Distribution of carp edema virus in organs of infected juvenile common carp
Published Online: Sep 20, 2023
Page range: 333 - 337
Received: Mar 08, 2023
Accepted: Aug 10, 2023
DOI: https://doi.org/10.2478/jvetres-2023-0049
Keywords
© 2023 Marek Matras et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Carp edema virus (CEV) was first detected in Japan in the 1970s, when mass mortality was observed in the fry of koi, the coloured variety of
Common carp with clinical signs of KSD were collected from a traditional carp farm. After confirmation of carp edema virus by real-time PCR, the fish were experimentally cohabited with common carp fry. In the cohabitation experiment 30 fry (45–50 g) were introduced to tanks with 10 KSD-affected common carp (320–350 g). As a negative control, another group of 30 fry were put together in a tank with naïve (CEV-negative) carp. After 12 h of cohabitation both groups were moved to separate tanks of 600 L capacity. The water in the tanks was filtered, aerated, and maintained at a temperature of 14 ± 1°C. Three experimental and three control fish were euthanised by immersion in a bath of 0.5 g L−1 tricaine solution (Sigma-Aldrich, St. Louis, MO, USA) at one-week intervals (7, 14, 21 and 28 days post cohabitation commencement). Tissue samples of the brain, gills, spleen, kidney, intestines and skin were collected from all euthanised fry. The carp were examined daily for clinical lesions of KSD. The experiment was performed in two identical parallel iterations.
Total DNA was extracted from samples of the brain, gills, spleen, kidney, intestines and skin using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Before testing, genetic material was eluted in 100 μL of acetate ethylenediaminetetraacetic acid buffer and stored at −80°C.
Genetic material of CEV was detected by real-time PCR using a protocol developed originally by the Centre for Environment, Fisheries and Aquaculture Science (CEFAS) in Weymouth, UK, which was successfully used previously in a study concerning infected samples from koi and common carp (8). The assay was performed using CEV qFor1 (5′-AGTTTTGTAKATTGTAGC ATTTCC-3′) and CEV qRev1 (5′-GATTCCTC AAGGAGTTDCAGTAAA-3′) respective forward and reverse primers. The 20 μL reaction volume contained 500 nM of forward and reverse primers, 200 nM of CEV qProbe1 (5′-AGAGT TTGTTTCTTGCCAT ACAAACT-3′), 1 × PCR buffer mix and 5 μL of the template DNA. A CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used, and the programme of amplification was initial incubation at 50°C for 2 min, then incubation at 95°C for 10 min, and 50 temperature cycles at 95°C for 15 s and 55°C for 1 min.
In the first experimental trial, the presence of genetic material of carp edema virus was confirmed by the real-time PCR method in tissue samples obtained from the brain, gills, intestines and skin of fish on the 7th day post cohabitation with CEV-affected fish. Carp edema virus nucleic acid was detected in the samples of fry brains, gills and skin on the 14th day of the experiment. At the third sampling interval the presence of carp edema virus in the brain, gills, kidney, intestines and skin was observed, whereas the last sampling confirmed the presence of CEV nucleic acid only in the gills and skin (Table 1). In the second experimental challenge, the presence of CEV DNA was noted in 11 out of 18 samples of the brain, gills, kidney, intestines and skin on the 7th day post cohabitation with CEV-infected common carp. The presence of carp edema virus was also confirmed in all sampled tissue types on day 14 of the second experimental trial. On the 21st day samples were positive in the cases of brain, gill, kidney, intestine and skin samples, whereas 28 days post infection, CEV DNA was only detected in a sample of skin (Table 2).
Real-time PCR results in fish experimentally infected with carp edema virus and maintained at 14°C (first iteration)
| Dpi | Fish number | Cycle threshold for a given tissue type | ||||||
|---|---|---|---|---|---|---|---|---|
| Brain | Gills | Kidney | Intestines | Skin | Spleen | |||
| 7 | 1 | +/40.60 | − | − | − | +/34.88 | − | |
| 2 | +/39.16 | +/38.62 | − | − | +/38.04 | − | ||
| 3 | +/39.79 | +/39.09 | − | +/40.77 | +/30.89 | − | ||
| 4 | − | +/39.31 | − | − | +/32.86 | − | ||
| 14 | 5 | +/40.56 | +/40.27 | − | − | +/35.84 | − | |
| 6 | − | +/38.56 | − | − | +/40.28 | − | ||
| 7 | +/40.11 | +/39.78 | +/41.29 | +/39.45 | +/38.49 | − | ||
| 21 | 8 | − | +/35.84 | +/40.09 | − | +/32.12 | − | |
| 9 | − | +/38.56 | − | − | +/40.67 | − | ||
| 10 | − | − | − | − | +/32.12 | − | ||
| 28 | 11 | − | +/39.67 | − | − | +/40.22 | − | |
| 12 | − | − | − | − | − | − | ||
Dpi – day post infection; – negative test result; + positive test result
Real-time PCR results in fish experimentally infected with carp edema virus and maintained at 14°C (second iteration)
| Dpi | Fish number | Cycle threshold for a given tissue type | ||||||
|---|---|---|---|---|---|---|---|---|
| Brain | Gills | Kidney | Intestines | Skin | Spleen | |||
| 7 | 1 | − | +/34.37 | − | − | +/29.98 | +/38.01 | |
| 2 | +/36.24 | +/36.06 | − | +/39.97 | +/24.19 | − | ||
| 3 | +/31.97 | +/34.36 | +/36.94 | +/40.06 | +/26.66 | − | ||
| 4 | − | +/38.13 | +/40.83 | − | +/32.54 | − | ||
| 14 | 5 | +/38.93 | +/39.12 | − | − | +/32.33 | − | |
| 6 | +/32.95 | +/33.74 | +/35.64 | − | +/28.10 | − | ||
| 7 | +/40.21 | +/38.52 | − | +/38.83 | +/37.18 | − | ||
| 21 | 8 | +/38.42 | +/37.75 | +/34.87 | +/37.97 | +/28.21 | − | |
| 9 | +/39.10 | +/35.67 | − | +/40.57 | +/36.79 | − | ||
| 10 | − | − | − | − | − | − | ||
| 28 | 11 | − | − | − | − | − | − | |
| 12 | − | − | − | − | +/33.79 | − | ||
Dpi – day post infection; – negative test result; + positive test result
Clinical symptoms were observed in common carp fry after contact with CEV infected fish, but no mortality was observed in either experiment. The first KSD symptoms were detected on the 8th day post infection in the first experimental trial as skin lesions (Fig. 1) and on the 7th day in the second experiment also as skin lesions and additionally as swollen gills (Fig. 2).
Fig. 1.
Skin lesions (petechiae) in common carp fry experimentally infected with carp edema virus
Fig. 2.
Swollen gills and skin petechiae in common carp fry experimentally infected with carp edema virus
In no samples originating from control fish in either experimental challenge was the presence of carp edema virus genetic material detected, and in clinical examination no signs of disease or mortality in these fish were noted to the end of the experiments.
Viral DNA was not always found in all organs or the skin of each individual fish. The percentage of positive results in a sample depended on the specific tissue from which the sample was collected. In both iterations of the experiment, the highest percentages of positive results were observed in skin and gill samples, whereas all samples from spleens were negative in the first experiment and only one was positive in the second (Fig. 3).
Fig. 3.
Percentages of positive real-time PCR results in tissue samples of carp edema virus–infected common carp fry
The results of our study on cohabitation of common carp fry with infected carp showed CEV to have high infectivity for this fish species. Common carp with confirmed koi sleepy disease syndrome were able to infect naïve counterparts, and the infection caused similar clinical signs,
The disease incubation period, or in other words the time interval from experimental infection until instances of clinical signs could be observed, was seven days (with the fish maintained at 14°C) according to observations made in our experiment. This result confirms the findings of other authors (2).
Our experiments also showed that the real-time PCR method using CEFAS primers (8) is very efficient in identifying CEV genetic material in tissue samples from moribund and asymptomatic KSD-affected fish. According to the “Infection with carp edema virus (CEV)” technical disease card published by the World Organisation for Animal Health, this diagnostic method can be used for presumptive and confirmatory purposes (16).
In our experimental cohabitation trials, we particularly sought to investigate the distribution of CEV DNA in the tissues of infected common carp in order to determine which organs were useful for sampling during CEV infection research. On the seventh day post infection, CEV DNA was most often found in the skin, gills and brain, and less frequently in the kidneys and intestines. Generally in infected common carp, CEV DNA could usually be found in several organs of each individual fish, although it was only detected in one sample of spleen tissue. The investigations by real-time PCR after the 28th day post infection led to the conclusion that carp edema virus genetic material is detectable more frequently in the skin and gills than in samples of the brain, kidney, intestines or spleen. The lowest cycle threshold value of 24.19 was noted in the skin. This contrasts with the finding of the study by Adamek
Concluding, in our cohabitation experiment focused on the pathogenesis of the CEV infection process, we confirmed the high infectivity of CEV. We nominated the most relevant organs which should be taken into consideration during sampling to establish CEV infection prevalence. The real-time PCR method used in the experiments was shown to be useful at the clinical and asymptomatic stage of virus infection and it can be used for presumptive and confirmatory purposes. This knowledge concerning the pathogenesis of CEV may be useful in diagnosis, an essential element in disease control.