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Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

There is an impressive global trade in live tropical freshwater ornamental fish. These consignments may contain potentiallyharmful bacteria and contaminants of therapeutics, a potential public health risk when professionals have direct contact with fish and transport water. In 2014–2015 we sampled and tested fifty consignments from 13 countries outside Europe at arrival in the Netherlands. Potential zoonotic bacteria were detected in 11 of 50 ornamental fish consignments. Aeromonasspp. (n = 59) isolated from fish showed resistance to oxytetracycline (85 % of strains), flumequine (53 %), trimethoprim-sulphamethoxazole (30 %), neomycin (34 %), florfenicol (9 %), and to nitrofurantoin (17 %). Isolates from fish consignments from Singapore and Congo showed multi-resistance against various antibiotics. In total 11 Escherichia coliisolates suspected of ESBL (extended-spectrum beta-lactamase)-production were found in 2 of 50 freshwater ornamental fish and 9 of 50 transport water samples, from Singapore (4×), Indonesia (2×), Congo (2×), Thailand (1×), and Hong Kong Special Administrative Region ofthe People's Republic of China (HKSAR) (1×). OXA-48-like carbapenemase gene variants of limited public health riskwere frequently found in Shewanella spp. Forty-nine of fifty water samples contained residues of one or more antibiotics, mostly tetracyclines and fluoroquinolones, but also chloramphenicol and nitrofurans, and of malachite green. Our findings are of concern since the current EU border inspections for import control do not consider these human health risks. It is therefore recommended to regularly screen consignments from more countries for the presence of antimicrobial resistant bacteria, residues of antibiotics, and potential zoonotic bacteria.

A multi-laboratory study was performed to investigate the most suitable incubation time for susceptibility tests of fish pathogens Vibrio anguillarum and Vibrio vulnificus performed at 28 degrees C. An isolate set consisting of 30 V. anguillarum and 26 V. vulnificus was used by four participating laboratories in Denmark, France, Sweden, and the Netherlands. Inhibition zone diameters were determined by agar disc diffusion for eight agents and Minimum Inhibitory Concentration (MIC) values were determined for seven agents using the standard CLSI testing protocols for non-fastidious organisms that specify 24-28 h incubation. In this work an additional set of readings was made after 48 h incubation. In total, 1120 paired zone sizes and 399 paired MIC observations were made at the two incubation times. Examination of the data demonstrated that incubation time had a small but statistically significant effect on the numerical values of susceptibility measures. However, the effects of incubation time on the precision of the data sets and the categorisation of isolates based on the application of epidemiological cutoff values were slight and statistically non significant. These analyses suggest that the susceptibility of these Vibrio species could be established using protocols that specify either 24-28 h or 44-48 h incubation.This study does not provide evidence that prolonged incubation to 48 h improves the quality of data generated by the tests. Therefore, it is recommended that the existing standard CLSI protocols with 24-28 h at 28 degrees C should be adopted for susceptibility testing of V. anguillarum and V. vulnificus. This study was performed in the frame of CoVetLab partner Insitutes https://www.covetlab.org/c5/ peer-reviewed

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15–23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts., Sub-lineages of the fish-pathogenic bacterium Aeromonas salmonicida display specific host preferences.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV-3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV-3-specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV-3-infected carp. French CyHV-3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV-3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post-infection. The results suggest that this non-lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV-3 disease.

Open workshop at the 18th EAFP Conference, Belfast, UK, 4th September 2017; International audience; Worldwide, viral diseases pose a serious challenge to the fish farming industry and wild fish stocks. In order to prevent the spread of serious fish diseases and to contain and control fish disease outbreaks at a global scale, the World Organisation for Animal Health (OIE) has published relevant data on these fish diseases, both in the Aquatic Animal Health Code (OIE, 2017a), including the OIE listed fish diseases, and in the Manual of Diagnostic Tests for Aquatic Animals (OIE, 2017b), including recommended diagnostic methods. The list of viral OIE notifiable fish diseases currently includes: Epizootic haematopoietic necrosis disease (EHN), Infection with HPR-deleted or HPR0 infectious salmon anaemia virus (ISA), Infection with salmonid alphavirus, causing Pancreas disease (PD) and Sleeping disease (SD), Infectious haematopoietic necrosis (IHN), Koi herpesvirus disease (KHVD), Red sea bream iridoviral disease (RSIVD), Spring viraemia of carp (SVC), and Viral haemorrhagic septicaemia (VHS). At European Union level, Commission Decision 2006/88/EC and Council Implementing Decision 2015/1554/EC provide specific regulation for surveillance and control of listed infectious aquatic diseases in Europe which include the fish viral diseases VHS, IHN, EHN, ISA, and KHVD (European Commission 2006, 2015). Due to the frequent emergence of new serious fish viral diseases, the OIE list of fish viral diseases is regularly being extended. However, apart from these well studied viral diseases, other non-notifiable serious fish viral diseases occur in freshwater fish farming. To focus on these new viral threats for freshwater fish farming, an open workshop was organized at the EAFP Conference at Belfast, 4th September 2017. The workshop consisted of five short lectures and a discussion, involving an audience of 69 international experts, originating from 25 countries, of which 6 outside Europe. The main topics presented included issues related to:i) the difficulty of preventing the global spread of cyprinid herpesvirus 2 (CyHV-2), ii) perhabdoviruses as a threat for percid farming, iii) pathogenesis and diagnostics of piscine orthoreoviruses in farmed rainbow trout, iv) Carp Edema Virus (CEV) in Europe, and v) the potential role of fish endogenous retroviruses in disease emergence. The general aim of the workshop was to identify potential collaborative approaches to carry out multidisciplinary studies aiming to define risks, diagnostic methods and suggest adequate prophylactic measures.

Carp edema virus disease (CEVD), also known as koi sleepy disease, is caused by a poxvirus associated with outbreaks of clinical disease in koi and common carp Cyprinus carpio. Originally characterised in Japan in the 1970s, international trade in koi has led to the spread of CEV, although the first recognised outbreak of the disease outside of Japan was not reported until 1996 in the USA. In Europe, the disease was first recognised in 2009 and, as detection and diagnosis have improved, more EU member states have reported CEV associated with disease outbreaks. Although the structure of the CEV genome is not yet elucidated, molecular epidemiology studies have suggested distinct geographical populations of CEV infecting both koi and common carp. Detection and identification of cases of CEVD in common carp were unreliable using the original PCR primers. New primers for conventional and quantitative PCR (qPCR) have been designed that improve detection, and their sequences are provided in this paper. The qPCR primers have successfully detected CEV DNA in archive material from investigations of unexplained carp mortalities conducted > 15 yr ago. Improvement in disease management and control is possible, and the principles of biosecurity, good health management and disease surveillance, applied to koi herpesvirus disease, can be equally applied to CEVD. However, further research studies are needed to fill the knowledge gaps in the disease pathogenesis and epidemiology that, currently, prevent an accurate assessment of the likely impact of CEVD on European koi and common carp aquaculture and on wild carp stocks.

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis in goldfish Carassius auratus auratus. However, the virus has also been detected in Prussian carp C. gibelio and crucian carp C. carassius from European and Asian countries. To prevent spread of the causative virus to other areas, investigation of the risk factors of spread of this virus is important. In this study, 8 batches of goldfish imported into the Netherlands by airfreight from Asia and the Middle East were investigated for the presence of the virus. CyHV- 2 DNA was detected by PCR in the pooled kidneys of 4 of the 8 imported goldfish batches, of which 1 was from a CyHV-2 disease case at a Dutch importer's quarantine facility. Sequence analysis of the CyHV-2 strains from this study and from previous reports showed that there were at least 6 different lengths in the mA region, resulting in tentatively at least 4 genotypes. Virus isolation was positive for only 1 (Amsterdam Schiphol-1 [AMS-1]) of the 8 samples. It was shown that the AMS-1 isolate was highly virulent to Ryukin goldfish after 100.3 TCID50 fish-1 intraperitoneal injection. The viral titre of the AMS-1 isolate for goldfish fin cells at several temperatures was similar to that of a Japanese CyHV-2 isolate. Our results prove that one of the routes of spread of various CyHV-2 strains is through the global trade of apparently healthy infected goldfish.

Infectious hematopoietic necrosis (IHN) — a highly lethal infectious salmonid disease — has caused substantial economic losses in the European production of rainbow trout (Oncorhynchus mykiss) since the late 1980s. The causal agent of IHN is the IHN virus (IHNV) introduced from overseas. However, until today, its phylogeographic spread in Europe remains poorly understood. We therefore sought to elucidate this unresolved topic by using the largest ever compiled dataset of European IHNV isolates (E isolates) (193 GenBank E isolates and 100 isolates from this study) for the complete glycoprotein (G) gene sequence. Our results clearly revealed that the active trout trade has left its traces in the E phylogeny. For example, the spread by trade of IHNV-infected trout was apparently the cause for the exposure of the E lineage to different local scenarios of selection and genetic drift, and therefore has led to the split of this lineage into various subordinated lineages. Accordingly, we also found evidence for E isolates being mixed Europe-wide by cross-border introduction events. Moreover, there were indications that this propagation of the E lineage within Europe corresponded with an extensive and rapid spread event, already during or shortly after its formation. Finally, in accordance with the high substitution rate of IHNV determined by previous studies, our dataset indicates that the mean period of occurrence of a single E haplotype is typically not longer than one calendar year.

One of the most valuable aquaculture fish in Europe is the rainbow trout, Oncorhynchus mykiss, but the profitability of trout production is threatened by a highly lethal infectious disease, viral hemorrhagic septicemia (VHS), caused by the VHS virus (VHSV). For the past few decades, the subgenogroup Ia of VHSV has been the main cause of VHS outbreaks in European freshwater-farmed rainbow trout. Little is currently known, however, about the phylogenetic radiation of this Ia lineage into subordinate Ia clades and their subsequent geographical spread routes. We investigated this topic using the largest Ia-isolate dataset ever compiled, comprising 651 complete G gene sequences: 209 GenBank Ia isolates and 442 Ia isolates from this study. The sequences come from 11 European countries and cover the period 1971–2015. Based on this dataset, we documented the extensive spread of the Ia population and the strong mixing of Ia isolates, assumed to be the result of the Europe-wide trout trade. For example, the Ia lineage underwent a radiation into nine Ia clades, most of which are difficult to allocate to a specific geographic distribution. Furthermore, we found indications for two rapid, large-scale population growth events, and identified three polytomies among the Ia clades, both of which possibly indicate a rapid radiation. However, only about 4% of Ia haplotypes (out of 398) occur in more than one European country. This apparently conflicting finding regarding the Europe-wide spread and mixing of Ia isolates can be explained by the high mutation rate of VHSV. Accordingly, the mean period of occurrence of a single Ia haplotype was less than a full year, and we found a substitution rate of up to 7.813 × 10−4 nucleotides per site per year. Finally, we documented significant differences between Germany and Denmark regarding their VHS epidemiology, apparently due to those countries’ individual handling of VHS.

Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based bla OXA-48 -like variants in Shewanella spp., including two new alleles, bla OXA-514 and bla OXA-515 . Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae .

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put-and-take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put-and-take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co-infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV-positive farms. Mortalities related to IHNV were mostly

Diseases are an important cause of losses and decreased production rates in freshwater eel farming, and have been suggested to play a contributory role in the worldwide decline in wild freshwater eel stocks. Three commonly detected pathogenic viruses of European eel Anguilla anguilla are the aquabirnavirus eel virus European (EVE), the rhabdovirus eel virus European X (EVEX), and the alloherpesvirus anguillid herpesvirus 1 (AngHV1). In general, all 3 viruses cause a nonspecific haemorrhagic disease with increased mortality rates. This review provides an overview of the current knowledge on the aetiology, prevalence, clinical signs and gross pathology of these 3 viruses. Reported experimental infections showed the temperature dependency and potential pathogenicity of these viruses for eels and other fish species. In addition to the published literature, an overview of the isolation of pathogenic viruses from wild and farmed A. anguilla in the Netherlands during the past 2 decades is given. A total of 249 wild A. anguilla, 39 batches of glass eels intended for farming purposes, and 239 batches of farmed European eels were necropsied and examined virologically. AngHV1 was isolated from wild A. anguilla yellow and silver eels from the Netherlands from 1998 until the present, while EVEX was only found sporadically, and EVE was never isolated. In farmed A. anguilla AngHV1 was also the most commonly isolated virus, followed by EVE and EVEX.

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non-salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold-conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13-nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody-coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 104 CFU mL-1, and results were obtained within 45 min. The antibody-coated gold nanoparticles were stable for at least 2 months at 4°C. The immunoassay using antibody-coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.

Production of European flat oysters Ostrea edulis in the Netherlands has been hampered by the presence of the haplosporidian parasite Bonamia ostreae, which is now an enzootic species following its establishment after 1980. We analyzed histopathological data from annual shell- fish disease monitoring from 1988 to 2006 to quantify prevalence of B. ostreae in flat oyster stocks of the marine Lake Grevelingen. In addition, we estimated prevalence of B. ostreae on a monthly basis with field surveys in 2003. The parasite was detected with PCR, using specific primers for B. ostreae. Prevalence of B. ostreae was analyzed relative to O. edulis density, biomass and a range of environ- mental parameters. B. ostreae was detected in flat oysters throughout the year with a higher preva- lence in spring than in autumn, possibly due to termination of spawning and the onset of oyster growth in autumn. Although B. ostreae was detected in all oyster weight classes, prevalence was highest in the largest oysters in spring and declined disproportionately in autumn, possibly due to high mortality of large oysters before autumn, suggesting that prevalence depends on oyster age. Parasite prevalence was independent of oyster density and total biomass, but appeared to be higher after a warm autumn. Abundance of the flat oyster (infected or non-infected with B. ostreae) was neg- atively related to the temperature of the preceding period, suggesting that mortality in flat oysters increased at higher water temperatures. Furthermore, O. edulis appeared to be more susceptible to B. ostreae after years with lower food availability and lower salinities (

In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities. Bacteria were produced in growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs. 2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the drumfilter effluent was different from those found in the reactors. However, all major community components were present in the effluent and reactor broths. HRT influenced the bacteria community, resulting in a DGGE profile dominated by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT compared to 7 h HRT, where bands indicative of alpha-proteobacterial populations most closely related to Rhizobium and Shinella spp. were most abundant. Molasses influenced the bacterial community. It was dominated by an Aquaspirillum serpens-related population. Providing total ammonia nitrogen (TAN) in addition to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacterium mizutaii and Jonesia spp. It was concluded from these results that a 6-7 h HRT is recommended, and that the type of substrate is less important, and results in communities with a comparably low pathogenic risk.

A clinical infection in post-larval (glass) European eels Anguilla anguilla was successfully induced after artificial bath immersion with Herpesvirus anguillae (HVA), isolated from diseased European eel. HVA caused a clinical infection after 7 d post-inoculation (pi); virus was detected by polymerase chain reaction (PCR) from Day 1 pi; virus isolation was positive from Day 7 pi, and HVA antigen was detected by immunohistochemistry in gills and stomach from Day 4 pi. Tissue changes were found by histological examination in gills and skin from Day 4 pi. In general, there was good correlation in the timing of the clinical signs, PCR, virus isolation, immunohistochemistry and histopathology results, although PCR, histopathology and immunohistochemistry were the first positive tests. HVA was first detected in skin and stomach, followed by gills, and later heart and intestine, whereas HVA was detected persistently in gills and skin. Koch's postulates were fulfilled. For diagnosis of HVA infections, clinical pathology combined with virus isolation and/or PCR are recommended.

Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2–3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes ofAnguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.

Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.

Dover sole (Solea solea) is an obligate ectotherm with a natural thermal habitat ranging from approximately 5 to 27°C. Thermal optima for growth lie in the range of 20 to 25°C. More precise information on thermal optima for growth is needed for cost-effective Dover sole aquaculture. The main objective of this study was to determine the optimal growth temperature of juvenile Dover sole (Solea solea) and in addition to test the hypothesis that the final preferendum equals the optimal growth temperature. Temperature preference was measured in a circular preference chamber for Dover sole acclimated to 18, 22 and 28°C. Optimal growth temperature was measured by rearing Dover sole at 19, 22, 25 and 28°C. The optimal growth temperature resulting from this growth experiment was 22.7°C for Dover sole with a size between 30 to 50 g. The temperature preferred by juvenile Dover sole increases with acclimation temperature and exceeds the optimal temperature for growth. A final preferendum could not be detected. Although a confounding effect of behavioural fever on temperature preference could not be entirely excluded, thermal preference and thermal optima for physiological processes seem to be unrelated in Dover sole.

Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.

Eel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r2-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel.

The worldwide decline of the eel population is thought to be caused by several factors, among which eel diseases. To investigate diseases of European silver eels Anguilla anguilla in the Netherlands, in Nov–Dec 2004 12 silver eels, and in Aug–Dec 2005 80 eels were caught in downstream parts (rivers) of the River Rhine and in Lake IJsselmeer. The eels were measured and weighed, necrotized, and individually checked for presence of external and internal parasites, bacterial infections and viruses, and blood smears were made for haematology. This is the first multidisciplinary study of the health of Dutch silver eels. In the small (2 × 6 eels) pilot study of 2004 in the River Rhine and Lake IJsselmeer respectively, most eels showed aspecific fin haemorrhages, some had ectoparasites, nearly none had parasites in the intestine, half of the groups had Anguillicola crassus in their swimbladder, only few had Trypanosoma 's in their blood, and no primary virus or bacterial infections were found, although one eel from Lake IJsselmeer was positive for anguillid herpesvirus 1 (AngHV-1, former Herpesvirus anguillae, HVA) in the PCR test only. The blood of all 6 and 2/6 of the eels respectively was considered abnormal, and the eels had a proper condition. In 2005, in 50 eels from the River Rhine and 30 from Lake IJsselmeer respectively, again aspecific fin haemorrhages were often seen, some of the eels had ectoparasites and parasites in the intestine, most eels had A. crassus in their swimbladder, 32% and 53% had Trypanosoma 's in their blood, from 44% and 13% of the eels AngHV-1 was isolated, and 44% and 27% were tested positive by PCR, with a peak in August, 10% of both groups of eels had an internal bacterial infection, mostly due to Aeromonas spp. The blood of about half of the eels was considered abnormal, but the eels had a proper condition. It was concluded, that the silver eels of this study had a proper Fulton condition factor (values 2.00–2.26), with aspecific fin haemorrhages, often were Trypanosoma -, A. crassus- and AngHV-1-infected, dependent on the season, and often showed an abnormal haematology. A. crassus causes injuries and is a chronic stress factor, more than the other parasites, which were mostly found less prevalent. In fact, lymphocytosis was directly related to A. crassus infection. Stress and injuries by A. crassus might induce disease through the presence of virus (AngHV-1), relevant in the health status of the silver eels during their spawning migration if ambient water temperatures would enhance a clinical infection of AngHV-1 disease. Moreover this virus might potentially decrease the survival of the silver eels by itself, because spawning migration to the Sargasso Sea takes wild eels to temperate/tropical areas in which the clinical infection by AngHV-1 is surely enhanced.

Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.

Three regions of koi herpesvirus (KHV) genomic DNA were compared for 34 samples from Japan, six from Indonesia, two from Taiwan, one from the Philippines, 13 from the Netherlands, one from the UK, one from the USA and one from Israel. The analyzed genomic regions included known PCR-detection targets (Sphl-5, 9/5 and the thymidine kinase gene). The KHVs from Asian countries were very homogeneous, although two variants were noted based on a single nucleotide polymorphism. In contrast, seven variants were found in KHVs from outside of Asia, and although closely related to one another, they were clearly distinct from those from Asian. The results suggest that a clear genetic distinction exists between Asian and European (including each single isolate from the USA and Israel) types of KHV, and that unique types of KHV were independently introduced or emerged in the respective geographic locations.

KOI HERPES VIRUS WORLD WIDE: RESULTS OF GLOBAL KHV QUESTIONNAIRE 2007-2009 O. Haenen*,1 and N. J. Olesen2 (and our colleagues who completed the questionnaire) 1 Central Veterinary Institute of Wageningen UR, NRL for Fish and Shellfish Diseases, P.O. Box 65, 8200 AB Lelystad, The Netherlands. 2 National Veterinary Institute, Technical University of Denmark (DTU), Hangøvej 2, DK-8200 Århus N, Denmark EPIZONE is a big EU network of excellence project within FP6 (www.epizone-eu.net), with 20 partners from Europe, and China, Turkey, FAO and DiVa. Its mission is to improve research on preparedness, prevention, detection, and control of epizootic diseases within Europe to reduce the economic and social impact of future outbreaks of emerging/notifiable diseases, like Foot-and-mouth disease through increased excellence by collaboration. Within EPIZONE, Work package 6.1 covers emerging diseases of fish, including Koi Herpes virus (KHV), which causes the notifiable KHV disease (KHVD) in koi and carp (Cyprinus carpio). In Sept 2007, at the last EAFP Conference at Grado, results of the detailed EPIZONE questionnaire on KHV disease in 2006-2007 were presented. In March 2009 a follow up KHV questionnaire was sent to > 65 countries world wide. By the start of May 2009, 40 countries had responded, i.e. > 60%. The results of the KHV questionnaire will be presented as a poster, and hand-out. Questions of the questionnaire were about koi (1), cultured (2) and wild carp (3), all Cyprinus carpio: • Prevalence of KHV in your country: year of first detection;? Number of outbreaks in 2004-2009 in 1, 2 or 3? • Clinics: what clinical signs were present in KHV outbreaks ? • Outbreaks: Was there disease and mortality in small and/or big fish? • Diagnosis: Which diagnostic tests were used for KHV detection, screening and confirmation? Did/do you participate in the KHV PCR ring test of CEFAS (UK); • Susceptible fish species: Was KHV isolated from other species than koi/carp? • Latent carriers: Do you have any experience with latency of KHV in koi/carp? • Measures (stamping out, temp change, therapy) and effects in 1, 2, and 3? • Vaccination: Is a KHV vaccine used in your country? • Any research on KHV in your country and laboratory? • National legislation in your country? • Any Further points? A full updated literature list on KHV on request: please E-mail olga.haenen@wur.nl

In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities. Bacteria were produced in growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs. 2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the drumfilter effluent was different from those found in the reactors. However, all major community components were present in the effluent and reactor broths. HRT influenced the bacteria community, resulting in a DGGE profile dominated by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT compared to 7 h HRT, where bands indicative of alpha-proteobacterial populations most closely related to Rhizobium and Shinella spp. were most abundant. Molasses influenced the bacterial community. It was dominated by an Aquaspirillum serpens-related population. Providing total ammonia nitrogen (TAN) in addition to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacterium mizutaii and Jonesia spp. It was concluded from these results that a 6-7 h HRT is recommended, and that the type of substrate is less important, and results in communities with a comparably low pathogenic risk.

We show that European eels infected with the rhabdovirus EVEX (Eel Virus European X) virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1000-1500 km. In contrast, virus-negative animals swam 5500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. Virus-positive eels showed a decline in hematocrit, which was related to the swim distance. Virus-negative eels showed a slightly increased hematocrit. Observed changes in plasma lactate dehydrogenase (LDH), total protein and aspartate aminotransferase (AAT) are indicative of a serious viral infection. Based on these observations, we conclude that eel virus infections may adversely affect the spawning migration of eels, and could be a contributing factor to the worldwide decline of eel.

Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation.

Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.

Herpesvirus of eel Herpesvirus anguillae (HVA) was isolated repeatedly from farmed eel of an outwardly healthy stock, but virus isolation was much greater in an experimental group of fish that were injected with dexamethasone. The results suggest that HVA can establish a latent infection in eel. Previous exposure of these eels to HVA virus was shown by detection of HVA-specific antibodies. These eels did not show clinical signs after a secondary infection with HVA. Tracing of seropositive eel stocks, which had previous contact with HVA, and of HVA carrier fish can be useful to control disease outbreaks due to HVA infection.

Dust and endotoxin exposure was measured in the Spring and Autumn in 96 veterinary practitioners, subdivided in four professional groups ((predominantly working with either swine, cattle, poultry, or companion animals). Furthermore, peak expiratory flow (PEF) variability was measured four times a day over a week in Spring and Autumn in order to detect occupation-related respiratory disorders. The participants kept a log book in which they recorded their activities during these periods. Practitioners working with swine, cattle, or poultry had a significantly higher exposure to dust (+/- 5 times) and endotoxin (+/- 10 times) than companion animal practitioners. PEF variability measurements gave no indications for an increased prevalence of bronchial hyperreactivity related to occupational activities. Respiratory protective devices (RPD) were used selectively. Only a small proportion of the practitioners used them at any time when entering a animal confinement building. Swine and cattle practitioners worked for approximately 3.6 hours per day in animal buildings, whereas poultry practitioners worked in the environment of poultry buildings for approximately 2.8 hours per day.

La migration des larves du 3eme stade de A. c. et les effets pathologiques ont ete etudies chez A. a. apres l'ingestion de P. f. (hote intermediaire infeste par des larves de A. c.). L'histologie des anguilles et des parasites a ete examinee a differents moments apres l'infestation

Pharmacokinetics, metabolism and clearance of sulphadimidine (SDM) were studied after a single intraperitoneal injection of SDM in carp at 20 degrees C. SDM was acetylated and hydroxylated to a small extent. The main metabolite was N4-acetyl derivative amounting only 2% of the total drug dose excreted; hydroxylation was less important (0.41% of the dose). The elimination half-life for SDM in carp was 17.5 h. The clearance values for SDM and its metabolites were equivalent. The importance of pharmacokinetic studies in different fish species is discussed.

The Planning Group on the Monitoring of Eel Quality met at the Research Institute Nature and Forest (INBO) Brussels, Belgium, on the 20–22 January 2015 during its first Workshop (WKPGMEQ) under the subject “Development of standardised and harmonised protocols for the estimation of eel quality”, chaired by Claude Belpaire (Belgium) and Olga Haenen (The Netherlands). There were 31 participants (21 attendees and 10 remote participants) representing 13 countries.Reliable assessment of the eel stock quality and its quantitative effect on the reproductive stock is currently not possible, due to insufficient spatial and temporal data coverage (ICES 2009). This has emphasised the urgent need to establish a comprehensive overview with improved spatial coverage of the quality of the eel population across Europe as an essential and urgent requirement. Understanding the reproductive potential of the international spawning stock is a key component to predicting the effects on stock recovery of changes to silver eel escapement, arising from management actions implemented within Eel Management Plans.To address this need, ICES 2012 recommended that Member States implement routine monitoring of lipid levels, contamination and diseases. Many countries have started compiling data on the health status of eels in their water bodies. Objectives for these monitoring actions are diverse and are not restricted to the framework of eel recovery. Eel quality is also monitored for different purposes, which include human health considerations and to meet requirements of the Water Framework Directive. Hence, there is a large amount of information collected by EU member countries. However, procedures with respect to sampling, analysis and reporting are not harmonised, hindering stock wide assessments and risking inefficient use of resources. Consequently, ICES (2009) identified the need to develop standardised and harmonised protocols for the estimation of eel quality, so that national data would be comparable between Member States and could be reliably incorporated in international stock assessments.The objective of WKPGMEQ was to document standardised and harmonised protocols for the estimation of the quality of the European eel Anguilla anguilla, with regard to the bioaccumulation of contaminants and the presence of diseases, including parasites.WKPGMEQ took advantage of the preparative work of the participants who in advance of the workshop drafted reports describing the framework and methods used in their countries for the assessment of contaminants and diseases in the eel. At the start of the meeting Member States’ country reports were presented. Two subgroups, covering contaminants and diseases respectively, further discussed the practical issues surrounding the sampling, assessment procedures, diagnostic approaches and reporting related to measuring contaminants and diagnosis of eel parasites and other diseases. As far as possible, common procedures and guidelines were described.The report starts with an overview of the current eel quality assessments in the Member States, and further discusses general issues on sampling of eel quality assessments. It includes a chapter on the assessment of eel condition in terms of fitness and lipid levels. In further chapters best practices to (sub)sample, analyse, report and visualize contaminants in the eel are described. The disease sections focus on parasitic diseases (including the swimbladder parasite Anguillicoloides), and on viral and bacterial diseases. Possible ways to integrate data and to implement them into eel quality indices have been suggested. The workshop also discussed the future perspectives of using biomarkers of effects to assess eel health. Finally the report concludes describing the international context and future perspectives in eel health assessments.Several recommendations were made to facilitate the further development of a framework to integrate eel quality assessments into the quantitative management of the eel stock. Member States should apply harmonised methods for eel quality assessments and reporting, and routine monitoring and reporting of lipid levels, contamination and diseases needs to be integrated in the requirements within the Eel Regulation. Raw data should be made available to the international community and the management of the Eel Quality Database needs a structural basis. There is an urgent need for an internationally coordinated research project aiming at improving the understanding and quantification of the effects of contaminants on the reproductive success of the European eel, to allow integration of quality indicators in stock wide assessments.ICES, 2009. Report of the 2009 Session of the Joint EIFAC/ICES Working Group on Eels, FAO European Inland Fisheries Advisory Commission; International Council for the Exploration of the Sea, Göteborg, 7–12 September 2009, EIFAC Occasional Paper No. 45, ICES CM 2009/ACOM: 15. Rome, FAO/Copenhagen, ICES. 2010. p. 540 (Online).ICES, 2012. Report of the 2012 Session of the Joint EIFAAC/ICES Working Group on Eels, Copenhagen, Denmark, 3–9 September 2012; ICES CM 2012/ACOM:18, EIFAAC Occasional Paper 49, 828 pp.ICES, 2015. Report of the Workshop of a Planning Group on the Monitoring of Eel Quality under the subject “Development of standardized and harmonized protocols for the estimation of eel quality” (WKPGMEQ), 20–22 January 2015, Brussels, Belgium. ICES CM 2014/SSGEF:14. 274 pp.