Koi herpesvirus (KHV) has infected farmed common carp in Poland clinically and asymptomatically since 2004. The role of non-carp species as vectors of virus transmission is well known except for in the case of KHV. The aim was to better understand this virus’ infection and transmission pathways in common carp, looking at the potential vector role of fishes kept with them.
Eight species were experimentally infected with KHV by immersion in a suspension at 20°C ±1 and transferred to a tank after 45 minutes. Specimens were euthanised at intervals up to 56 days post infection (dpi) and tissue was examined for KHV DNA. Surviving infected fishes were introduced at intervals, each time into a separate tank, to naïve common carp for experimental infection. These were observed daily for symptoms, sacrificed along with controls after three months, and dissected to provide tissue samples. Also fish from 14 species collected from a farm with a history of KHV were sampled from 3 to 22 months after disease was confirmed. Organ sections from single fish were collected in a single tube.
Viral DNA was detected in tench and roach samples up to 49 dpi, but in three-spined stickleback and stone maroko samples only up to 14 dpi. Transmission of KHV to naïve carp occurred after cohabitation. KHV DNA was detected in three fish species three months after the farm outbreak.
We confirmed that grass and Prussian carp, tench, roach, and brown bullhead can transfer the virus to naïve common carp.
Keywords
- fish
- common carp
- koi herpesvirus
- vectors
The problem of losses due to infection with koi herpesvirus (KHV) disease (KHVD) occurred in the 1990s. Since the connection of mass mortality of koi in the United States and Israel with the presence of a new virus of the
A factor having a huge impact on the rate and extent of spread of the infection has been unrestricted trade in koi, often over very large distances (Fig. 1). Besides the possibility of horizontal transmission of the virus directly from fish to fish, the ability for the virus to spread by vectors like water (it being the major abiotic vector) should also be taken into consideration. However, hypothetically, animate vectors,
Fig. 1
Shipment of live koi

The investigations presented in this article were initiated for better understanding the pathways of CyHV-3 infection transmission in
Fish from eight different species were experimentally infected with CyHV-3 by immersion and viral DNA was assayed in samples. It could be detected in two species, tench and roach, in samples taken up to 49 days post infection (dpi). We confirmed KHV DNA up to 42 dpi in grass carp and Prussian carp samples. However, viral DNA was found in samples from three-spined stickleback and stone maroko only up to 14 dpi (Table 1). No clinical symptoms or mortality in fish experimentally infected with KHV were observed to the end of the experiment.
Results of experimental infection of fish from eight different species with CyHV-3
Dpi | Species | |||||||
---|---|---|---|---|---|---|---|---|
Three-spined stickleback | Stone moroko | European bitterling | Grass carp | Prussian carp | Tench | Roach | Brown bullhead | |
7 | + | + | + | + | + | + | + | + |
14 | + | + | + | + | + | + | + | + |
21 | − | + | − | + | − | + | − | − |
28 | − | − | − | − | + | + | − | + |
35 | − | − | − | + | − | − | + | − |
42 | − | − | − | + | + | + | + | − |
49 | − | − | − | − | − | + | + | − |
56 | − | − | − | − | − | − | − | − |
+ positive test results, – negative test results
In the second experimental challenge, where fish from different species were transferred at seven dpi to naïve common carp, the first clinical symptoms of KHV occurred after 28 days of cohabitation. Moribund fish presented enophthalmia, over-production of mucus on the skin, and necrosis of the gills (Fig. 1). Carp started dying on the 30th day after transfer of fish from different species and their cumulative mortality reached 90%. In samples from moribund carp the presence of KHV nucleic acids was detected. During three months’ observation of carp from the remaining three groups (where fish from different species were transferred after 14, 21, and 28 dpi), neither clinical symptoms nor mortality were observed. In samples from euthanised fish from the three groups mentioned above, the presence of virus DNA was confirmed (Table 2). In both experimental trials, no clinical symptoms or mortality in fish from different species experimentally infected with KHV were observed and in samples originating from control fish, the presence of KHV was not detected.
Species of fish experimentally infected with KHV and results of virus transfer trial to naïve carp
Species of fish experimentally infected with KHV |
Grass carp, Prussian carp, tench, roach, brown bullhead | |||
---|---|---|---|---|
Time of transfer of different fish species (two fish per species) to naïve common carp (dpi) |
||||
7 | 14 | 21 | 28 | |
Mortality in common carp (%) | 90 | 0 | 0 | 0 |
qPCR results | + | + | + | + |
Symbols as in Table 1
In samples from different fish species from a traditional common carp farm with a history of KHV, KHV DNA was detected in common carp three months after confirmation of a KHV outbreak. Viral nucleic acids were detected in samples from four fish (two Prussian carp, one tench, and one brown bullhead). All samples collected at the 4, 10, 13, 16, and 22 month time points were negative (Table 3), and in samples collected from channels which supply water to and drain it from the farm no presence of KHV DNA was detected.
Results of examination of different fish species taken from farm with KHV history
Sampled species: | Bleak, brown bullhead, European perch, freshwater bream, goldfish, grass carp, ide, northern pike, Prussian carp, roach, rudd, tench, wels catfish, white bream | |
---|---|---|
Time of sampling after KHV outbreak (months) | Number of positive samples | Number of negative samples |
3 | 4 (tench, brown bullhead, Prussian carp) | 27 |
4 | 0 | 25 |
10 | 0 | 48 |
13 | 0 | 45 |
16 | 0 | 56 |
22 | 0 | 28 |
Fig. 2
Clinical symptoms of KHVD in common carp

The results of our experimental and field investigations showed the presence of KHV DNA in species of fish which had contact with infected common carp. According to published data (7), we have confirmed the presence of KHV DNA in 10 different fish species. Bergmann
Concluding,
Fig. 1

Fig. 2

Species of fish experimentally infected with KHV and results of virus transfer trial to naïve carp
Species of fish experimentally infected with KHV |
Grass carp, Prussian carp, tench, roach, brown bullhead | |||
---|---|---|---|---|
Time of transfer of different fish species (two fish per species) to naïve common carp (dpi) |
||||
7 | 14 | 21 | 28 | |
Mortality in common carp (%) | 90 | 0 | 0 | 0 |
qPCR results | + | + | + | + |
Results of examination of different fish species taken from farm with KHV history
Sampled species: | Bleak, brown bullhead, European perch, freshwater bream, goldfish, grass carp, ide, northern pike, Prussian carp, roach, rudd, tench, wels catfish, white bream | |
---|---|---|
Time of sampling after KHV outbreak (months) | Number of positive samples | Number of negative samples |
3 | 4 (tench, brown bullhead, Prussian carp) | 27 |
4 | 0 | 25 |
10 | 0 | 48 |
13 | 0 | 45 |
16 | 0 | 56 |
22 | 0 | 28 |
Results of experimental infection of fish from eight different species with CyHV-3
Dpi | Species | |||||||
---|---|---|---|---|---|---|---|---|
Three-spined stickleback | Stone moroko | European bitterling | Grass carp | Prussian carp | Tench | Roach | Brown bullhead | |
7 | + | + | + | + | + | + | + | + |
14 | + | + | + | + | + | + | + | + |
21 | − | + | − | + | − | + | − | − |
28 | − | − | − | − | + | + | − | + |
35 | − | − | − | + | − | − | + | − |
42 | − | − | − | + | + | + | + | − |
49 | − | − | − | − | − | + | + | − |
56 | − | − | − | − | − | − | − | − |
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