Potential vector species of carp edema virus (CEV)

Abstract During a PCR‐based CEV survey in Poland in 2015–2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV‐infected carp yielded positive results. These species of fish were experimentally cohabited with CEV‐infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR.

vectors of CEV infection. A better understanding of the mechanisms of transmission of koi sleepy disease is important and the knowledge of animate vectors will be helpful in measures aiming to limit the spread of the virus.

| Experimental trials
Fish from six different species were experimentally cohabited with CEV-infected common carp (Cyprinus carpio) in two separate trials. In the first cohabitation experiment, 30 specimens each of bleak (Alburnus alburnus), crucian carp (Carassius carassius), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) were introduced to tanks with 20 CEV-infected carp. Control fish (30 in each species) were cohabited with naïve carp. Both groups were transferred after 12 hr of cohabitation with CEV-infected and naïve fish to separate 600-L tanks with filtered and aerated water at a temperature of 16 ± 1°C. At 3, 7, 14 and 21 days post-exposure, two fish per species were killed by immersion into a 0.5 g/L tricaine solution (Sigma-Aldrich) and samples of brain, gills, spleen, kidney, intestine and skin were collected. At the same time intervals, samples from control group fish were collected.
In the second experimental trial, bleak, crucian carp, European perch (Perca fluviatilis), Prussian carp and roach were cohabited with CEV-infected carp for 72 hr. At 7-day intervals (at 7, 14, 21, 28, 35 and 42 days post-exposure), three fish per species were killed and samples were taken as described above. At the same time intervals, samples from control group fish were collected.

| DNA extraction
The tissue samples were homogenized and total DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer instructions. The DNA was eluted in 100 µl of acetate ethylenediaminetetraacetic acid (AE) buffer and stored at − 80°C before testing.

| Molecular identification
All samples were initially tested by real-time PCR using a QuantiTect Probe commercial PCR kit and a Rotor-Gene Q thermal cycler (Qiagen, Germany). The protocol adopted was developed originally by the Centre for Environment, Fisheries and Aquaculture Science (CEFAS) in Weymouth, UK. Assays were performed in a 20 µl reaction volume consisting of 500 nM forward CEV qFor1 (5′-AGTTTTGTAKATTGTAGCATTTCC-′3) and reverse CEV qRev1 (5′-GATTCCTC AAGGAGTTDCAGTAAA-′3) primers, 200nM CEV qProbe1 (5′-AGAGT TTGTTTCTTGCCATACAAACT-′3), 1 × PCR buffer mix and 5 µl of the DNA extracted above. Assays were performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The samples were initially held at 50°C for 2 min followed by 10 min at 95°C and were then put through 50 temperature cycles of 15 s at 95°C and then held for 1 min at 55°C. The CEFAS primers and the above real-time PCR method have been successfully used in infected tissue samples from koi and common carp (Matras et al., 2017). CVI NL-13013096 and CVI NL-14009726 genetic materials (courtesy of Dr Olga Haenen from the NRL for Fish, Crustacean and Shellfish Diseases in the Netherlands) were used as a positive control in the molecular identification.

| RE SULTS
The presence of carp oedema virus nucleic acid after 12 hr of cohabitation was detected by the qPCR method on the 7th-day postexperimental exposure in tench and crucian carp, in both cases in the gills. On the 14th day post-exposure, the presence of CEV nucleic acid was found the gills of in two tench and one Prussian carp. We observed the presence of virus on the 21th day post-cohabitation in two Prussian carp, also only in samples from the gills (Table 1). In the second experimental challenge with 72 hr of cohabitation, we observed the presence of CEV DNA in 7 out of 15 killed fish (one crucian carp, one Prussian carp, one European perch, two roach and two bleak) on the 7th day post-exposure. In samples from this period of infection, we obtained positive results in gills and in the skin (Table 2). On day 14 after the transfer of the fish to separate tanks, the presence of CEV was confirmed in nine fish, the sample tissue being the gills of two Prussian carp, one roach, one bleak and one European perch and also in the skin of one crucian carp and one European perch. In one crucian carp, we detected virus nucleic acids in both samples: from gills and skin. On the 21st day post-exposure, only two samples were positive: one from the gills of a crucian carp and the second from the skin of a roach. CEV DNA was detected 28 days post-infection in the gills of three fish (two crucian carp and one European perch) and in the skin of four fish (one Prussian carp, one roach and two bleaks). In samples originating from the next time interval, we obtained positive results in 12 fish. We observed the presence of virus DNA in the gills and skin of roach, but we also had positive samples from the skin in 11 fish (Table 2). In samples collected 42 days post-exposure, we confirmed CEV DNA in five fish. In samples originating from control fish, we did not detect the presence of carp oedema virus. In samples from bleak and roach, in trials with 12-hr exposure, we did not detect carp oedema virus.
But in the experimental challenge with 72 hr cohabitation, we confirmed virus DNA in samples collected from all species used in the experiment. We did not observe any clinical signs or mortality in fish after cohabitation with CEV-infected common carp to the end of the experiment.

| D ISCUSS I ON
The results of our investigation confirmed that CEV-infected common carp are able to transmit the virus to other fish species. In the literature, there are published results indicating that KSD-afflicted common carp, as well as the ornamental koi variety, are able to transmit the viral disease to other fish from the Cyprinus carpio species . However, we showed that during cohabitation an infective agent is transmitted from CEV-infected common carp to heterospecific fish of the bleak, crucian carp, European perch, Prussian carp, roach and tench species. In fish from six species used in our study, we observed neither clinical signs nor mortality. In  This knowledge concerning the mechanism of spread of the CEV will be useful for veterinary services and breeders to limit the spread of CEV to naïve populations, making future measures for the eradication of the disease more reliable.