 1991).
Genetic predisposition seems to influence the appearance, frequency and
severity of CP (Schperclaus, 1941; Hines et al., 1974). Some inbred carp strains
are particularly susceptible.
An effective treatment for HEPC is not known. Replacement of inbreeding
by cross-breeding can significantly reduce the incidence and severity of disease
(Schperclaus, 1941, 1979; Hines et al., 1974). Sloughing of lesions can be
speeded up by raising the water temperature above 20C. Liming of pond water
in spring was practised in European carp culture to accelerate recovery. Cross-
216
N. Fijan
breeding and the above prophylactic measures have almost completely
eliminated HEPC from carp farms in Europe (Schperclaus, 1979; N. Fijan,
unpublished data).
Cyprinid herpesvirus Icarp interactions were first described by Sano et al.
(1991, 1993b). An IFAT demonstrated the virus in infected 2-week-old carp after
221 days in gills, liver, kidney, intestinal mucosa, oesophagus and lamina
propria. Subsequently, virus could not be detected until the appearance of
tumours, when it reappeared in skin (and in neoplastic tissue) but not in intestine.
Search for the viral genome by in situ hybridization (Sano et al., 1993b) in
asymptomatic fish 819 weeks after infection revealed its latent establishment in
gills, cranial nerve ganglia, subcutaneous tissue and spinal nerves. The genome
is associated with the induction and recurrence of HEPC.
There are no references about the specific immune response. Recurrence of
infection indicates the absence of immunity.
Additional information is needed for the evaluation of health risks in farmed and
feral fish populations. Further studies should include the confirmation of CHV
isolation from skin lesions in coloured, farmed and wild carp strains, a search for
cases of naturally occurring CHS in fry and the determination of its range and
economic significance. In addition, the elucidation of persistent infection with
CHV and mechanisms of its reactivation and the influence of the genetic make-
up of carp strains on hostvirus interactions (including immune response) and
the comparison of isolates from geographical regions and the metabolism of
minerals in induced and natural cases of EHPC are also needed. Methods should
be developed for virus isolation from fish pox in other species.
GRASS CARP HAEMORRHAGIC DISEASE
Introduction
Grass carp haemorrhagic disease is a severe acute disease caused by the grass
carp reovirus (GCRV). The disease has been known in the People
s Republic of
China since the 1950s (Mao et al., 1988). The GCRV was isolated during
197884 (Mao et al., 1988) and described by Chen and Jiang (1984). Nie and
Pan (1985), Wolf (1988) and Winton (1989) reviewed the status of knowledge on
the virus and on the disease. Additional data are presented by Jiang and Ahne
(1989) and Jiang et al. (1994). Some papers written in Chinese have an English
abstract.
Grass carp haemorrhagic disease affects young fish and yearlings of grass carp
(Ctenopharyngodon idella) and of black carp (Mylopharyngodon piceus). It is
widespread in the southern regions of mainland China and causes severe,
economically devastating losses for the largest national aquaculture production
in the world. Mao et al. (1988) reported mortality rates of 5070% in yearlings.
Gudgeon (P. parva) and Gobiocypris rarus (Wang et al., 1993, 1994) are
susceptible to experimental infection. The disease is not registered outside
mainland China.
The disease
Outbreaks of GCHD occur in summer at water temperatures above 25C.
Affected fish have exophthalmia and severe haemorrhage at the base of fins and
in gills, opercula, eyes, oral cavity, musculature, liver, kidney, spleen and
digestive system. Mortality can exceed 80%. Fingerlings are more susceptible
than yearlings. The disease is serially transmissible by injection of cell-free
filtrates (Chen and Jiang, 1984). Guo and Jiang (1993) described differences in
gross lesions and histopathological alterations caused by two GCRV isolates,
which indicated the possible existence of serological and virulence types.
Experimental infection with grass carpvirus isolate 873 (GV-873) causes severe
pathology in liver and spleen, while GV-9014 induces hyperaemia and haemor-
rhage in muscles and oral cavity, with a low level of pathology in visceral organs.
The virus
The GCRV (fish reovirus, grass carp haemorrhage virus (GCHV), haemorrhagia
virus of grass carp) is an aquareovirus (Jiang and Ahne, 1989; Jiang et al., 1994).
Icosahedral virus particles examined by several authors show a double capsid
layer and a diameter of about 6570 nm (Jiang and Ahne, 1989; Zeng and He,
1992). Infectivity is not affected by organic solvents, acidic or alkaline pH or
trypsinization but is sensitive to freezethaw cycles and heat (Jiang and Ahne,
1989; Zeng and He, 1992). Chen and Jiang (1984) noted virus replication in
three grass carp cell lines. Deng et al. (1985) found the cell line from grass carp
kidney (GCK-84) to be the most sensitive among six lines from various fish and
tissues. The cell line yielded titres of over 10
8
TCID
50
ml
1
. Several authors
successfully used other grass carp cell lines (Li et al., 1988; Ke et al., 1990; Yang
et al., 1992; Luo et al., 1993) and a clear cytopathic effect was reported in
several of them. Of 12 fish, one reptilian and one mammalian cell line inoculated
in experiments of Jiang and Ahne (1989), none developed a cytopathic effect
even after 30 blind passages. Electron microscopy demonstrated latent infection
only in lines from grass carp fin (CF), kidney (CK) and ovary (CO) and two of
them produced numerous virus particles of 6575 nm in diameter. Positive
immunoelectron microscopy with convalescent grass and black carp sera
218
N. Fijan
confirmed their identity. Isolates from grass and black carp were pathogenic for
both species. Infection by bath and injection induced typical GCHD signs, as
well as mortality between 50 and 95%. CF and CO cells produced simul-
taneously an interferon-like substance.
Distinct strains may be present in some geographical areas. An isolate from
Hunan province differed from those present in three other areas (Ke et al., 1990).
Comparison of two GCRV isolates (Jiang et al., 1994) showed similarity in
morphology and size, but also differences in RNA profile, antigenicity,
characteristics in cell cultures and virulence for grass carp fingerlings.
Aquareoviruses from carp with pox and from a sick eel did not cause disease in
grass carp.
Mao and coauthors (Mao et al., 1988, 1989; Mao and Shao, 1989; Shao et
al., 1990) reported two viruses in tissues of grass carp with GCHD examined by
electron microscopy. Together with the reovirus, authors found an ssRNA virus
measuring 2030 nm in diameter and temporarily assigned it to Picornaviridae.
Both viruses were isolated and purified from sick fish by polyethylene glycol
(PEG) and protamine sedimentation. They were pathogenic to grass carp
yearlings (mortality around 65%). The pathology of both infections was
dominated by haemorrhage, but its predominant location was distinctive. The
reovirus affected the gut, while the picornavirus induced severe haemorrhage in
the musculature. Wang et al. (1993) expressed the view that these two particle
sizes represented different developmental stages of GCHV.
Diagnostic methods
Diagnosis of GCHD is based on clinical signs, isolation of GCRV in a
susceptible cell culture and its identification. An ELISA assay for identification
is described by Min et al. (1986) but more recent data are not available.
Immunoelectron microscopy was also used for identification of the virus (Chen
and Jiang, 1984; Jiang and Ahne, 1989). Yang et al. (1991) provided preliminary
data on rapid and simple GCHV identification in purified preparations, cells and
fish tissue by coagglutination, using antibody-sensitized staphylococci.
Data on predisposing factors are limited to water temperature. Non-specific
prevention methods are not known. Breeding for resistance, using G. rarus as a
model, was suggested by Wang et al. (1994). Immunization seems to be a
promising approach for prevention. Injection of an inactivated virus prepared
from infected tissue induced a relatively long-lasting, specific, protective
immunity (Nie and Pan, 1985). Methods of large-scale virus production were
improved (Yang et al., 1992; Ye et al., 1992; Luo et al., 1993) and the selection
of a virus strain for the vaccine was also reported (Luo et al., 1993). Zhu et al.
(1993) found that the Kelieao-Yufukang, a combination of two drugs,
possessed in vitro and in vivo anti-GCRV activity.
Disease signs appear in about 710 days after infection with cell culture-grown
virus (Zhu et al., 1987; Jiang and Ahne, 1989). In G. rarus incubation lasts 5
days and mortality peaks at 68 days reaching 80% (Wang et al., 1994). A
significant reduction in number of red blood cells, total plasma protein and urea
nitrogen starts 45 days after experimental infection and progresses until the
onset of external signs. The virus causes liver dysfunction and changes in blood
levels of several enzymes, as well as of albumin and cholesterol (Zhu and Xie,
1993). The targeted visceral and muscular blood capillaries have damaged cell
organelles (Zheng et al., 1991). Gills may be the site of virus entry and shedding
(Wang et al., 1993).
Grass carp reovirus can induce specific resistance (Nie and Pan, 1985) and
the convalescent sera contain antibodies (Jiang and Ahne, 1989). Other aspects
of the immune response were not reported.
Future studies should include the clarification of the putative picornavirus
presence and its involvement in disease, comparison and grouping of all
accessible isolates, improvement of serological and development of other
methods for identification of virus(es). Research is also required on
epizootiology (carriers, shedding mechanisms, vectors, role of farming
practices, etc.) and on the immune response. Fish farmers require methods for
prevention and control of GCHD, including a vaccine and a practically
applicable vaccination procedure.
GOLDEN SHINER VIRUS DISEASE
Introduction
Golden shiner virus disease is an acute to subacute disease usually causing
protracted and low mortality in pond populations of golden shiner (Notemigonus
crysoleucas). Golden shiner virus (GSV) is an aquareovirus which was isolated
during investigation of low-level mortality (Plumb et al., 1979).
Pathogenicity of GSV seems to be limited to a single species of fish. The virus is
distributed throughout the golden shiner farming areas in the south-eastern and
midwestern USA. It has not been reported from feral populations. Hedrick et al.
(1989) isolated GSV from healthy grass carp of unspecified age.
220
N. Fijan
The disease
Affected shiners do not feed and they swim lethargically near the surface unless
disturbed. Severe haemorrhage affects the dorsal musculature. Petechial
bleeding can be seen in the cornea, ventral portion of skin, visceral fat and
intestinal mucosa.
The virus
Golden shiner virus is not sensitive to ether, chloroform or heating to 56C, but
is stable at pH 310 and retains its infectivity after treatment with nucleases. It
retains infectivity in water kept between 4 and 30C for 15 days and this is
sufficient for passive transmission from fish to fish (Brady et al., 1993). The
cell-culture medium maintains its infectivity for several months at 4 and 70C.
The virus has an icosahedral shape and measures about 7075 nm in diameter
(Plumb et al., 1979; Winton et al., 1987). It is serologically related to the
aquareovirus isolate from European cyprinids (Ahne and Klbl, 1987) and does
not have common antigens with IPN virus (Schwedler and Plumb, 1980). The
genome of GSV is composed of 11 segments of dsRNA belonging to three size
classes and the virion contains five major structural proteins (Winton et al.,
1987).
Fathead minnow cells support the replication of GSV and several other lines
(including BB and chinook salmon embryo (CHSE-214)) are also susceptible to
infection. Bluegill fry, CCO, RTG-2 and some other cell lines do not support
replication of GSV (Plumb et al., 1979; Winton et al., 1987). The optimum
temperature for in vitro replication is 30C (Schwedler and Plumb, 1982a). The
cytopathic effect is characterized by the formation of vacuolated syncytia with
intact nuclei 1224 h after inoculation of susceptible cell cultures incubated at
30C. Affected cells condense and detach. A progressive infection of cells results
in destruction of the whole cell monolayer. In a regressive infection, the focal
cytopathic effect can be overgrown by surrounding cells and the virus is not
demonstrable by subcultivation.
Golden shiners support replication of aquareoviruses isolated from channel
catfish, chum salmon and oyster at 23 and 28C, but such infections are
unknown in natural conditions (Brady and Plumb, 1991).
Diagnostic methods
Presumptive diagnosis is based on clinical signs of GSVD. Virus isolation and
identification is carried out by inoculation of FHM cells, incubation at 30C and
virus neutralization.
All ages of shiners in ponds are susceptible to GSV infection but mortality is
highest among 56-month-old fish. The disease is generally mild, and
cumulative losses are below 5%. Crowded conditions in tanks exacerbate GSVD
incidence and precipitate losses up to 75% (Plumb et al., 1979).
The main predisposing factors for disease outbreaks are high water
temperatures (2732C) and overcrowding. Schwedler and Plumb (1982b)
reported higher virus titres, prevalence and duration of infection in fish under
natural exposure conditions kept at higher densities. The incidence in
production-pond population was 2.8% and in high-density population it
increased to 50%.
Experimental infection by cohabitation and by i.p. injection of cell culture-
grown virus causes variable mortality and indicates a low pathogenicity of GSV.
The initially high virus titres in infected fish decrease after 2 weeks (Plumb
et al., 1979). Hedrick et al. (1989) could not induce overt signs of infection in
fingerlings of golden shiner and grass carp infected by bathing, but virus was
detectable in both species after 7 days and in some shiners at 4 weeks.
There are no prevention and treatment against GSV. Economic losses are
tolerable and farmers are not stimulated to prevent the dissemination of infection
by avoiding the purchase of infected shiners or by disinfection (facilities,
equipment and eggs).
Topics for further studies
A cloned DNA probe for rapid detection of GSV RNA in fish tissues by nucleic
acid hybridization could speed up diagnosis and contribute to studies on the
pathogenicity of PFRD. Research on epizootiology should encompass host
range, influence of age, physiological states (other than temperature and varying
densities in tanks) and pond management practices, sources of infection and
vectors other than water, virus shedding and vertical transmission.
SYSTEMIC IRIDOVIRUS DISEASES CAUSED BY
RANAVIRUSES
Introduction
Iridoviridae of the genus ranavirus are becoming increasingly important as
pathogens of feral, cultured and ornamental teleosts. They are endotheliotropic
and can induce severe disease in susceptible fish species, characterized by
necrotic lesions in vascular walls and visceral organs. The group consists of the
epizootic haematopoietic necrosis virus (EHNV) (Langdon et al., 1986b; Eaton
et al., 1991; Hedrick et al., 1992), isolates from two ictalurid fishes in Europe
(Ahne et al., 1989; Pozet et al., 1992), turbot (Scophthalmus maximus)
iridovirus-like agent (Bloch and Larsen, 1993), an isolate from two freshwater
222
N. Fijan
tropical ornamental species (Hedrick and McDowell, 1995) and largemouth bass
virus (LMBV) (Plumb et al., 1996). The present discussion considers mainly
diseases caused by EHNV and ictalurid viruses, which can cause 100% mortality
in young susceptible warm-water hosts. The economic damage is rare but
serious.
The type strain of ranavirus is the frog virus 3 (FV3) (Granoff et al., 1965;
Essani and Granoff, 1989; Aubertin, 1991). Hedrick et al. (1992) suggested that
EHNV and ictalurid isolates were strains of FV3. The amphibian group in
ranavirus genus includes amphibian viruses from North America (Essani and
Granoff, 1989), the European green frog virus (Fijan et al., 1991; Ahne et al.,
1995) and the Australian Bohle (or burrowing frog) iridovirus (Speare and
Smith, 1992; Hengstberger et al., 1993). The North American tadpole oedema
virus and the European frog virus are not pathogenic for fish tested in
experiments (Wolf et al., 1968; Fijan et al., 1991), but the Bohle iridovirus
induced 100% mortality in bath infected barramundi (Lates calcarifer) (Moody
and Owens, 1994). The latter report indicates the possible importance of
amphibian ranaviruses for finfish aquaculture.
Epizootic haematopoietic necrosis and EHNV were reviewed by Wolf
(1988) and McAllister (1993a). This chapter describes EHN in redfin perch.
Members of the ranavirus genus were isolated from freshwater fishes on three
continents. Epizootic haemopoietic necrosis virus affects redfin perch (Perca
fluviatilis) and rainbow trout (Langdon et al., 1986a,b, 1988; Langdon and
Humphrey, 1987) in Australia (Victoria, New South Wales, and South Australia).
Sheatfish iridovirus (or IW for iridovirus wels) was isolated in Germany (Ahne
et al., 1989). Black bullhead iridovirus (or ICF for iridovirus of catfish) caused
mortality of Ameiurus melas (syn. Ictalurus melas) in France (Pozet et al., 1992)
and in Italy (Bovo et al., 1993). Iridovirus of ornamental tropical fish (IOTF)
was isolated from imported guppy (Poecilia reticulata) and doctor fish
(Labroides dimidatus) in California, USA (Hedrick and McDowell, 1995).
Largemouth bass virus was isolated from a mortality among adult largemouth
bass in a reservoir in South Carolina, USA (Plumb et al., 1996).
The redfin perch and rainbow trout isolates of EHNV are serologically
indistinguishable. Langdon (1989) experimentally infected 11 teleosts. The
mosquito fish (Gambusia affinis) is the only species besides redfin perch which
is susceptible to horizontal transmission. Four native species are highly
susceptible to the virus and at least one of them (Macquarie perch, Macquaria
australasia) is in decline. Atlantic salmon (Salmo salar) develops clinical
disease but the infection is not lethal. Barramundi is refractory and so are two
exotic cyprinids, which do not seem to be the original host (Langdon, 1989).
Whittington et al. (1996) demonstrated the spread of EHN in redfin perch to
river systems and impoundments in Australia.
In outbreaks of sheatfish and black bullhead diseases (Ahne et al., 1989;
Pozet et al., 1992), the water environment contained several other warm-water
species, including cyprinids, pike and perch, but they remained unaffected. The
ornamental fish strain is pathogenic for rainbow trout (Hedrick and McDowell,
1995). Largemouth bass virus has not induced mortality in infected adult
largemouth bass (Plumb et al., 1996).
The reported geographical ranges of fish ranaviruses are so far restricted to
respective continents of isolation. However, that from exotic ornamental fish is
suspected to be a part of putative transcontinental transmission (Hedrick and
McDowell, 1995). The Bohle iridovirus, which is present only in Queensland, is
geographically distinct from EHNV (Speare and Smith, 1992).
Diseases
Fish ranaviruses are endotheliotropic and cause haemorrhagic diathesis, oedema
and peripheral circulatory failure.
Epizootic haematopoietic necrosis appeared during the spring of 1984 in a
lake and caused severe mortality among juvenile redfin perch. The disease is less
serious in farmed rainbow trout. Experimentally infected 3545-day-old redfin
perch develop depression, skin darkening and erratic swimming and die after
45 days (Langdon, 1989). Some have erythema around the brain and nostrils.
Clinical signs in disease outbreaks described by Langdon and Humphrey (1987)
are identical, except for skin ulcers invaded by fungus in some fish. Focal
necrosis is a consistent finding in haematopoietic kidney and liver of naturally
and experimentally infected fish. In the spleen and pancreas, this sign is variable.
Necrotic haemopoietic cells are disseminated in all vessels. Lesions in other
susceptible species are inconstant (Langdon, 1989). The most consistent is the
necrosis in haemopoietic kidney and liver. A description of gross lesions in
redfin perch by Reddacliff and Whittington (1996) includes haemorrhage around
bases of fins, focal haemorrhage in gills, oedema and multiple necrotic foci in
liver. Microscopic changes consist of focal to extensive necrosis in
haematopoietic kidney, liver, spleen, heart, pancreas and lamina propria of the
intestine. Thrombosis, haemorrhage and fibrinous exudate are common in gills.
Lesions in rainbow trout are similar but milder.
The sheatfish iridovirus disease (iridovirous wels disease (IWD)) is
characterized by loss of appetite, apathy, ataxia (including rapid spiral
swimming), petechial haemorrhage in skin and internal organs and generalized
destruction of haematopoietic tissues in the kidney and spleen. The cumulative
fry mortality in a recirculation system was 100% (Ahne et al., 1989). Fry
infected by bath in virus suspension and by cohabitation also succumbed to high
mortality within 8 and 11 days, respectively (Ahne et al., 1990). Adult fish are
also susceptible but mortality does not exceed 30% (Ahne et al., 1991).
Histopathology and electron microscopy in experimentally infected sheatfish of
45 cm revealed alterations in all organs (Ogawa et al., 1990). Endothelium and
reticuloendothelial cells are the main target. Periarteriolar necrosis of the
haemopoietic tissue and degeneration of tubular and duct epithelia are
prominent in kidneys. Gill epithelium and chloride cells are hyperplastic and
oedematous. The lumen of the circulatory system is congested and the
224
N. Fijan
proliferating cells contain eosinophilic inclusions. Alterations in the skin include
proliferation and necrosis of epithelial cells, hyperplasia of monocellular glands
and zonal haemorrhage in hypodermis. Myocarditis and endocarditis are diffuse.
Small necrotic foci are seen in the liver and spleen. Glial proliferation and
spongiosis in the brain are also pronounced. The pathology and incubation most
closely resemble those of EHN.
The black bullhead iridovirus disease (iridovirus of catfish disease (ICFD))
can induce total acute mortality in pond populations and is highly lethal to
experimentally infected subadults and adults. Loss of appetite is evident 12
days before the onset of clinical signs. Other signs include oedema and
haemorrhage around pectoral and pelvic girdles and in internal organs. The
kidney is the principal target organ and both the haematopoietic and the
excretory part are severely altered. Blood vessel walls are damaged. Necroses in
the spleen and kidney can be severe. Interlamellar spaces in gills are obliterated
and lamellar fusion is evident (Pozet et al., 1992).
Iridovirus of ornamental tropical fish was isolated from carriers but the
experimental infection of rainbow trout induced low mortality, considerable
lesions in the kidney and spleen and virus titres greater than 10
8
TCID
50
g
1
(Hedrick and McDowell, 1995).
Experimental infection of barramundi with Bohle iridovirus resulted in
mortality and focal necrosis in the liver (Moody and Owens, 1994).
Viruses
Fish ranaviruses are distinct agents, although they have similar hexagonal
morphology and size (slightly over 130140 nm), number and weight of
structural polypeptides and common antigens (Hedrick et al., 1992). They
replicate in cytoplasm and induce inclusion bodies as centres of virus
production. The cytopathic effect in cell cultures is characterized by focal
rounding up of cells from substrate and cell lysis, expansion of these changes
and destruction of the cell monolayer. According to Essani and Granoff (1989),
the pattern and number of polypeptides in ranaviruses differ from those in
lymphocystis virus and in two goldfish iridoviruses isolated by Berry et al.
(1983). The PCR with primer sets for EHNV amplified a genomic DNA segment
from IOTF (Hedrick and McDowell, 1995).
Iridoviruses are presumably internalized by the cell during a receptor-
mediated endocytosis. Budding and envelopment at the plasma membrane
secure the exit of mature IW (Granzow et al., 1997).
Epizootic haemopoietic necrosis virus replicates in BB, BF-2, FHM, RTG-2
and several other cells. Langdon and Humphrey (1987) and Langdon et al. (1988)
obtained highest virus yields in FHM cells. Bluegill fry cells kept at
2022C are recommended in the OIE Manual (1995b) for the isolation of EHNV.
Sheatfish iridovirus is readily isolated in BF-2 and FHM cells at
2030C. Black bullhead iridovirus grows in EPC, CCO and BF-2 cells, with
optimum plaque development in the latter. Iridovirus of ornamental tropical fish
replicates in the same cell lines. Largemouth bass virus was isolated in FHM cells.
Perch and trout isolates of EHNV differ in the molecular weights of some
structural proteins (Hengstberger et al., 1993).
The range of cells susceptible to Bohle iridovirus and EHNV is very similar.
The two viruses exhibit a high degree of cross-reactivity in ELISA tests, have
very similar morphology and structure but differ in size, cytopathic effect on
host cells, molecular weights of proteins, polypeptide profiles, Western blots and
site of virus release from cells (Speare and Smith, 1992; Hengstberger et al.,
1993).
The persistence of EHNV in water and cell-culture medium at optimum
temperature for EHN outbreaks in redfin perch is important in epizootiology.
Diagnostic methods
Procedures recommended by the OIE Manual (1995b) for EHNV are applicable
to other viruses in the ranavirus group considered in this chapter. Presumptive
diagnosis is based on clinical signs, virus isolation in BF-2 or other susceptible
cells and electron microscopy. Identification of EHNV is based on IFAT or
ELISA tests (OIE Manual, 1995b) and PCR (Gould et al., 1995). Other viruses
in the group show antigenic relatedness to EHNV and FV3 demonstrable by
IFAT (Hedrick et al., 1992; Ahne et al., 1995; Hedrick and McDowell, 1995),
Western blotting and nucleic acid hybridization (Hedrick and McDowell, 1995).
Epizootic haematopoietic necrosis virus is detectable by ELISA, immuno-
histochemistry and electron microscopy (Hyatt et al., 1991). An improved
antigen-capture ELISA (Whittington and Steiner, 1993) can recognize EHNV in
clarified fish tissue and in cell-culture supernatant. The lowest detectable level
of the virus in supernatant is 10
3.5
TCID
50
ml
1
. Sensitivity and specificity of this
method for tissue samples are about 81 and 99%, respectively, and for cell-
culture supernatant 96 and close to 100%. Manual grinding with a pestle in a
tube, followed by vortexing in the same tube with 3 mm glass beads and
clarification in a microcentrifuge, is the most efficient method for releasing
EHNV from tissue samples (Whittington and Steiner, 1993).
Natural epizootics of EHN in early summer among young redfin perch last for
23 weeks. They are recurrent in several major waterways in Victoria, Australia
(Langdon, 1989). Adults are rarely affected. Virus isolation from juveniles and
adults immediately after an epizootic is infrequent (Langdon and Humphrey,
1987). Hundred-day-old survivors of disease outbreaks are resistant to challenge
(Langdon, 1989). Redfin perch carriers were not detected and there is no
evidence for vertical transmission. An unknown reservoir and carrier host
are suspected. Silver gulls (Larus novaehollandiae) and great cormorants
(Phalacrocorax carbo) can spread EHNV by the regurgitation of ingested
material (Whittington et al., 1996). Other means of spread include transportation
of fish by humans, transfer on boats, nets and other equipment as well as water
226
N. Fijan
flow and migration of carrier fish in a catchment area (Whittington et al., 1996).
The epizootiology of other diseases in the group was not studied.
The close relatedness of fish and amphibian ranaviruses and the
pathogenicity of Bohle iridovirus for barramundi should be kept in mind in
programmes for avoidance of pathogens in aquaculture (Hedrick et al., 1992;
Ahne et al., 1995). Frogs are ubiquitous on large farms for warm-water fishes.
These could be reservoirs and vectors of fish pathogens. Hedrick et al. (1992),
Hedrick and McDowell (1995) and Hedrick (1996) consider transcontinental
movements of amphibians and exotic ornamental fish as possible reasons for the
appearance of similar viruses in Australia and Europe. It was suggested that
control be extended to aquatic amphibians and tropical aquarium fishes (Ahne et
al., 1995; Hedrick, 1996).
Ultraviolet sterilizing units are effective in neutralizing EHNV at flow rates
used in hatcheries (Miocevic et al., 1993).
Australia enforces stringent quarantine restrictions for the import of
ornamental fish and restriction of sale and movement of rainbow trout from
EHN-affected hatcheries (Doyle et al., 1996). Only general preventive measures
are applicable for diseases in this group. More data on epizootiology of these
diseases may lead to better targeting of control measures. Treatment is not
available.
Adult redfin perch is extremely susceptible to EHNV infection by bath and i.p.
inoculation. As little as 0.08 TCID
50
ml
1
was lethal at 1921C. The incubation
period at this temperature is 11 days and at 1319C up to 28 days. Disease does
not develop below 12C (Whittington and Reddacliff, 1996). Virus replication in
endothelial cells results in necrosis and consequent haemorrhage.
The immunological response in fish and rabbits does not generally include
neutralizing antibodies. One survivor of ICFD had neutralizing antibodies
(Pozet et al., 1992). However, rabbits react to these viruses by producing
antibodies suitable for IFAT and ELISA. Redfin perch survivors from natural
disease outbreaks are resistant to challenge with EHNV (Langdon, 1989). The
epizootiology of EHN in rainbow trout suggests that this species is incapable of
developing long-lasting resistance (OIE Manual, 1995b).
Topics for further studies
The development of methods for distinguishing ranavirus isolates requires
further attention. Such methods are needed to study epizootiology and taxonomy.
Surveys of sheatfish, bullhead, ornamental and other fish production sites
for ranaviruses in fish and amphibians are needed to assess the range, extent and
significance of currently known and possible other diseases in this group.
Studies on host ranges and reciprocal pathogenicity of fish and amphibian
ranaviruses for early life stages could help to detect virus sources and reservoirs.
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CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Main host Reference

hyp, hyperplastic or proliferative lesions; hs, haemorrhagic

Main host Reference

hs, haemorrhagic septicaemia; hyp, hyperplastic or proliferative lesions; nhs,

EM, demonstrated by electron microscopy, but not isolated in cell culture; I,

tancl, Z., Kezic, N. and Teskeredzic, E. (1977b) Vaccination of

Z. and Fijan, N. (1981) Swimbladder inflammation in carp

Culjak, K. and Miyazaki, T. (1986) Spring

Rehulka, J., Hrdonka, M. and Konsov, V. (1984) Summarized

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