Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 11 WORKSHOP Novel viral inections threatening Cyprinid sh O. Haenen1*, K. Way2*, B. Gorgoglione3, T. Ito4, R. Paley2, L. Bigarré5 and T. Walek6* 1 Central Veterinary Institute of Wageningen UR, The Netherlands; 2 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth DT4 8UB, England; 3 Clinical Division of Fish Medicine, ¢ȱȱ¢ȱǰȱ§ĵȱŗǰȱŗŘŗŖȱǰȱDzȱ4 Tamaki Laboratory, Aquatic Animal Health Division, National Research Institute of Aquaculture (NRIA), Fisheries Research ¢ǰȱȱśŗşȬŖŚŘřȱ Dzȱ5ȱǰȱ£·ǰȱDzȱ6 College of Veterinary Medicine, Department ȱ ȱȱȱ¢ǰȱ¢ȱȱǰȱ ǰȱȱřŘŜŗŗȬŖŞŞŖǰȱ * Workshop co-organizer Introduction In 2007 koi herpesvirus disease (KHVD), caused an open workshop was organized at the 17th by the alloherpesvirus Cyprinid herpesvirus 3 EAFP International Conerence on Diseases o (CyHV-3), was listed by the World Organiza- Fish and Shellsh. Seven short lectures were ol- tion or Animal Health (OIE) and was quickly lowed by a discussion, involving an audience o ollowed by listing as a non-exotic disease in the approximately 60 international experts. New eld European Union (EU), related to the Directive observations and preliminary research on novel 2006/88/EC (EC, 2006). Up until the listing o cyprinid viral inections were presented, aiming KHVD, Spring Viraemia o Carp (SVC) was to illustrate their impact in various geographic the only viral disease o cyprinids listed by the regions. The main topics presented included OIE (OIE, 2012). issues related to the current emergence and detec- tion o Cyprinid iridoviruses, Carp Edema Virus Apart rom CyHV-3 and SVCV, in the last ve (CEV), Cyprinid Herpesvirus 2 (CyHV-2), and a years, several novel non-notiable cyprinid novel myxo-like virus. Additionally, the promo- viruses have been detected in Europe and their tion o a collaborative approach to be undertaken relation to clinical disease and mortality out- or the genetic characterization o novel cyprinid breaks is a cause or concern. viruses was highlighted. The general aim o the workshop was to identiy potential collaborative To gain a comprehensive overview o current approaches to carry out multidisciplinary studies potential new viral threats or cyprinid sh, and aiming to dene risks, diagnostic methods and to provide an update on their characterization, suggest adequate prophylactic measures. * Corresponding author and workshop leading organizer’s email: [email protected] 12, Bull. Eur. Ass. Fish Pathol., 36(1) 2016 Overview of Carp Edema Virus in A eature o CEV/KSD outbreaks in common Europe carp in Europe, and particularly in the UK, is K Way and DM Stone that many have occurred during periods o low water temperatures (6-9°C) in winter and Carp Edema Virus (CEV) is the etiological agent early spring. Outbreaks o KSD in koi ponds o Koi Sleepy Disease (KSD), originally de- are generally seen at higher temperatures (15- scribed in Japan in the 1970’s as a viral edema 25°C) in late spring or early summer. In the UK o juvenile color Cyprinus carpio koi and later a link with Spring Carp Mortality Syndrome morphologically identied as a poxvirus. The (SCMS) was suspected (Way and Stone, 2013). virus causes severe damage to gill lamellae, When archived DNA and preserved gill tissue leading to hypoxia and lethargy, which man- samples, including paran-embedded ormalin iests as sleepy behavior, and mortality can xed gill tissue, were analyzed by PCR, CEV reach 80-100% (Ono et al., 1986; Lewisch and was detected in samples rom SCMS cases rom Gorgoglione et al., 2015). 2004 and 1998/99. The disease is known to be widespread across CEV/KSD is the latest example o a virus disease Japan where koi are cultured and, as a conse- o cyprinids that has emerged, largely unno- quence, the disease has been managed rather ticed, in Europe and where the true extent o than aempting to eradicate the virus in koi the spread o the virus is not known. The cir- populations. International trade in koi has un- cumstances o the emergence and spread o doubtedly led to global spread o CEV/KSD but CEV/KSD are similar to those o KHVD almost the occurrence o disease outbreaks has oten 20 years ago. not been recognized and consequently, rarely reported. In the USA, CEV has been detected Possible reasons or why this virus has only in association with disease outbreaks in koi at rarely been identied in carp mortality inves- import sites and in hobby ponds since 1996 tigations include: 1) the virus is, at present, (Hesami et al., 2015). In Europe, outbreaks o non-culturable and not isolated during routine KSD and PCR detections o CEV-like virus screening; 2) the disease may not always were reported rom 2009. At a CEV workshop cause high mortalities (extensive outbreaks) in Copenhagen in January 2015 (meeting report in common carp, and 3) CEV/KSD is possibly available rom hp://www.eurl-sh.eu/reports), misdiagnosed as KHVD without conrmation delegates rom Austria, Czech Republic, France, o the true cause. Also, until recently, ew ade- Germany, Italy, The Netherlands and the UK quate diagnostic tools have been available. In reported multiple KSD outbreaks in imported the USA in 1996 the virus was identied in koi koi and also outbreaks in common carp. Follow- by transmission electron microscopy (TEM) ing the workshop inormation was received on (Hedrick et al., 1997). KSD/CEV cases in Poland. A CEV alert paper giving the emergence o CEV in Europe and The knowledge gaps that, it is suggested, need its signicance to European aquaculture is cur- to be addressed to assess the true impact o the rently in preparation (Keith Way, pers.comm.). disease on European carp populations include: Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 13 1) the mechanisms o virus survival outside the negative trade eects, threatening the high-val- host; 2) are there susceptible aquatic species ued ornamental sh industry and carp arming. other than carp (carriers); 3) does the virus exist as a low-level persistent inection and are there In Austria, the occurrence o KSD was demon- aquatic vectors; 4) what is the prevalence in strated or the rst time during the spring o carp populations; 5) what are the important 2014, rom two unrelated cases rom common environmental actors responsible or triggering carp and koi carp (Lewisch and Gorgoglione et CEV/KSD outbreaks al., 2015). In 2015, the rst Austrian case arrived in late February rom a spontaneous KSD out- More tools are needed to provide early warning break aecting koi rom a large garden pond o emerging pathogenic viruses o aquaculture (> 250 m3), which allowed preliminary trans- and resolve aquatic/sh diseases o unknown mission and susceptibility investigations to be aetiology. Possible improvements include: 1) carried out. A marked dierential susceptibility a beer characterization o known viruses and was seen between various individuals rom the more use o next generation sequencing, partic- same pond, providing evidence o clinically ularly to allow eective design o generic PCR asymptomatic carriers. An adult koi (20 cm primers that detect a range o DNA viruses; total body length) was hospitalized at 17 °C 2) development o qPCR assays or sample showing a severe inection with the ectopar- screening; 3) beer mechanisms in Europe asites Trichodina sp. and Gyrodactylus sp., and (with support rom the EU) or collaboration multiple skin inections with opportunistic bac- and networking to tackle serious or potentially teria (eg Shewanella putrefaciens). Surprisingly, serious epizootics. 6 hours cohabitation was enough to transmit the CEV inection to other, nave koi and SPF common carp, showing typical clinical paerns Preliminary investigations on and mortality by day 7 post-inection (d.p.i.), transmission of and susceptibility to (Figure 1). CEV viremia was already visible and Carp Edema Virus CEV / Koi Sleepy detectable at 1 d.p.i., associated with a sleepy Disease KSD behavior, and possibly showing highest DNA B Gorgoglione, E Lewisch, O Schachner and levels at 7 d.p.i. (see the thicker band at the gel M El-Matbouli electrophoresis: Figure 2). Koi carp are traded worldwide as valuable or- CEV was detected by PCR (using the nested namental sh, particularly appreciated as pets PCR protocol rom CEFAS, Stone Way, pers. in European garden ponds, which are highly comm. 2013), and was only detectable in DNA popular in Austria. Common carp Cyprinus rom specically skin mucus, gills laments carpio is oten armed in poly-cultural systems, and blood. Thereore, we suggest these tissues representing the main reshwater sh produc- are the best to be sampled or KSD diagnostics. tion in the world, especially relevant in Asian DNA rom internal organs (including rom parts and Central-East European countries (FAO, o the kidney (pronephros and mesonephros), 2014). The spreading o CEV/KSD may have spleen and swim bladder) gave aint positivity, 14, Bull. Eur. Ass. Fish Pathol., 36(1) 2016 Figure 1. KSD aected common carp, inected with CEV. The carp died at 7 d.p.i. ollowing cohabitation with a CEV-inected koi carp at 17°C. Clinical changes included emaciation, sunken eyes, mucus aggregations on the body and on the pale gills (let operculum removed). Figure 2. CEV detection in DNA samples rom a common carp (Figure 1), which died at 7 d.p.i. ollowing cohabitation with a KSD aected koi carp. Heparinised blood was sampled during sh hospitalization, while urther tissues were aseptically sampled at necropsy. Gel electrophoresis o amplicons ollowing the second step o a nested PCR (using CEFAS, Stone Way 2013 nested PCR protocol), with 478 bp band indicating positivity. Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 15 usually at the second step o the nested PCR, Discussion and exclusively rom sh in terminal condition T.