Journal of Fish Diseases 2009, 32, 1035–1039
Short communication
Comparison of the resistance of selected families of
common carp, Cyprinus carpio L., to koi herpesvirus:
preliminary study
P F Dixon1, C L Joiner1, K Way1, R A Reese1, G Jeney2 and Z Jeney2
1 Cefas Weymouth Laboratory, Weymouth, Dorset, UK
2 Research Institute for Fisheries, Aquaculture and Irrigation (HAKI), Szarvas, Hungary
Keywords: Cyprinus carpio, disease resistance, koi
herpesvirus, resistant strain.
Koi herpesvirus disease (KHD), caused by koi
herpesvirus (KHV), is a highly infectious disease of
carp, Cyprinus carpio L., usually causing high
mortality (reviewed by Dixon 2008). The disease
has a worldwide distribution and affects ornamental
carp (koi carp) as well as common carp in both
aquaculture and open waters (rivers, lakes, etc.).
The disease is economically very important, having
an impact on the trade in high value ornamental
fish, sport fisheries and food production. Attempts
have been made to reduce the economic impact of
the disease by vaccination (Ronen, Perelberg,
Abramowitz, Hutoran, Tinman, Bejerano, Steinitz
& Kotler 2003; Perelberg, Ronen, Hutoran, Smith
& Kotler 2005), but as yet no vaccine has been
approved for use outside Israel. An alternative
method of controlling the disease by use of resistant
strains has been investigated (Shapira, Magen, Zak,
Kotler, Hulata & Levavi-Sivan 2005; Zak,
Perelberg, Magen, Milstein & Joseph 2007). The
latter authors bred three hybrid and three pure
crosses from two Hungarian and one Israeli strain
of fish and reported survival rates after challenge
with KHV of between 4% and 20%. Shapira et al.
(2005) crossed two strains of domesticated carp
with one strain of wild carp and exposed the
Correspondence P Dixon, Cefas Weymouth Laboratory,
Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK
(e-mail: peter.dixon@cefas.co.uk)
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doi:10.1111/j.1365-2761.2009.01081.
progeny to KHV both in the laboratory and in the
field. The mortalities ranged from 8.9% to 89.7%
in the field trial and from 8.6% to 68.5% in the
laboratory trial, and with one exception there was
good comparability of survival rates of the different
crosses in the two trials.
To further explore the potential of producing
strains of carp resistant to KHV, we have
investigated the survival of a large number of
carp families following exposure to the virus, and
present the results here. This study is part of a
larger study, which includes investigating produc-
tion characteristics, the resistance of the families to
Aeromonas hydrophila, post-infection changes at
the genomic level (Williams, Li, Hughes,
Gonzalez, Vernon, Vidal, Jeney, Jeney, Dixon,
McAndrew, Bartfai, Orban, Trudeau, Rogers,
Matthews, Fraser, Gracey & Cossins 2008) and
proteomic level, heritability characteristics, the
genetic correlation between different traits and
gene linkage mapping.
The production and other characteristics of the
fish families will be described in full in a separate
communication (Z. Jeney, unpublished data).
Briefly, 96 families were produced by diallele
crossing of four strains of carp (Duna, Amur, Tata
and HAKI 15) maintained in a live gene bank at
HAKI, Hungary. Five females and 10 males from
each strain were used to produce six families per
strain cross. The families were maintained in
separate tanks until they were large enough to be
injected with a passive integrated transponder (PIT)
tag. Twenty fish from each of 94 families (there
 Journal of Fish Diseases 2009, 32, 1035–1039 P. F. Dixon et al. Comparison of KHV resistance of selected families of common carp

were insufficient fish from the remaining families) clinical signs of disease were more apparent as the
were transported to the Cefas Weymouth Labora- disease progressed, and included exophthalmia, pale
tory, UK, where the fish were allocated to 900 L rough patches on the skin and excess mucus
tanks as follows: one tank contained five fish from production. The overall mortality was 92.5% when
each family (infected in trial 1), and the remaining the trial was terminated at day 21. All fish in the
fish were mixed and randomly allocated to three majority (66) of the families died, most of them by
further tanks (infected in trial 2). The fish were day 9 although for some families extinction took
maintained at 15 C until approximately 10 days longer. In 20 families four of the five fish died and
before challenge when the temperature was increased again the majority of the deaths occurred between
to 21 C at the rate of 1 C day)1. For the days 7 and 9. However, in family 62, one of three
challenges, KHV isolate UK H361 2.3 was grown fish in the trial died and in families 50 and 64 only
in koi fin (KF) cells at 20 C, as described by two mortalities occurred. In family 20, there were
Hedrick, Gilad, Yun, Spangenberg, Marty, four mortalities, but they occurred over a longer
Nordhausen, Kebus, Bercovier & Eldar (2000). A time scale (final mortality on day 17).
cohabitation challenge was used in which common Trial 2 commenced when the fish were approxi-
carp from a KHV-free source in the UK were given mately 10 months old. Eighty-one fish died prior to
an intraperitoneal injection of 100 lL of KHV at a the trial, 61 of which died over one night but no
concentration of 103 tissue culture infectious dose infectious agents were detected in the fish. Twelve
(TCID50) mL)1 and kept at 21 C until the first survivors of the KHV infection had no tag. The
signs of disease were apparent. The fish were then number of infected fish introduced into the tanks was
introduced into the tank containing the test carp reduced to reduce the mortality rate so that a better
families at the rate of 1:10 of the test fish in trial 1. In differentiation between sensitive and more-resistant
trial 2, the three tanks were linked and water at fish might be obtained. In the first 2 days
21 C was re-circulated between them, and the post-introduction of the KHV-infected fish, four
infected fish for cohabitation were introduced at the fish died but KHV was not detected. A single
rate of 1:20 of the test fish in each tank. The fish were KHV-induced mortality occurred on day 6 followed
examined twice daily and dead fish were removed by a rapid increase of KHV-induced mortality,
and identified by scanning for the PIT-tag. At least peaking on days 9 and 10 and resulting in an
10% of the dead fish were tested for the presence of overall mortality of 93.9% (Fig. 2). Although the
KHV by isolation in KF cells (Hedrick et al. 2000) overall mortalities in the two trials were similar,
and/or by polymerase chain reaction (OIE, 2007). the rate of mortality was slightly slower in trial 2. At
Trial 1 was a prechallenge to test the infection the end of the experiment on day 21, five fish
parameters, and commenced when the fish were from each tank (which represented approximately
approximately 9 months old. Four fish died before 20% of the surviving fish) were randomly selected,
the trial, four fish were not accounted for at the end bled under anaesthesia, and sera collected. All
of the trial and four of the survivors had no tag. The sera were positive for antibody against KHV when
first mortalities in the test fish started on day 6 post- tested by an enzyme-linked immunosorbent assay as
introduction of the infected fish, and the bulk of the described by St-Hilaire, Beevers, Way, Le Deuff,
mortalities occurred on day 7 (Fig. 1). External Martin & Joiner (2005).

400 100
300 100
Cumulative mortality (%)

90
Cumulative mortality (%)

90 350
250 80
80 300
Daily mortality
Daily mortality

70 70
200 250 60
60
150 50 200 50
40 150 40
100 30 30
100
50 20 20
10 50 10
0 0 0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Days post-cohabitation challenge Days post-cohabitation challenge

Figure 1 Koi herpesvirus (KHV) infection trial 1. KHV-infected Figure 2 Koi herpesvirus (KHV) infection trial 2. KHV-infected
carp were introduced into the tank at day 0. Combined data for all carp were introduced into the tanks at day 0. Combined data for
families showing daily (bars) and cumulative mortality (d). all families showing daily (bars) and cumulative mortality (d).

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 Journal of Fish Diseases 2009, 32, 1035–1039 P. F. Dixon et al. Comparison of KHV resistance of selected families of common carp

100
90

Cumulative mortality (%)

80
70
60
50
40
30
20
Figure 3 Koi herpesvirus (KHV) infection 10
trial 2. Cumulative mortality of families 0
most sensitive to KHV exhibiting mortality 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
pattern 1, acute mortality. For clarity, mor- Days post-cohabitation challenge
tality of only 20 families (numbered in key) 4 5 6 15 17 18 31 32 41 42
are shown. 53 54 55 58 59 60 68 71 85 86

100
90
Cumulative mortality (%)

80
70
60
50
40
30
20
10
Figure 4 Koi herpesvirus (KHV) infec-
tion trial 2. Cumulative mortality of 0
families exhibiting mortality pattern 2, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Days post-cohabitation challenge
high mortality but over a protracted
2 12 13 25 27 28 29 33 38 43
period. For clarity, mortality of only 20
families (numbered in key) are shown. 44 48 50 61 63 64 66 76 90 93

When the mortality curves of the individual pattern; Fig. 3), (b) an extended period of mortality
families were plotted, three different patterns were reaching between 70% and 100% by the end of the
apparent (Figs 3, 4 & 5): (a) acute mortality with trial on day 21 (43 families were in this pattern;
all family members dead within 6 days of the start
of the mortalities on day 6 (46 families were in this
Table 1 Crosses of common carp producing the most resistant
families in either trial 1 or trial 2 with koi herpesvirus
100
90
Cumulative mortality (%)

Cumulative Cumulative
80 mortality in mortality in
70 Family no. trial 1 (%) trial 2 (%) Crossa
60
50 8 66.6b 53.8c A1 · A2
40 9 80.0 53.8c A1 · D7
20 80.0 57.1d A3 · D2
30
49 80.0 46.6 T4 · T7
20
50 40.0 73.3 T4 · T8
10
62 33.3e 53.8c D3 · D6
0 64 40.0 73.3 D3 · T4
1 2 3 4 5 6 7 8 9 101112131415161718192021
Days post-cohabitation challenge a
Amur(A), Duna (D) and Tata (T); fish number (no.), first named fish is
8 9 20 49 62 female.
b
Two fish died and one survived following infection. Two fish in this
Figure 5 Koi herpesvirus (KHV) infection trial 2. Cumulative family were not accounted for at the end of the trial.
mortality of families exhibiting mortality pattern 3, most c
Two fish died pretrial.
resistance to KHV. All families in this group are shown d
One fish died pretrial.
e
(numbered in key). Two fish died pretrial. One fish died and two survived following infection.

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 Journal of Fish Diseases 2009, 32, 1035–1039 P. F. Dixon et al. Comparison of KHV resistance of selected families of common carp

Fig. 4), (c) an extended period of mortality not
exceeding 60% by the end of the trial; five families
(8, 9, 20, 49 and 62; Fig. 5) were in this pattern.
Differences in the survival results between the
two trials may be attributable to the challenge
pressure, as twice the density of infected fish was
added in the first trial. Trial 1 was a prechallenge
trial to test the challenge procedure, and the two
trials were not intended to be replicates. Numbers
of fish in each family were much smaller in trial 1.

1.0 Amur
Duna
Haki-15
0.8
Tata

0.6
Survival

0.4

Figure 6 Koi herpesvirus (KHV) infec-

0.2 tion trial 2. Kaplan–Meier survival esti-
mates collated according to female
0 parents. The lines (see key on graph)
show the survival of all families with
0 6 9 12 15 21
Days post-cohabitation challenge a female parent from the named carp
strain.

1.0
Amur
Duna
Haki-15
0.8
Tata

0.6
Survival

0.4

0.2
Figure 7 Koi herpesvirus (KHV) infec-
tion trial 2. Kaplan–Meier survival
0 estimates collated according to male
0 6 9 12 15 21 parents. The lines (see key on graph)
Days post-cohabitation challenge show the survival of all families with a
male parent from the named carp strain.

Table 2 Hazard ratios (Cox regression using Breslow method for ties) for parentage of the four carp strains

Parentage Hazard ratioab SE zc-Value P > |z| 95% Confidence interval

Amur female 0.84 0.07 )2.25 0.02 0.72–0.98

Duna female 0.99 0.08 )0.10 0.92 0.85–1.16
HAKI 15 female 1.056 0.08 0.67 0.50 0.90–1.23
Amur male 1.18 0.10 2.01 0.04 1.00–1.39
Duna male 0.97 0.08 )0.38 0.70 0.82–1.14
HAKI 15 male 1.66 0.14 6.09 0.00 1.41–1.96
a
Tata was taken as the baseline, so has a hazard ratio of 1.
b
Hazard ratios >1.0, positive relationship; hazard ratios <1.0, negative relationship.
c
Ratio of each regression coefficient to its standard error (SE).

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 Journal of Fish Diseases 2009, 32, 1035–1039 P. F. Dixon et al. Comparison of KHV resistance of selected families of common carp
Both these considerations preclude making a
definitive comparison between the two trials and,
for example, chi-squared tests were not applied. On
a qualitative basis, however, we note that, in
general, the same families had surviving members
at the end of each trial. Family 62 was found to be
amongst the most resistant families in both trials.
Family 20 had 80% mortality in trial 1, but over a
longer time-scale than the other families and was
one of the most resistant families in trial 2. Families
50 and 64 both had 40% mortality in trial 1, and
both had 73.3% mortality in trial 2; they were both
in mortality pattern 2 (Fig. 4).
The crosses that produced the most resistant
families are shown in Table 1. The carp strains
Amur and Duna are both ÔwildÕ strains and are
parents of five of the seven families in Table 1.
Hence it appears that crosses including wild strains
of carp may be important for production of KHV-
resistant carp. These findings reflect the results of
Shapira et al. (2005),who reported that the most
resistant carp crosses in their trials were those with a
wild strain of carp, i.e. Sassan, which coincidently
also comes from the river Amur. However, the
individual parent of the strains of carp is important
as the majority of the crosses in this study using the
wild strains did not produce significantly resistant
families, and pure crosses of the domesticated Tata
strain also produced a highly resistant family.
The mortality data were analysed blindly and the
presence of three clusters of families confirmed by a
log-rank test (StataCorp 2005). The effects of
parentage were then investigated as fixed factors
using a log-rank test and Cox regression (StataCorp
2005; Figs 6 & 7). Table 2 shows the hazard ratios
for each parentage. Overall, male parentage had a
higher effect on mortality [v2 = 51.7 comparing
four male families; 7.7 (not significant) comparing
females], but the numbers tested were not precisely
balanced. When both main effects were tested it
appears that having a female from the Amur strain
confers an almost significant benefit whereas a male
from family HAKI 15 confers a substantially
increased hazard.
Overall our results are in agreement with those of
Shapira et al. (2005) and also suggest that breeding
for KHV-resistance in carp is feasible. This will be
explored in further trials.
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Acknowledgements
This study was funded by the European Commis-
sion 6th Framework Programme (EUROCARP,
Project 0022665) and Defra contracts F1167 and
F1170.
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Received: 12 December 2008
Revision received: 2 April 2009
Accepted: 8 April 2009