Carp Edema Virus Research Articles

Carp oedema virus disease outbreaks in Czech and Slovak aquaculture.

This work describes the first confirmed cases of carp oedema virus disease (CEVD) in Slovakia and the Czech Republic and the phylogenetic analysis of Czech and Slovak carp oedema virus (CEV) isolates. Four cases of disease outbreak in the Czech Republic are described, the oldest dating from mid-May 2013 and one case from Slovakia dating from May 2019. In all cases, virus presence was confirmed using nested PCR. PCR products were sequenced and compared with 357-bp nucleotide sequences encoding the CEV P4a protein in GenBank. In four cases of disease outbreak (three common carp breeding facilities and one koi garden pond), CEV detected belonged to genogroup I. In one case (koi garden pond), fish were confirmed as infected with CEV from genogroup II. This work complements data on CEV occurrence in European countries and contributes to a better understanding of the pathways leading to transmission of the virus throughout Europe.

Current findings on carp edema virus, control challenges, and future outlook

The carp edema virus (CEV) is a pox virus that infects common carp and koi. Its infection causes koi sleepy disease (KSD) that is one of the most important common carp diseases with considerable risk to the productivity of common carp and koi all over the world. In 1970s, KSD was first time reported in Japan but recent increase in worldwide trade of the common carp is causing the spread of virus to various other countries and it is becoming a major threat to the common carp and koi industries. Previous research revealed that the CEV usually required suitable water temperature between 20 and 25 °C and disease outbreaks mostly occur in months of June and July in reported countries. At present, no suitable cell line has been reported for the in vitro replication of CEV; thus, it is difficult to study the CEV in vitro. This review is aimed to summarize the current findings on the known characterization of CEV, KSD pathogenesis, disease diagnostic techniques, its control, and preventive measures. The future prospective about CEV research and development is also discussed in this review.

Koi herpesvirus disease outbreak: Input for the implementation of a surveillance program in South Tyrol – Italy

A severe episode of high and abnormal mortality was observed in the population of Cyprinus carpio of Lake Caldaro (South Tyrol, Italy) in summer 2016. The diagnostic investigation carried out led to the identification of Koi Herpesvirus (KHV) as the etiologic agent.Following this disease outbreak and its socio-economic consequences, the local authorities, in agreement with the local Fishing Association, decided to implement a surveillance program for the achievement of KHV-free health status (Category I) in the Province, in accordance to the Implementing Decision (EU) 2015/1554. The selected area was a defined geographical compartment (the Monticolo lakes compartment, South Tyrol, Italy), which is located near Lake Caldaro, where the Koi Herpesvirus disease (KHVD) outbreak had occurred. This area is of particular interest because it supplies other water bodies with juvenile C. carpio individuals; with the achievement of a KHV-free health status, South Tyrol could possibly become independent in the breeding of this fish species.Suitable samples were collected and processed during a two-year period in order to detect the presence/absence of KHV. The same samples were tested for other viruses that can affect carp, namely spring viraemia of carp (SVCV) and carp edema virus (CEV). According to the results, the authors conclude that the Monticolo lakes area should be classified as KHV-free, as no sample has tested positively for the presence of this specific virus (KHV).

Detection of Koi Herpesvirus (KHV) and Carp Oedema Virus (CEV) in Invasive Round Goby, Neogobius Melanostomus Pallas, 1814, from Poland and Germany.

IntroductionThe aim of the study was to determine the transmission potential of carp edema virus (CEV) and koi herpesvirus (KHV) introduced to Europe by the invasive round goby (Neogobius melanostomus).Material and MethodsA total of 70 round goby specimens were collected from the Szczecin Lagoon, Poland, and locations in Germany in the third and fourth quarters of 2018. The fish were analysed to detect KHV and CEV by PCR.ResultsSix fish specimens were positive for the presence of KHV, while none of the gobies examined showed the presence of CEV.ConclusionThe CEV genome was detected in the goby specimens from Germany and from Poland. Considering the high pace of the spread of the round goby and its effectiveness in acquisition of new ecological niches, it should be kept out during refilling of carp ponds. Further studies should focus on experimental cohabitation of CEV-infected round gobies and specific-pathogen-free (SPF) carp to investigate the potential for active virus transfer.

Koi herpesvirus and carp edema virus threaten common carp aquaculture in Croatia.

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.

Carp edema virus in ornamental fish farming in india: a potential threat to koi carps but not to co-cultured Indian major carp or goldfish

Koi sleepy disease caused by carp edema virus (CEV) of pox virus is a potential carp killer and poses a constant threat to co-cultured fish. Only limited studies are available on its occurrence from the eastern part of India. Here, we report a large-scale mortality of koi carps ( Cyprinuscarpio koi )in ornamental fish farm of Choudwar, Cuttack, Odisha. The fish showed clinical signs of ulcers on body, patchy haemorrhages, massive necrosis of gills, and observed sleeping at the bottom of tanks before death. However, the goldfish maintained in the same tanks did not show any clinical signs or mortality. We noticed Trichodina infestation in the gills of affected animals and secondary bacterium Aeromonashydrophila in kidney tissue. The gills, liver and kidneys of koi carp were found positive for carp edema virus (CEV) (Poxviridae) in PCR followed by sequencing and blast search, and phylogenetic analysis. the damages to the gills and kidney along with the presence of intracytoplasmic eosinophilic inclusions in gill epithelial cells upon histopathology suggested viral association. Experimental challenge with CEV failed to reproduce the disease in Indian major carp and goldfish, the commonly co-cultured fish species with koi carp, though, the disease was reproduced in koi carp. To our best knowledge, it is the first report of CEV infection from the farm of the eastern part of the country. Another mortality incidence in ornamental shop retailer in the same locality also revealed the presence of CEV in koi carp and pointed possibly towards its wide spread. Observations from this study suggest the need for CEV disease surveillance in the country

First detection of koi herpesvirus and carp oedema virus in Iraq associated with a mass mortality in common carp (Cyprinus carpio).

At the end of October 2018, a mass fish mortality occurred in Iraq, involving thousands of tons of cultured and wild common carp (Cyprinus carpio) along Euphrates and Tigris rivers. Fish were found dead or moribund along rivers coasts, showing lethargy, dyspnoea and flared gills. At necropsy, discoloration patches were noticed on the gills. Wet preparations showed rare metacercariae and Dactylogyrus spp. Samples were subjected to bacteriological tests and virological investigation through real-time PCR and nested PCR. Both were positive for koi herpesvirus (KHV) and carp oedema virus. Results obtained were confirmed by the OIE reference laboratory of KHV disease (KHVD) at Cefas (UK) and by sequence analysis. This is the first report on the detection of both viruses in Iraq.

Outbreak of carp edema virus disease in cultured ornamental koi in a lower temperature in China

Carp edema virus (CEV) is the pathogen of carp edema virus disease (CEVD), an emerging infectious disease with high morbidity and mortality worldwide. In the winter of 2016, a disease outbreak occurred in a koi pond in Chengdu, located in the southwest of China. The water temperature at that time was 11–13 °C. The clinical signs of the diseased koi were lethargy, unresponsiveness, enophthalmos, swollen gills, and hemorrhagic skin. Histopathological changes were observed in moribund fish, such as significant cell hyperplasia in the gill lamellae, and swelling and vacuolar degeneration in epithelial cells of the renal tubules and hepatocytes. Electron microscopy of renal tissues showed virions of a pox-like virus (200–235 nm diameter). The presence of specific 528 bp and 478 bp fragments was detected by nested PCR. An experimental infection demonstrated cumulative mortality of 15% (3/20) at a water temperature of 13 ± 2 °C. Furthermore, sequence analysis of the partial 4a genes showed that the CEV isolate (MG189372) in this study had 100% homology with a UK strain (KX254027). The evidence showed that the pathogen involved in this outbreak was CEV. To the best of our knowledge, this is the first report of carp disease caused by CEV at low temperature in China, demonstrating that CEV disease should be monitored in the cold season and at low temperatures. The findings are of profound significance to reducing the risk of CEVD outbreaks in carp aquaculture.

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States.

Koi herpesvirus (KHV; cyprinid herpesvirus-3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real-time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.

Identification of new genogroups in Austrian carp edema virus isolates.

Carp edema virus disease (CEVD), also known as koi sleepy disease (KSD), is an emerging viral disease caused by the carp edema virus (CEV). It is characterized by lethargic behavior, gill necrosis, and generalized edema, leading to significant morbidity and mortality in common carp and koi Cyprinus carpio. Accurate diagnosis of CEVD relies on amplification of a P4a protein-encoding DNA segment from the CEV genome. A phylogenetic analysis of amplified fragments revealed 3 distinct CEV genogroups: I, IIa, and IIb. We explored the phylogenetic relationship between Austrian CEV isolates with existing CEV genogroups. The phylogenetic analysis (n = 18) established the presence of the 3 extant CEV genogroups as well as 2 new CEV genogroups (IIIa and IIIb) classified to identify the Austrian isolates that were distinct from the existing CEV genogroups. It is evident that CEV infection cases are growing in number each year, which may be due to development of sensitive diagnostic assays, while information regarding the virus is scarce. National and international efforts are required to study the epidemiology of the CEV in major carp-producing countries.

Quantitative diagnostics of gill diseases in common carp: not as simple as it seems.

During a disease outbreak, affected fish exhibit particular clinical signs, and the task in veterinary diagnostics is to identify the causative agent(s) as a prerequisite for appropriate treatment measures. In this study, we present an outbreak of a multifactorial gill disease in a cohort of ornamental koi Cyprinus carpio with gill necrosis as the main exterior clinical sign. By means of pathogen identification and determining pathogen abundance in various tissues, mortality of individual fish was found to be caused by different agents. Three out of 5 diseased individuals suffered from koi herpesvirus disease (KHVD) associated with a systemic infection with cyprinid herpesvirus 3 (CyHV-3), 1 fish succumbed to koi sleepy disease (KSD) caused by a high carp edema virus (CEV) load in the gills co-infected with CyHV-3 and flavobacteria, and the last fish had low loads of both viruses but high flavobacteria and Ichthyobodo burdens and most likely died from an interaction of these bacterial and parasitic agents. The results indicated that correct identification of the agent responsible for the observed clinical signs or mortality during co-infection might require quantitative determination of the abundance of the pathogens as well as detailed knowledge of the infection biology of these pathogens.

First report of carp edema virus in the mortality of wild common carp Cyprinus carpio in North America.

Carp edema virus (CEV) is an unclassified poxvirus that infects skin and gill tissue to cause koi sleepy disease. In the USA, CEV was first detected in 1996 in a California koi wholesaler, and has since been reported sporadically only within imported and domestic koi. Common carp Cyprinus carpio are a non-native species now present in most waterways in the USA. In May 2017, >526 large adult common carp in spawning condition died in Mill Pond, Park Ridge, NJ, USA. The water temperature during the kill was 15°C and the affected fish displayed marked lethargy prior to death. The presence of CEV was confirmed by endpoint PCR, real-time quantitative PCR (qPCR), and transmission electron microscopy (TEM), making this the first report of CEV associated with a wild carp kill in North America. Phylogenetic analysis of a region of the 4a gene encoding the major core protein clustered the CEV strain among others in genogroup I, which includes CEV strains previously detected in common carp cultured in Europe. Gill histopathology included severe lamellar fusion and apoptosis in the interlamellar region and TEM identified cytoplasmic virions consistent in morphology with CEV in the branchial epithelial cells. Five months following the mortality, surviving fish were collected and screened for CEV by purifying and concentrating virus from the gills and testing with qPCR. No evidence of CEV was found, supporting previous studies showing CEV is not detectable in gills after abatement of clinical signs.

OUTBREAK AND TREATMENT OF CARP EDEMA VIRUS IN KOI ( CYPRINUS CARPIO) FROM NORTHERN CALIFORNIA.

Carp edema virus (CEV) is the causative agent of carp edema virus disease (CEVD), also referred to as koi sleepy disease, which is an emerging disease of global concern that may cause high rates of morbidity and mortality in common carp and ornamental koi ( Cyprinus carpio). This article reports the third confirmed outbreak of CEVD in California. In June 2015, three koi presented with clinical signs of cutaneous lesions, severe lethargy, and signs of hypoxia. All fish tested positive for CEV by polymerase chain reaction (PCR). Euthanasia and complete necropsy were performed on two fish. The most significant necropsy findings included necrotizing branchitis with marked interstitial edema, multifocal cutaneous ulcerations, and severe cutaneous edema. Treatment of the pond with 0.3-0.5% salt was recommended to the owner. Approximately 7 wk later, a recheck visit was made to the pond. No mortalities had been noted since the initiation of the salt treatment. Physical examination revealed a vast improvement but not complete elimination of the clinical signs of hypoxia and intermittent lethargy in the affected fish. Gill biopsy samples from the two most affected fish were tested and remained PCR positive for CEV. Subsequent recheck visits over 11 mo postdiagnosis and initiation of treatment showed continued improvement in most fish. Gill samples from all fish in the pond ( n = 9) were repeatedly tested by quantitative PCR for CEV, and all samples were negative. This case series further confirms the global spread of CEV and the need for practitioners to be vigilant for outbreaks of this disease. If CEVD is suspected, treatment with 0.3-0.5% salt can be recommended to potentially mitigate the effects of this disease. However, fish may remain potential carriers of this pathogen, and strict biosecurity measures should continue to be enforced for any pond that has had a confirmed CEV outbreak.

Flavobacteria as secondary pathogens in carp suffering from koi sleepy disease.

Koi sleepy disease (KSD) is a disease with increasing importance in global common carp aquaculture. Despite the fact that carp edema virus (CEV) is most likely the causative agent of KSD, the disease often presents itself as multifactorial with several parasites and bacteria species present on gills, skin or in internal organs. Therefore, in this study, we analysed and presented initial results on an interaction of flavobacteria and CEV in the development of clinical KSD in carp suffering from proliferative gill disease. We examined selected field samples from Germany and Hungary and confirmed the presence of CEV and flavobacteria co-infections in subset of the samples. In several infection experiments, we studied the transfer and dynamics of both infections. Furthermore, we analysed which Flavobacterium species could be isolated from KSD-affected fish and concluded that Flavobacterium branchiophilum is a possible copathogen. Antibiotic treatment experiments showed that CEV seems to be the primary pathogen causing an insult to the gills of carp and by these enabling other pathogens, including F.branchiophilum, to establish co-infections. Despite the fact that F.branchiophilum co-infection is not required for the development of clinical KSD, it could contribute to the pathological changes recorded during the outbreaks.

First detection of carp edema virus in association with cyprinid herpesvirus 3 in cultured ornamental koi, Cyprinus carpio L., in China

An outbreak of a carp disease occurred in a koi pond in early October 2016, Chengdu, southwest of China. Characteristic signs of infected fish include erratic swimming of moribund fish at the water surface, respiratory distress, presence of thick and foggy mucus covering the body and gills. Skin ulcers were also noticed in a few cases. Microbiological, parasitological and virological investigations were carried out with fish samples collected from various localities. Histologically, the gills, kidney and liver showed discernible changes. By analyzing gill tissues in six samples by PCR and nested-PCR for detection of cyprinid herpesvirus 3 (CyHV-3) and carp edema virus (CEV) respectively, PCR products of 292 bp and 478 bp (by using nested primers) could be amplified. The outbreak of carp disease caused by CEV and CyHV-3 mixed infection was confirmed by sequence analysis of the PCR products. The viral load in the gills and kidney were quantified by real-time TaqMan PCR, which in turn would aid the evaluation of the significance of the virus to the etiology of the disease. The result demonstrated that CEV plays the major role to the death of fish. Phylogenetic analyses revealed that the CEV isolate (GenBank accession no. MG189371) showed closest identity to the CEV isolated from UK (KX254027) with 99% homology. To our knowledge, this is the first report a carp disease caused by CEV and CyHV-3 mixed infection in China.

Rapid detection and differentiation of carp oedema virus and cyprinid herpes virus-3 in koi and common carp.

Carp oedema virus (CEV) and koi herpes virus (KHV) are of major concern to common carp breeders and koi enthusiasts worldwide. The viruses cause diseases that exhibit similar external signs; thus, it is difficult to distinguish between them clinically. In this study, we developed and optimized rapid and accurate single- and multiplex isothermal diagnostic tools, based on recombinase polymerase amplification (RPA), for detection and differentiation of CEV and KHV. The assays were combined with a lateral flow dipstick to enable visual detection of amplification products and simplify post-amplification analysis. Both CEV- and KHV-RPA assays were specific for their target virus. The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50min, compared to 10 and 7hr for CEV- and KHV-PCR tests, respectively. The assays can be performed in field situations to improve screening of fish and reduce spread of these viruses and thereby enhance the common carp and koi industries.

Histopathology and Detection of Cyprinid Herpesvirus Infection in Two Koi Farms in Tianjin City (China)

Koi herpesvirus (KHV) is the etiologic agent of koi herpesvirus disease (KHVD) causing mass mortalities in common carp (Cyprinus carpio carpio), koi (Cyprinus carpio koi), and ghost carp (Cyprinus carpio goi) populations. In this study, we report new outbreaks of the koi herpesvirus (KHV) disease in a koi carp farm in Tianjin city, northern China. From June to September 2017, severe mortalities of koi juvenile and broodstock occurred in ornamental fish, and infected koi carp which showed signs of lethargy and loss of appetite, and clinical signs including sunken eyes, gill necrosis, and excessive secretion of mucus. Histopathologically, the gills showed hyperplasia and degeneration of epithelial cells and fusion of the lamellae, and foci necrosis was found in gills, liver, kidney, and spleen. In addition, KHV and Carp edema virus (CEV) were detected in the diseased koi fish by diagnostic PCR tests for the viruses. The results showed that the nucleotide sequences of thymidine kinase (TK), DNA polymerase (SphI-5), and four open reading frame (ORF 25, ORF 56, ORF 72, ORF 81) genes confirmed the highest identity with other KHV strains such as KHV-I, KHV-U and KHV-J, but the CEV was negative. The phylogenetic tree showed KHV-TJ1708 and KHV-TJ1709 clustered with the Asian strain KHV-J (AP008984). Results of the present study confirmed the prevalence of Carp herpes disease (KHVD) in China koi populations by means of clinical examination, histopathology and molecular technique. These data will provide a reference for diagnosis, quarantine, and surveillance of KHV in China.

First report of carp oedema virus infection of koi (Cyprinus carpio haematopterus) in the Republic of Korea.

Twenty-five koi (Cyprinus carpio haematopterus) bought from a wholesale fish market in Korea, showed lethargic behaviour and 100% mortality within 20days. Carp oedema virus (CEV) was detected by PCR in all 25 koi. Results of detailed histopathological and clinical examinations of 17 koi indicated loss of body balance, severe infiltration of inflammatory cells into the inter-lamellar spaces of the gills and vacuolization and inclusion bodies in gill epithelial cells. Sequence analysis of PCR products of these koi showed up to 99% identity to the previously reported sequences, suggesting that the observed mass mortality resulted from koi sleepy disease (KSD) due to CEV infection. To the best of our knowledge, this study is the first report of KSD in the Republic of Korea. Partial sequences of 4a protein from the virus indicated that the present CEV detected in koi from Korea is more closely related to that from the UK and Poland than from Japan. The present findings indicate that the prevalence and spread of KSD must be closely monitored in both European and Asian countries to avoid potential economic losses to the global koi industry.

Carp edema virus from three genogroups is present in common carp in Hungary.

Hungary is an important carp producer with intensive trading relationships with farms in other carp-producing areas in Europe. Carp in Europe were recently found infected with carp edema virus (CEV), a poxvirus which causes the koi sleepy disease (KSD) syndrome. Moribund carp were collected from 17 fish farms and angling ponds in different regions of Hungary. Histological analysis of gills from these carp revealed a proliferation of the interlamellar epithelium and an infiltration by eosinophilic cells. In 13 of 17 of these carp, CEV DNA was detected by qPCR and in seven fish more than 1×104 copies of virus-specific DNA sequences per 250ng of DNA, which could be considered as clinically relevant and a cause of disease. A phylogenetic analysis of the sequences revealed that all three genogroups of CEV were present in Hungarian common carp with genogroup I being most abundant. These results support the hypothesis of a prolonged presence of CEV in European carp populations and suggest that previous outbreaks of KSD were not recorded or misdiagnosed. Hence, a testing of carp and koi for infection with CEV should be included into disease surveillance programmes to prevent further spreading of this disease.

Viral infections in common carp lead to a disturbance of mucin expression in mucosal tissues

In response to the constant challenge by potential pathogens, external surfaces of fish, their skin, gills and intestinal tract, are coated with mucus, a gel like substance which largely prevents the entry of pathogens. This mucus gel consists mainly of water and mucins, large O-glycosylated proteins, which are responsible for forming a gel like mixture. A modulation of the mRNA expression of mucins, was described in viral diseases in mammals however there is a knowledge gap about the regulation of mucins during viral infection in fish. Therefore, novel sequences for common carp mucins were located in an early version of the common carp genome and their mRNA expression measured in carp under infection with three different viral pathogens: (i) the alloherpesvirus cyprinid herpesvirus 3, (ii) the rhabdovirus spring viremia of carp virus and (iii) the poxvirus carp edema virus. The results showed a downregulation of mucin mRNA expression in gills and gut of common carp under infection with these pathogenic viruses. This could be a sign of a severe distress to the mucosal tissues in carp which occurs under viral infection. The reduced expression of mucins could help explaining the increased susceptibility of virus-infected carp to secondary bacterial infection.