CARP EDEMA VIRUS DISEASE IN SERBIA – A DISEASE OUT OF CONTROL

A poxvirus named carp edema virus (CEV) is the causative agent of carp edema virus disease (CEVD), which is an emerging disease of global concern that may cause high rates of morbidity and mortality in ornamental koi and common carp. Common carp ( Cyprinus carpio ) is the most important fi sh species for warm‐water aquaculture in Serbia. CEVD was fi rst detected in Serbia in 2017. During the 2017-2020 period, an increasing number of CEVD outbreaks in carp farms was reported. Th e carp were collected from farms in diff erent regions of Serbia from 2017 to 2020. Th e fi sh were sampled for disease diagnosis because they exhibited lethargy and anorexia, which eventually led to mortality. Mortality started with clinical signs of hypoxia and the fi sh swam slowly and were unresponsive. Th e gills were pale and covered with a thick mucus layer. In later stages of the disease, the lesions in the gills turned into a necrotizing form. A moderate to high amount of opportunistic fresh-water bacteria were isolated from the gills of the diseased fi sh. By performing real-time PCR, CEV was detected in 38 samples of the diseased carp taken from 21 carp farms. Th ese outbreaks further confi rm the spread of CEVD and the need for practitioners to be vigilant in the event of an outbreak of this disease. To prevent further spreading of the disease, it is very important to introduce CEV testing before moving fi sh. To avoid further transmission of the virus to common carp populations in Serbia, the testing of CEV should become part of fi sh disease surveillance programs. Fish health service should be aware of the presence of CEV in Serbia which may result in high losses in carp aquaculture. Action should also be taken to prevent transmission of CEV to carp populations in open waters.


INTRODUCTION
Common carp (Cyprinus carpio) is the most important fi sh in Serbian aquaculture, with annual production of approximately 11,000 tons (Marković et al. 2011). In order to maintain and intensify the production, one of the main goals is to prevent the occurrence and spreading of the diseases, which could limit the sustainability of fi sh production. With that goal, annual control of listed fi sh diseases, namely koi herpesvirus (KHV) and spring viremia of carp virus (SVCV) has been carried out. However, the occurrence and spread of a new disease which may signifi cantly aff ect the health of carp is a disturbing fact which requires attention of all the parties involved in the production chain. Carp edema virus disease (CEVD) is an emerging disease considered to be a potential risk for the carp aquaculture and for global food security as well (Kurita et al. 2009;Way et al. 2017), with new data about its spread, economic and biological properties being published rapidly over the past few years (Rehman et al. 2020; Machat et al. 2021).
Th e fi rst outbreak of a disease caused by carp edema virus (CEV) was reported in 1974 in Japan, and for long time this disease was detected exclusively in ornamental koi carp (Murakami et al. 1976), but recently it was confi rmed as a causative agent of a disease in koi and common carp in Europe, USA and many Asian countries ; Marsella et al. 2021).
In common carp, the disease was initially detected in the United Kingdom and in the Netherlands in 2012 ( In the majority of cases, farmed common carp were involved. However, there are also reports of mortality events involving wild fi sh. For the fi rst time in Europe, CEV was detected in wild common carp in Italy ( Marsella et al. 2021). A wide temperature range from 6 to 24 °C has been recorded for the reported cases, although most of the outbreaks occurred at the temperature between 19 and 24 °C (Divya et al. 2019). Th e disease generally has the characteristics of an acute infection at water temperatures between 15 and 25 °C, but it also occurs at a lower temperature (6 -10 °C) when aff ecting common carp, resulting in a chronic infection characterized by a lower mortality (Lewisch et al. 2015;Way et al. 2017; Toff an et al. 2020).
Diseased fi sh become lethargic or show sleepy behavior and eventually die due to anoxia. Th e juvenile carp usually congregate near the surface of a pond or water inlet, whereas the older fi sh tend to stay at the bottom of the pond. Th e most frequently observed external lesions consist of increased skin mucous production, enophthalmos, erosion or hemorrhages at the base of the fi n and gill necrosis . Gross lesions in gills (hypertrophy, hyperplasia and lamellae clubbing) and subsequent necrosis of secondary gill lamellae may lead to the death of the infected fi sh (Machat et al. 2021).
Carp edema virus disease (CEVD) is caused by carp edema virus (CEV), which belongs to the subfamily Chordopoxvirinae in the Poxviridae family. CEV has double-stranded DNA genome and has a strong affi nity to infect the gill epithelial cells. Phylogenetic analysis of the partial core protein p4a has revealed the existence of three genogroups: I, IIa, and IIb showing 6 -10% genetic diversity Soliman et al. 2019).
Genogroup I consists exclusively of viral samples obtained from common carp collected mostly from European aquaculture countries, namely: Th e United Kingdom, Germany, Poland, Hungary and Balkan countries.
Genogroup IIa contains viruses isolated predominantly, but not exclusively from koi carp showing clinical signs of the disease with Asian and European isolates of the virus being included.
Genogroup IIb includes viral isolates from koi and common carp and was classifi ed by phylogenetic analyses between the two aforementioned genogroups. Th e genogroup IIb is discovered in various carp samples in Poland .
Th e CEV spread is most probably the result of lack of availability of eff ective diagnostic and preventive measures in the global trade. Th e detection of the virus is diffi cult at subclinical level in specimen. Th erefore, prevention of the disease is diffi cult and needs extra care at diff erent stages such as transportation, transfer of fi sh from one pond to another, etc. It is likely that the establishment of an eff ective monitoring system and development of a highly specifi c diagnostic method could be helpful for the control of disease outbreaks (Rehman et al. 2020). Adequate measures for the prevention and disease control need to be taken in order to minimize the chances of disease outbreak such as avoiding transport and restocking of susceptible fi sh in diff erent farming systems when the water temperature becomes suitable for virus replication Currently, no treatment is available for CEVD. Diff erent research showed that the mortality rate reduces the number CEV-infected carp when infected fi sh are immersed in 0.5% salted water.
Aft er the fi rst occurrence of carp edema virus disease (CEVD) in 2017 (Radosavljevic et al. 2018), an increasing number of CEV positive carp farms was detected. In this paper we report the increase in the incidence, disease in severity and mortality of CEVD at a carp farms during a four-year period, from 2017-2020.

Fish samples
Th e carp were collected from 21 fi sh farms in diff erent regions of Serbia from 2017 to 2020. Th e carp were sampled for disease diagnosis because the fi sh exhibited lethargy and anorexia, which eventually led to mortality. Th e total of 38 samples (the gill and kidney tissues of fi ve individuals were pooled to form one sample) were examined including one-year-old (C1), two-year-old (C2) or three-year-old (C3) carps (5 fi sh from each group). External examination and sampling were performed for further investigation. Aft er gill clipping and skin scraping, the presence of external parasite infection was inspected with an optical microscope. For bacterial culture, the organ surface was sterilized using a heated blade and punctured to perform parenchymal swab sampling. Bacterial cultures of the gills, liver, spleen, and kidney were kept for 48 h using tryptic soy agar plates at 20 °C. Th e gill and kidney tissue were sampled for virus isolation and DNA extraction.

Virus isolation
Th e post-mortem collected pooled gills and anterior kidney were homogenized in Earle's salt-based Minimal Essential Medium (MEM) (Sigma-Aldrich). Th e homogenate was inoculated in an array of cell lines (including CCB, EPC, BF-2, RTG-2, CHSE) for three passages, according to standard procedures.

Molecular detection by PCR
Th e samples were tested for CEV according to real-time PCR protocol described by Way et al. (2017). Th e sample RNA was extracted from the homogenates of the gill and anterior kidney tissues of diseased fi sh with the QIAamp Cador Pathogen Mini Kit (Qiagen), according to the manufacturer's instructions. Th e DNA was then stored at −20 °C before PCR amplifi cation.

RESULTS
Th e total of 190 fi sh from 21 carp farms were examined, and 38 outbreaks of CEVD in common carp of various age were noticed in 6 regions in Serbia (Table 1, Figure 2). During CEVD outbreaks, no behavior changes or clinical signs of disease were noticed in other fi sh species present in the same pond with carp. Most outbreaks occurred between late spring and early summer, when water temperature was between 14 °C and 25 °C. But, in a few cases, the occurrence of the disease with mortalities was noticed in autumn and early spring when temperature was just 7 -12 °C (Table 1). Th e highest mortality rates were recorded during an outbreak at the beginning of May (with water temperature of 18 °C) and during three outbreaks in September (water temperature 21 °C and 22 °C) ( Table 1). Various clinical signs of the disease were observed in CEV positive carps (Figure 3). Usually, fi sh farmers reported an increasing mortality of the fi sh that were unresponsive, gasping for air and swimming slowly.
Dominant clinical signs noticed during CEVD episodes includes: 1. Lethargic behavior with reduced food intake ( Figure 3A). 2. Various cumulative mortality -mostly low < 20%, but in severe cases > 50%. 3. Overproduction of mucus on gills and skin (Figures 3B, 3D). 4. Pale swollen gills, sometimes with irregular discoloration and gill necrosis, but never as drastic as with KHVD ( Figure 3E). 5. No pathologies in internal organs, except fi brinous peritonitis ( Figure 3F). 6. Various secondary bacterial and fungal gill and skin infections (motile aeromonads, pseudomonads, Flavobacteriae, B. mucoides, Saprolegnia spp, Branchiomyces spp.) ( Figure 3C). Besides these, various clinical signs were noticed in individuals, including sunken eyes, hyperemia or hemorrhages at fi n base, fi brinous peritonitis, the perianal region, and the abdominal body surface, gill hemorrhages, skin erosions and ulcerations. In majority of the cases, the bacteria were not isolated from internal organs (liver, kidney and spleen), but always present on gills and skin lesions of the aff ected fi sh.
By performing real-time PCR, CEV was detected in samples from 38 outbreaks of disease from 21 carp farms.
No cytopathic eff ect was observed in fi ve cell lines (CCB, EPC, BF-2, RTG-2, and FHM) following inoculation of fi ltrate from gill and kidney tissue homogenate of CEV positive carp, aft er 10 days of inoculation and even aft er three blind passages.

DISCUSSION
Th e present study revealed that CEV was widespread in Serbian common carp farms and the virus was detected during 38 outbreaks on 21 carp farms. Water temperature is a signifi cant factor for development of CEVD. Th e disease in common carp has been reported at water temperatures between 7 and 15 °C (Lewisch et al. 2015), and also at lower water temperatures between 6 and 9 °C (Way and Stone, 2013). Important predisposing factor is rapid temperature change and primary rapid rise in temperature. A majority of detected CEVD outbreaks in Serbia occurred when water temperature ranged between 13 and 24 °C, but in 5 cases, the disease occurred between 7 and 12 °C. Th is corresponds with fi ndings of other authors (Lewisch et  . Th e cumulative mortality rate in majority of the cases described here (34 out of 38) was below 20%, but in three outbreaks, the mortality was higher than 50%, and in one outbreak around 35%.
In 17 outbreaks, co-infection with bacteria or fungi was present, as reported by other authors Machat et al. 2021). Th ese isolated bacteria and fungi are known to be opportunistic pathogens for carp, and they had probably contributed to increase the severity of the disease.
No cytopathic eff ect was observed in fi ve cell lines following inoculation of fi ltrates of gill and kidney homogenate of CEV positive carp, aft er three blind passages. Th e results are in accordance with earlier reports, where the virus was not isolated on various cell lines (Oyamatsu et al. 1997 Having in mind an obvious increase in the number of CEVD cases in common carp and spread to new locations, adequate measures which will enable timely detection of the causative agent in infected fi sh and possible vectors should be adopted as soon as possible, since there are still many unsolved questions regarding potential asymptomatic vector species, modes of transmission, virus isolation, etc.
Th e emergence of infectious diseases is usually triggered by ecological changes, oft en associated with human interventions, such as transfer of organ-isms, environmental degradation, agricultural practices or technology (Jones et al. 2008). Introduction of new pathogens into naive populations can cause mass mortalities, as happened with common carp aft er introduction of KHV and with Prussian carp aft er introduction of CyHV-2. Usually, the fi rst data about occurrence of new disease comes from fi sh in aquaculture, and the acquired data should be used as valuable information for prediction of the impact of the disease on susceptible free-living fi sh populations. Based on the presented information, it is obvious that there is a need for adequate legislation in order to control and prevent further spread of the virus. Th e implementation of control measures which will slow the spread of the disease is needed urgently, having in mind that CEV is detected in wild common carp populations in Europe and USA (Lovy et

CONCLUSION
CEVD is slowly becoming one of the biggest threats to carp aquaculture, due to increasing mortality in infected fi sh, and also due to reduced growth and the fact that secondary bacterial and fungal infections are regularly found in diseased fi sh, causing additional pathologies and mortality. Fish health service should be aware of the presence of CEVD, which may result in substantial losses in carp aquaculture. Based on the great importance of carp aquaculture in Serbia, a detailed surveillance and control program for CEVD is warranted, especially due to absence of international regulations regarding this disease. Internal movements of carp between aquaculture facilities are of particular concern because large numbers of live carp are moved and, since infections in aquatic animals are frequently subclinical, they would not be directly noticed before, during and immediately aft er the movement of the infected fi sh. Having in mind that movement of live aquatic animals always carries a risk of transferring aquatic animal pathogens, the controls should also involve internal fi sh movements between carp farms. In addition, disease quarantine inspections of imported/exported fi sh should be performed with greater precision. Th e quarantine procedure for CEVD should be performed in such way that carp is quarantined for a minimum of 30 days in the water with a constant temperature (between 15 and 25 °C). In order to avoid the spread of CEV infections, testing carp for CEV should become part of fi sh disease surveillance program.

Author's Contribution:
VR and VM made contributions to concept and design of the study, they collected data and draft ed the manuscript. DG and VM carried out the molecular diagnostic tests and prepared the alignment of nucleotide sequences and conducted the molecular genetic analysis. LJV carried out the data analysis. VM and KN revised the manuscript critically and together with VR prepared the fi nal draft of the manuscript. All the authors read and approved the fi nal manuscript.