Koi Sleepy Disease Research Articles

Stressing out-carp edema virus induces stress and modulates immune response in common carp.

Carp edema virus (CEV) is a fish poxvirus that primarily infects the gills of common carp. CEV causes koi sleepy disease (KSD), which is highly contagious and can result in mortality of up to 100%. In the present study, we analyzed the stress and immune responses during KSD in two strains of common carp with different resistance to CEV: susceptible koi and resistant Amur sazan. Experiments were performed at two temperatures: 12°C and 18°C. In the case of koi carp, we also analyzed the effect of supplementation of 0.6% NaCl into tank water, which prevents mortality of the CEV-infected fish (salt rescue model). We found that CEV-infected koi kept at 18°C had the highest viral load, which correlated with the most severe histopathological changes in the gills. CEV infection resulted in the activation of stress response reflected by the upregulated expression of genes involved in stress response in the stress axis organs and increased levels of cortisol and glucose in the blood plasma. These changes were the most pronounced in CEV-infected koi kept at 18°C. At both temperatures, the activation of antiviral immune response was observed in koi kept under freshwater and NaCl conditions upon CEV infection. Interestingly, a clear downregulation of the expression of adaptive immune genes was observed in CEV-infected koi kept under freshwater at 18°C. CEV induces a stress response and modulates adaptive immune response in koi, and this is correlated with the level of viral load and disease development.

Distribution of carp edema virus in organs of infected juvenile common carp.

The disease caused by carp edema virus (CEV) manifests with lethargy as a primary sign; this observation in koi in Japan gained the disease the name koi sleepy disease (KSD). In the years following the discovery of the virus in Japan, KSD cases have been noted in the UK in koi and common carp. Conducting research in order to expand knowledge of the processes of distribution of CEV in infected fish organs will be helpful for eradication and diagnostic purposes. Carp edema virus-affected fish with clinical signs of KSD were experimentally cohabited with common carp fry (30 fish). Three fish were euthanised by bath in a 0.5 g L-1 tricaine solution at one week intervals (7, 14, 21 and 28 days post cohabitation). Tissue samples from the brain, gills, spleen, kidney, intestines and skin were collected, and the total DNA was extracted and tested by real-time PCR. By the seventh day post infection, CEV DNA was most often found in the skin, gills and brain and less frequently in the kidney and intestines. In many of the common carp fry, CEV DNA could typically be found in several organs of each individual fish, although it was only found in one sample of spleen tissue. In this experimental study the pathogenesis of the CEV infection process was shown, the high infectivity of CEV was confirmed and the best organs were determined for sampling in CEV-infection experimentation. The real-time PCR method used in our cohabitation experiments was shown to be useful at the clinical and asymptomatic stage of virus infection.

Immune responses in carp strains with different susceptibility to carp edema virus disease.

Carp edema virus disease (CEVD), also known as koi sleepy disease (KSD), represents a serious threat to the carp industry. The expression of immune-related genes to CEV infections could lead to the selection of crucial biomarkers of the development of the disease. The expression of a total of eleven immune-related genes encoding cytokines (IL-1β, IL-10, IL-6a, and TNF-α2), antiviral response (Mx2), cellular receptors (CD4, CD8b1, and GzmA), immunoglobulin (IgM), and genes encoding-mucins was monitored in gills of four differently KSD-susceptible strains of carp (Amur wild carp, Amur Sasan, AS; Ropsha scaly carp, Rop; Prerov scaly carp, PS; and koi) on days 6 and 11 post-infection. Carp strains were infected through two cohabitation infection trials with CEV genogroups I or IIa. The results showed that during the infection with both CEV genogroups, KSD-susceptible koi induced an innate immune response with significant up-regulation (p<0.05) of IL-1β, IL-10, IL-6a, and TNF-α2 genes on both 6 and 11 days post-infection (dpi) compared to the fish sampled on day 0. Compared to koi, AS and Rop strains showed up-regulation of IL-6a and TNF-α2 but no other cytokine genes. During the infection with CEV genogroup IIa, Mx2 was significantly up-regulated in all strains and peaked on 6 dpi in AS, PS, and Rop. In koi, it remained high until 11 dpi. With genogroup I infection, Mx2 was up-expressed in koi on 6 dpi and in PS on both 6 and 11 dpi. No significant differences were noticed in selected mucin genes expression measured in gills of any carp strains exposed to both CEV genogroups. During both CEV genogroups infections, the expression levels of most of the genes for T cell response, including CD4, CD8b1, and GzmA were down-regulated in AS and koi at all time points compared to day 0 control. The expression data for the above experimental trials suggest that both CEV genogroups infections in common carp strains lead to activation of the same expression pattern regardless of the fish's susceptibility towards the virus. The expression of the same genes in AS and koi responding to CEV genogroup IIa infection in mucosal tissues such as gill, gut, and skin showed the significant up-regulation of all the cytokine genes in gill and gut tissues from koi carp at 5 dpi. Significant down-regulation of CD4 and GzmA levels were only detected in koi gill on 5 dpi but not in other tissues. AS carp displayed significant up-expression of Mx2 gene in all mucosal tissues on 5 dpi, whereas in koi, it was up-regulated in gill and gut only. In both carp strains, gill harbored a higher virus load on 5 dpi compared to the other tissues. The results showed that resistance to CEV could not be linked with the selected immune responses measured. The up-regulation of mRNA expression of most of the selected immune-related genes in koi gill and gut suggests that CEV induces a more systemic mucosal immune response not restricted to the target tissue of gills.

Characteristics of pathology and transcriptome profiling reveal features of immune response of acutely infected and asymptomatic infected of carp edema virus in Koi.

Koi sleepy disease (KSD) is a high mortality and infection viral disease caused by carp edema virus (CEV), which was a serious threat to aquaculture of common carp and export trade of Koi worldwide. Asymptomatic infection is an important cause of the difficulty in preventing KSD and its worldwide spread, because asymptomatic infection can be activated under appropriate condition. However, the understanding of the molecular correlates of these infections is still unknown. The purpose of this study was to compare the pathology change, enzyme activity, immunoglobulin activity, host and viral gene expression differences in acutely infected and cohabiting asymptomatic Koi infected with CEV. Healthy Koi were used as a control. The gross pathology, histopathology and ultrastructural pathology showed the difference and characteristics damage to the tissues of Koi under different infection conditions. Periodic Acid-Schiff stain (PAS), enzyme activity and immunoglobulin activity revealed changes in the immune response of gill tissue between acutely infected, asymptomatic infected and healthy Koi. A total of 111 and 2484 upregulated genes and 257 and 4940 downregulated genes were founded in healthy Koi vs asymptomatic infected Koi and healthy Koi vs acutely infected Koi, respectively. Additionally, 878 upregulated genes and 1089 downregulated genes were identified in asymptomatic vs. acutely infected Koi. Immune gene categories and their corresponding genes in different comparison groups were revealed. A total of 3, 59 and 28 immune-related genes were identified in the group of healthy Koi vs asymptomatic infected Koi, healthy Koi vs acutely infected Koi and asymptomatic infected Koi vs acutely infected Koi, respectively. Nineteen immune-related genes have the same expression manner both in healthy Koi vs acutely infected Koi and asymptomatic Koi vs acutely infected Koi, while 9 immune-related genes were differentially expressed only in asymptomatic Koi vs acutely infected Koi, which may play a role in viral reactivation. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative reverse transcription PCR (RT-qPCR), and the results were consistent with the RNA-Seq results. In conclusion, the data obtained in this study provide new evidence for further elucidating CEV-host interactions and the CEV infection mechanism and will facilitate the implementation of integrated strategies for controlling CEV infection and spread.

Carp edema virus in Ukraine – The evidence for the furthest east presence of CEV genogroup I in Europe

Carp edema virus (CEV) is a poxvirus infecting gills of common carp (Cyprinus carpio) and causes a deadly disease called koi sleepy disease (KSD). Currently thanks to higher vigilance and screening for its presence the virus was confirmed to be widespread on the European continent and affects both ornamental koi, farmed and feral common carp. Phylogenetic research of CEV is however in its infancy. Until recently due to the lack of available sequence information, the phylogenetic analysis was limited to a p4a gene. Based on this gene two main clades were established. The genogroup I clade is of European origin and can be found in farmed or feral common carp in Europe. However, the eastern border of the geographic range of CEV from genogroup I is not yet established, therefore we decided to obtain samples from clinically affected carp from Ukraine. Furthermore, we used sequences of the DNA binding viral core protein 8 (VP8) and of the uracil DNA glycosylase (UDGS) to confirm the existence of the genogroups I and II. The results indicate that CEV is highly prevalent in Ukraine. Phylogenetic analysis using the p4a gene showed that common carps in Ukraine are infected with CEV genogroup I. The two additional genes used for the phylogenetic analysis supported the phylogenetic findings. This confirms that common carps in continental Europe are hosts for CEV from the genogroup I and the geographic range for this virus is reaching to the most eastern parts of Ukraine.

Fish immune responses to natural infection with carp edema virus (Koi sleepy disease): An emerging fish disease in India

Emerging pathogen, carp edema virus (CEV) causes koi sleepy disease (KSD) in Koi and common carp causing severe mortalities worldwide. In the present study, a total of 150 fish species belonging to eight different families were sampled from the ornamental fish retailers and farms, located in Karnataka, India. The OIE protocol viz., level-I, II and III diagnoses confirmed the infection of CEV in 10 koi fish. Interestingly, other fish species belonging to different fish family including cyprinidae family were negative to CEV. Further, CEV infection was confirmed by sequencing (partial 4a gene); it showed the similarity with that of CEV reported from India and Germany strains with similarity of 97.4–99.94% and belonged to genogroup IIa. TEM analysis of purified CEV, in vivo cohabitation and tissue infection experiments confirmed the CEV infection. In addition, viral load was significantly higher (106−7 copies) in koi collected from Dakshina Kannada than of Bengaluru (103−4 copies). To understand the host–pathogen interaction, different organs such as gill, kidney, liver and spleen from naturally (CEV) infected koi were used to study the immune gene responses by using eight innate and one adaptive immune response. Results indicated that TNF-α, RohTNF-α, iNOS, IFN-γ and IL-10, and catalyze β-2M of MHC class I pathway genes were upregulated in koi. Higher expression of immune genes during the CEV infection may have inhibited viral replication and mount an antigenic adaptive response. Similar to other viral infections, interferon-γ play an important role during poxvirus infections. Quantification of immune genes in infected fish will provide insights into the host responses and provide valuable information to devise intervention strategies to prevent and control disease due to CEV.

Detection and phylogenetic analysis of carp edema virus in common carp (Cyprinus Carpio) in Iran; 2020–2021

Carp edema virus (CEV), a member of the family Poxviridae, is the etiologic agent of carp edema virus disease, also known as the koi sleepy disease (KSD). The disease is important across the world because it increases mortality rates in common carp and koi carp. From autumn 2020 to spring 2021, gills and internal organs were collected from suspected common carps cultured in twenty Iranian farms in Khuzestan, Gilan, and Mazandaran provinces. The presence of CEV was confirmed by molecular diagnosis. PCR was performed using specific primers to amplify the p4a gene, and positive samples were subsequently sequenced. Phylogenetic analysis assigned them to CEV genogroup I, and they shared high nucleotide sequence similarity with CEVs from Italy, Hungary, Iraq, Poland, the United States, and the Czech Republic. The results confirmed the presence of CEV in Iranian freshwater aquaculture after five years of observing clinical symptoms strongly reminiscent of CEV disease. The results also warned of the spread of the virus throughout the continent and point to the need to implement restrictions on the fish trade. Further studies are needed to determine the full-genome sequences of circulating viruses in order to provide insights into the epidemiology of fish viruses in Iran and the region.

It is everywhere-A survey on the presence of carp edema virus in carp populations in Germany.

Carp edema virus (CEV) is the causative agent of koi sleepy disease (KSD), a serious gill disease affecting common carp, Cyprinus carpio, and its ornamental variety, koi. After recent detections of the virus in various countries around the world, KSD has emerged as a new global disease in carp. However, the prevalence of the infection in carp populations in a given geographical region has not been studied thoroughly. The present communication reports an investigation into the presence of CEV in carp and koi populations in Germany. For this purpose, gill samples collected from carp and koi populations suffering from gill diseases or collected for a routine examination of their health status were tested for the presence of CEV by PCR. In total, 651 fish samples from 401 carp or koi cases were examined in 2015 and 2016, additional 118 samples from previous studies were included in the examination. CEV was detected in archive samples from carp dating back to 2007, and in koi samples dating back to 2009. From 2015 to 2016, CEV was detected in 69% of cases from carp populations examined from the main carp-producing areas in Germany, and in 41% of the examined cases from koi populations from all over Germany. Clinical KSD occurred mainly from April to June in carp populations at water temperatures ranging from 8 to 12°C and in koi populations at water temperatures ranging from 18 to 22°C. Most fish from clinically affected carp or koi populations harboured high virus loads of above 10,000 copies of CEV-specific DNA per 250 ng DNA, while gills from fish of other fish species from the ponds, including goldfish, grass carp and European perch were found CEV negative or harboured a low virus load. A phylogenetic analysis revealed the presence of multiple CEV variants from genogroup I in carp and genogroup II in koi populations in Germany. Genetically identical genogroup I isolates were detected in carp from different geographical locations in Germany and in other European carp populations. Some German genogroup II variants were identical to variants previously recorded from koi in Asian and other European countries. The data presented here show that CEV is highly prevalent in German common carp and koi populations and implies the spreading of this virus by intense trading of common carp and koi without necessary risk mitigating measures. As infections with this virus may induce serious disease, CEV diagnostic should be included in health surveillance and disease monitoring programmes.

Genetic diversity of the carp oedema virus in France.

The koi sleepy disease of carp caused by the carp oedema virus (CEV) was observed on farms and in ponds in France since the 2010s. Samples of CEV collected in France over a period of eight years were characterized at the molecular level by sequencing the partial p4a gene. All the sequences, except one, fell into two well-defined genogroups. Sequences obtained from CEV detected in common carp generally clustered in genogroup I and sequences from CEV detected in the koi were assigned to genogroup II. A particular sample was different to the others and represented a putative new genogroup possibly arose from a recombination event between a genogroup II sequence and one from an unknown genogroup. Compared with sequences from CEV of other countries, most of the French sequences exhibited high degree of DNA identities with those published previously, indicating identical sources of viruses. The sequence diversity suggests multiple introductions of the viruses in France. Among the French sequences, two genogroup-specific molecular markers were identified. One was an insertion/deletion identified within a microsatellite and other was a group of single nucleotide polymorphisms. CEV seems to generate genetic diversity via diverse mechanisms: substitutions, indels and recombination events.

Koi sleepy disease as a pathophysiological and immunological consequence of a branchial infection of common carp with carp edema virus

ABSTRACT Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, “koi sleepy disease” (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L−1, and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L−1. At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

Complete Genome Sequence of Carp Edema Virus Isolated from Koi Carp.

Carp edema virus disease (CEVD), or koi sleepy disease, is caused by CEV. Here, we report the complete genome sequence of CEV strain FTI2020, isolated from koi carp. This sequence information has great potential for improving our understanding of the genetic characteristics of this piscine poxvirus.

A Seasonal Study of Koi Herpesvirus and Koi Sleepy Disease Outbreaks in the United Kingdom in 2018 Using a Pond-Side Test.

Simple SummaryCyprinid herpesvirus (CyHV)-3 and carp edema virus (CEV), the causative agents of koi herpesvirus disease and koi sleepy disease, respectively, are emerging DNA viruses infecting koi and common carp. Similarities in their clinical presentation present difficulties for its on-site identification based on gross pathology. Fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for detecting CyHV-3 and CEV DNA were designed to use border inspection posts and local testing by national authorities for outbreak control. The limit of these tests’ detection (102 and 103 viral copies for CyHV-3 and CEV, respectively) allows for the amplification of viral DNA in clinical samples in less than 20 min. The assays’ field performance was tested with 63 common carp mucus swabs taken during disease investigations in 2018, and the results validated with the reference laboratory analysis. Overall, the good performance, ease of use, and cost-effectiveness of these tests make them good candidates for a point of care test. However, further work is required to incorporate reliable internal controls and improve the sensitivity of these tests’ asymptomatic testing.Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. The limit of detection was 102 and 103 viral copies under 25 min for CyHV-3 and CEV, respectively. The specificity of the CyHV-3 LAMP assay was 95.6% of 72 fish herpesviruses tested. Sixty-three non-lethal common carp mucus swabs were collected across 16 sites during disease investigations. DNA extractions were performed in under 10 min using the QuickExtract™ digestion buffer. The LAMP amplification of CyHV-3 DNA in mucus swabs from clinical cases was detected from 4 to 13 min in 13 sites, while a co-infection of CyHV-3 and CEV was confirmed by LAMP in a single site. The LAMP results agreed with the results of the reference laboratory. The common carp ef1a was amplified only in 61% of the mucus swabs collected, preventing its use as a robust internal control to distinguish false negatives from invalid tests. After further optimization, these tests could be implemented for border inspection posts surveillance and decentralizing testing during disease outbreaks.

Carp edema virus and immune response in carp (Cyprinus carpio): Current knowledge.

The importance of world aquaculture production grows annually together with the increasing need to feed the global human population. Common carp (Cyprinus carpio) is one of the most important freshwater fish in global aquaculture. Unfortunately, carp production is affected by numerous diseases of which viral diseases are the most serious. Koi herpesvirus disease (KHVD), spring viraemia of carp (SVC), and during the last decades also koi sleepy disease (KSD) are currently the most harmful viral diseases of common carp. This review summarizes current knowledge about carp edema virus (CEV), aetiological agent causing KSD, and about the disease itself. Furthermore, the article is focused on summarizing the available information about the antiviral immune response of common carp, like production of class I interferons (IFNs), activation of cytotoxic cells, and production of antibodies by B cells focusing on anti-CEV immunity.

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 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

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.