Egtved Virus: The Susceptibility of Brown Trout

and Rainbow Trout to Eight Virus Isolates

and the Significance of the Findings
for the VHS Control
P. E. v.
On the basis of limited experimental evidence (Rasmussen, 1965; J>rgensen, 1974;
de Kinkelin et al., 1974) brown trout have generally been considered to be almost
refractory to natural infection with Egtved virus. Nevertheless VHS in a few rare cases
has been observed in brown trout under trout farm conditions. The author has isolated
Egtved virus twice from such cases, once (1969) from brown trout of Italian origin,
once (1972) from brown trout in a Danish trout farm (J>rgensen, unpublished results).
Recently also de Kinkelin and Le Berre (1977) isolated Egtved virus from brown trout.
The latter isolate, designated strain 23/75, was found to be pathogenic to brown trout
as well as to rainbow trout in bath infection experiments.
The present experiments were carried out to make clear whether the pathogenicity
of strain 23/75 to brown trout was a unique feature of that strain or whether other
Egtved virus isolates were also brown-trout-pathogenic when tested by means of bath
infection.
A total of eight virus isolates was examined, among others the reference strain of
each of the three preliminarily identified serotypes, strain F 1 (serotype 1), strain He
(serotype 2; J>rgensen, 1972, 1974), and strain 23/75 (serotype 3; Le Berre et ai.,
1977).
Material and Methods
Groups of 20 brown trout (average body weight 6 g) and of 50 rainbow trout (average
body weight 7 g) were exposed to virus concentrations of approximately Ix 1 OS pfu per
ml of water for one hour. Negative controls received cell culture medium instead of virus.
The aquaria were supplied with running tap water at a temperature of 9 _10C. The
observation period was six weeks, during which all dead fish were examined for the
presence of VHS symptoms.
Results
The results of the experiments are shown in Table 1.
Among the eight virus isolates three were found to be pathogenic to brown trout
as well as to rainbow trout. The remaining five were pathogenic only to rainbow trout.
1 State Veterinary Serum Laboratory, Department for Jutland, 2,8200 Arhus N, Den-
mark
W. Ahne (ed.), Fish Diseases
Springer-Verlag Berlin Heidelberg 1980
4
P.E.V. ].prgensen
Table 1. Mortality in rainbow and brown trout after bath infection with 8 different strains of
Egtved virus
Virus Mortality in Mortality in Sera- 50% pnt. Origin of Virus inoculum
strain rainbow trout brown trout type against virus strain produced in
(%) (%) anti-Fl a
Fl 10 0 1 1,500 Rainbow trout FHM cells
He
b
64 0 2 500 Rainbow trout -
23/75 64 58 3 300 Brown trout
Jedsted 68 0 ? 1,200 Rainbow trout -
Ans.p 74 79 ? 600 Brown trout
N.543 96 0 ? 600 Rainbow trout -
Bidstrup 62 0 ? 600 Rainbow trout -
No S8 32 ? 300 Rainbow trout -
Controls 0 0
a SO% plaque neutralization titer
b Previously designated isolate 61 (J.prgensen, 1972)
Strain which caused the highest mortality in brown trout, was, like strain
23/75, originally isolated from brown trout 1972, unpublished results),
whereas strain No was isolated from rainbow trout.
The symptoms of the brown trout which died during the experiments Were clearly
those of VHS, and Egtved virus was readily isolated from affected individual fish. Sero-
logical identification of the reisolated virus strains was carried out by means of indirect
immunofluorescence utilizing a rabbit antiserum to strain F 1 1974).
In 50% plaque neutralization tests all eight isolates were neutralized by a rabbit
antiserum to strain F1 (Table 1).
Discussion
The results clearly show that brown trout are susceptible to some Egtved virus strains
under the conditions of bath infection at a high virus concentration. Brown trout were
not found to be susceptible to strain He as reported by de Kinkelin and Le Berre
(1977). The reason for this discrepancy may be differences in the virus passage levels
or virus cultivation techniques.
The above findings add an interesting detail to the knowledge of the epizootiology
of VHS. In spite of the pronounced difference between the virus titer in the water dur-
ing the bath infection experiments (10
5
pfu per ml) and during a natural infection (less
than 1 TCID
so
per ml) 1974) it must probably be expected that brown
trout can become VHS-infected in streams receiving the outlet from VHS-infected
trout farms at least under conditions of severe stress. It thus appears that brown trout
may make up a potential virus reservoir in the streams.
When the severity of the above fmdings with regard to VHS control in trout farms
is estimated it has also to be considered that the wild fish population of many (perhaps
Egtved Virus: The Susceptibility of Brown Trout and Rainbow Trout
most) European rivers includes a large number of rainbow trout, which have either es-
caped from trout farms or been stocked into the rivers for fishery purposes.
Since rainbow trout are more susceptible to VHS than brown trout it must probab-
5
ly be expected that the quantitatively most important virus reservoir exists in the wild
rainbow trout.
In addition to rainbow and brown trout, pike may also playa role as carriers of
Egtved virus. At least this must be expected on the basis of a recent report by Meier
and (1979) of a severe spontaneous outbreak of virologically verified VHS
in pike fry in Switzerland. The report in question represents the first demonstration
of VHS in nonsalmonid fish, and it may justify the feeling that more species of fish
may in time turn out to be susceptible to the disease.
In rivers with several VHS-infected trout farms situated next to each other along
the stream and with free movement of wild fish up and down the stream it is likely,
considering the susceptibility of several species of fish to the virus, that a smaller or
larger percentage of the wild fish are, at least periodically, harboring the virus. Against
that background it might appear necessary to remove the wild fish population before
prevention of VHS in the trout farms is attempted, since persistence of the infection in
the wild fish might lead to reinfection of sanitized trout farms.
In Denmark control of VHS in trout farms has been practiced for more than 15
years without measures being taken to remove the wild fish populations
1974). The basis for this policy was the recognition that it would be very difficult or
impossible to obtain permission for such steps from government authorities and priva-
te owners and that it would be extremely difficult to remove all wild fish from a
stream, even if permission was obtained.
The principle of the VHS control program is that all VHS-infected farms along a
stream, starting at the top of the stream, are emptied, disinfected, kept dry for one
A B C D E F G
A.
1968 0 + + + + + +
B.
1969 0 E+ E+ + + + +
1970 0 E E + + + +
C. 1971 0 0 0 E E E E
1972 0 0 0 0 0 0 0
1973 0 0 0 0 0 0 0
D.
1974 0 0 0 0 0 0 0
1975 0 0 0 0 0 0 0
E.
1976 0 0 0 0 0 0 0
1977 0 0 0 0 0 0 0
F. 1978 0 0 0 0 0 0 0
1979 0 0 0 0 0 0 0
G.
+ VHS infection; E "stamping out" procedure; 0 indicates freedom from VHS;
Two signs indicate two events in one year
Fig. 1. Development of the VHS control program in 7 trout farms (A-G) at the river "Lille A"
(The figure is based on information obtained from Dr. N. P. Kehlet, Veterinary Services, Copen-
hagen)
6
P.E.V. ~ r g e n s e n
A B C D E F G H
1966 + + + + + + + + +
1967 E + E + + + + + +
1968 0 E 0 E + + + + +
1969 0 0 0 0 + + + + +
1970 + 0 0 + + + + + +
1971 E 0 0 E + + + + +
1972 0 + +E + + + + + +
1973 0 E 0 E E E E E E
1974 0 0 0 0 0 0 0 0 0
1975 0 0 0 0 0 0 0 0 0
1976 0 0 0 0 0 0 0 0 0
1977 0 0 0 0 0 0 0 0 0
1978 0 0 0 0 0 0 0 0 0
1979 0 0 0 0 0 0 0 0 0
+ indicates VHS infection; E indicates "stamping out"
procedure; 0 indicates freedom from VHS; Two signs in-
dicate two events in one year
Fig. 2. Development of the VHS control program in 9 trout farms (A-I) at the river "Simested
A" (The figure is based on information obtained from Dr. N. P. Kehlet, Veterinary Services, Co-
penhagen)
month, and then repopulated with fish from VHS-free farms. As shown in Figs. 1
and 2 there are cases in which this policy has been very successful, since it has led so
far to freedom from VHS in all the farms involved for periods of 8 and 6 years respec-
tively.
I t is not known whether in those cases the wild fish in the streams were actually
carriers of Egtved virus or not. If they were it might be suspected that the duration of
the virus carrier state was short, since the VHS eradication program was successful.
Probably the removal of the common sources of virus, i.e., the densely populated
VHS-infected trout farms, caused a drop in the virus concentration in the streams as a
side effect of the sanitation. Perhaps the virus concentration reached a level at which
new infection of wild fish did not occur and at which already infected fish were able
to clear themselves of the infection.
Since there is no experimental evidence available which supports the above hypothe-
sis it is put forward only as a working theory. In other streams the VHS control pro-
gram has been less successful, since reinfection has sometimes occurred shortly after
the repopulation of the farms. It is not clear at present to what extent this has been
due to persistence of the infection in the wild fish and to what extent to other sources
of virus such as closely situated VHS-infected farms from which virus may be trans-
ferred by animals, personnel, trucks, and so on.
It would be very valuable to have experiments initiated which could help in
estimating the relative significance of each of those two fundamentally different
causes of VHS reinfection in sanitized trout farms.
Egtved Virus: The Susceptibility of Brown Trout and Rainbow Trout
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