Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

Virus infections of the European Eel in North Rhine Westphalian rivers

Viral infections have been suggested to play a role in the decline of the panmictic population of the European eel (Anguilla anguilla). However, despite the importance of knowledge about pathogenic eel viruses, little is known about their spread in the wild European eel population and only a few eel pathogenic viruses have been described so far. In this study, we aimed to investigate the health status of the A. anguilla stock in North Rhine Westphalia (NRW) State of Germany. For this purpose, we examined tissue samples of 16 elvers, 100 yellow eels and 6 silver eels, sampled from the rivers Rhine, Lippe and Ems. Virus detection was performed via a combination of cell culture and PCR. Next to the detection of frequently encountered pathogenic eel viruses (anguillid herpesvirus 1 and eel virus European X (EVEX)), we isolated the eel picornavirus 1 (EPV-1) from tissue of yellow eels and elvers and demonstrate the distribution of EPV-1 in wild eel population in NRW.     

Anthropogenic spreading of anguillid herpesvirus 1 by stocking of infected farmed European eels,Anguilla anguilla (L.), in the Schlei fjord in northern Germany

The Schlei fjord in northern Germany is the recipient water of a comprehensive eel, Anguilla anguilla (L.), stocking programme. Since 2015, stocked eels become alizarin red S marked, but to date no control mechanism is implemented in this stock enhancement measure to prevent anthropogenic spreading of diseases. Consequentially, it was possible that farmed stocking cohorts of 2015 and 2016 (in total ca. 1040 kg) were subsequently tested positive for anguillid herpesvirus 1 (AngHV 1). For this study, 100 eels [total length (TL) 24.3–72.9 cm, age ca. 1–6 years] were caught in 2016 and investigated with regard to AngHV 1 infection, parasite load (Anguillicoloides crassus) and body conditions. 68% of the eels were found to be virus positive while larger specimens were more often infected. In addition, a fitted generalized linear model (area under the curve = 0.741) demonstrated that an increase in individual TL is accompanied with an increased risk of clinically relevant virus loads. Anguillicoloides crassus turned out to be an important stressor for eels, because parasite and virus load revealed a significant positive correlation. The results of this study evidently show the urgent need of a disease containment strategy for eel stocking programmes.     

Presence of viruses in wild eels Anguilla anguilla L,from the Albufera Lake (Spain)

A virological analysis was conducted on wild eels from the Albufera Lake (Spain). A total of 179 individuals at different growth stages were collected in two different surveys (2004 and 2008). Presence of anguillid herpesvirus (AngHV‐1), aquabirnavirus and betanodavirus was confirmed by PCR procedures in both surveys, although the number of detections was clearly higher in 2008 (83% of the eels analysed resulted positive for virus presence). AngHV‐1 was the viral agent most frequently detected, followed by aquabirnaviruses. Betanodaviruses were detected by the first time in wild eels, and although the detections were only made by nested PCR, high percentage of positives were achieved. In addition, in 2008, seven aquabirnaviruses were isolated. Phylogenetic analysis performed using partial sequences of both genomic segments of aquabirnaviruses indicated that the seven isolates could be typed as WB (genogroup I) on the basis of segment A sequences, but when segment B was used six of them clustered with C1 strain (genogroup V) and one was typed as Ab (genogroup II). These results indicate natural reassortment between different strains of aquabirnaviruses in the eels. Although betanodaviruses were not isolated in cell culture, the analysis of the sequence of the nested PCR product indicated that they clustered with SJNNV genotype. The diversity of viral agents and the high level of viral detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought.     

The impact of Anguillicola crassus on European eels

The impact of the parasitic swimbladder nematode, Anguillicola crassus Kuwahara, Niimi & Itagaki, on European eel populations is assessed with reference to published research on its origin and rapid dissemination, life cycle and transmission dynamics, and its pathogenic effect. The parasite was originally endemic to East Asia, but has transferred from its native host, the Japanese eel, Anguilla japonica Temminck & Schlegel, to the European eel, Anguilla anguilla (L.) and American eel, Anguilla rostrata (Le Seur). Anguillicola crassus is a very successful colonizer and is now known to occur in four continents (Asia, Europe, Africa and America). The nematode can severely impair swimbladder function and has caused mortalities in both farmed and wild populations in the presence of other stressors. Anguillicola crassus may impair the capacity of European eels to complete the spawning migration, although direct evidence is not available to support this hypothesis. Areas for future research are recommended.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

Effect of aromatable androgen (17‐methyltestosterone) on induced maturation of silver European eels (Anguilla Anguilla): Oocyte performance and synchronization

The reproductive performances of silver European eel in term of gonad development and egg production, employing slow‐release implants with the androgen 17‐MT (1 mg) in combination with traditional weekly injection of carp pituitary extract (CPE) was evaluated. Wild female European eels (Anguilla anguilla) underwent a standard induction protocol with CPE and were randomly divided into three groups (N‐group, no implant; Y‐group, with implant; and control, C‐group, no treatment). The results showed that 17‐MT‐treated females (Y‐group) reproduced spontaneously about 6 weeks earlier than the N‐group females with a saving of almost 40% in CPE and time of induction. Concerning artificial induction of maturation in female silver eels, our study demonstrated that they positively respond to androgen exposure also in terms of eggs productivity. Indeed, Y‐group was more productive than N‐group: in Y‐group, 11 eels ensured an eggs production that exceeded 50% of initial body weight (BW), whereas in N‐group only three eels have exceeded this value. The results suggest that 17‐MT should be considered in future protocols for the improvement of the artificial reproduction of female silver European eels.     

Detection of Herpesvirus anguillae during two mortality investigations of wild European eel in England: implications for fishery management

Herpesvirus anguillae (HVA) was detected during disease investigations of European eel, Anguilla anguilla L. at two stillwater fisheries in central England. These represent the first records of HVA from UK eels. Both mortalities were eel‐specific and took place during August 2009 and July 2010 at water temperatures between 17 and 19.4 °C. Pathological changes consistent with HVA infection included haemorrhaging in the fins, skin lesions and necrosis within the gills and liver. Transmission electron microscopy revealed active virion replication within the gill tissue. An initial assessment of risk is presented, indicating that HVA represents a high disease risk to UK eel stocks. However, further studies are required to establish the distribution of HVA before a reliable assessment of impact may be obtained. Until then, the detection of HVA holds important implications for eel conservation and management, in particular eel stocking activity.     

鳗鲡疱疹病毒的分离与鉴定

为获知鳗鲡"脱粘败血病"与病毒的关系,实验用蔗糖密度梯度离心的方法从发病的欧洲鳗鲡内脏器官组织匀浆液中纯化了病毒粒子,负染后利用电镜观察;进一步用EO细胞对病毒进行了分离、培养,并对感染病毒的细胞进行超薄切片,电镜观察;然后,提取病毒DNA,利用鳗鲡疱疹病毒的PCR检测方法对其进行了鉴定。结果显示,接种匀浆上清液的EO细胞出现细胞融合的病变效应;分离病毒的病毒粒子具囊膜,大小约为200 nm;从感染病毒的细胞上清液DNA中扩增出特异性条带,序列测定与比对分析表明,该序列与鳗鲡疱疹病毒欧洲株(AngHV-1)的序列完全一致。研究表明,利用EO细胞分离了一株鳗鲡病毒,经形态观察和DNA分析,确认该病毒为鳗鲡疱疹病毒,命名为AngHV-FJ。该研究为深入开展鳗鲡疱疹病毒的致病机制及鳗鲡"脱粘败血病"的防控研究奠定了重要基础。     

Using a telemetry study to assess the boundary net efficiency of an enclosure system used for yellow eel density monitoring

To assess yellow eel (Anguilla anguilla L.) densities in non‐tidal waters, 1‐ha enclosure approach was developed. For unbiased density and biomass calculations, the catchability of the system needs to be evaluated. A telemetry study with 49 tagged eels was conducted in 2015 and 2016 to verify the effectiveness of the 1.8‐m high boundary net. Excluding recaptured eels (N = 10), 42.8% of the released eels (N = 15) escaped the enclosure within 48 hr. Regarding the movement frequency, similar movement patterns were observed for enclosed and escaped eels within 48 hr after release. Based on the telemetry study, it is concluded that the boundary net is recognised as an obstacle by yellow eels, and reduces the escapement of enclosed eels.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

Isolation of a novel polyomavirus,related to Japanese eel endothelial cell‐infecting virus,from marbled eels,Anguilla marmorata (Quoy & Gaimard)

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 10^7.0, 10^7.4 and 10^7.7 TCID₅₀ mL^?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.     

A size‐age model based on bootstrapping and Bayesian approaches to assess population dynamics of Anguilla anguilla L. in semi‐closed lagoons

A size‐age modelling technique is presented for assessing the vital rates, stock and recruitment of eel populations in semi‐closed lagoons with fully monitored migration of silver eels. Data for yellow and silver European eels (Anguilla anguilla L.) were obtained in 2011 from the Comacchio lagoon (Italy). The analysis was performed in three steps: (i) correction of yellow eel data, which are affected by the fyke nets selectivity during samplings, (ii) estimation of survival curve, stock, recruitment and metamorphosis rates of the population (calibration using data from 2011) and (iii) validation of the model using the observed amount of silver eel migrating population of the next year. A bootstrap procedure was used to assess the level of uncertainty for each parameter using the 95% intervals of the highest posterior density distribution HPDD (Bayesian approach). The measured abundance of silver eels was 0.56 ind·ha^?1, while the yellow eel abundance and recruitment were estimated by the model for 2011 at 8.77 ind·ha^?1 and 5.99 ind. ha^?1 respectively. The model performance during validation was satisfactory as the observed total mass of migrating population of 2012 (3777 kg) was inside the 95% HPDD intervals (3197–3839 kg) of model's predictions. The estimated stocks and recruitment were at least ten times lower from the respective estimations of previous studies of 1989 highlighting the crucial conditions of the population. The proposed modelling approach can provide significant information about eel population conditions, facilitating the evaluation of a range of management options in the context of eel conservation plans.     

Modification in Body Fat Content and Fatty Acid Profile of Wild Yellow Shortfin Eel,Anguilla australis,through Short‐term Fattening

Wild‐catch eels with low fat content are either not accepted at all or accepted as a lower‐quality and less valuable product in the global eel market. This study was undertaken with the goal of increasing the fat content of yellow shortfin eel, Anguilla australis (104 ± 11 g initial weight), captured in the wild and kept in captivity for a short period, as well as assessing their rate of growth. An 86‐d feeding trial was conducted indoors in a recirculation aquaculture system, at 25.5 ± 1.6 C. Two commercial extruded dry feeds were tested; one formulated for European eel and one not specifically intended for use on eels. Good values of specific growth rate (1.10 and 1.12%/d) and feed conversion ratio (1.01 and 1.10) were achieved with both tested feeds. At the end of the trial, eels achieved 20–22% total fat, starting from wild fish with 7% fat content. Fat quality of the fattened eel was appreciably superior (higher proportion of omega‐3 fatty acids) than the fat of eels captured in the wild. The results of this study are encouraging for the prospect of fattening yellow shortfin eels and obtaining a market‐oriented eel product.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

Long‐term records (1781–2013) of European eel (Anguilla anguilla L.) production in the Comacchio Lagoon (Italy): evaluation of local and global factors as causes of the population collapse

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Movements of the European eel (Anguilla anguilla) in a chalk stream

Abstract – The movements of wild European eels (Anguilla anguilla L.) were monitored continuously over a 2‐year period in a tributary of the River Itchen, Hampshire, UK, using a passive integrated transponder (PIT) antenna system. The time of these movements was then related to a number of monitored and calculated environmental parameters. No eel movements were recorded in the winter months when mean stream temperature was below 10 °C. The numbers of movements peaked in May, when mean water temperatures and day length were increasing. The movement of eels was significantly (P < 0.001, R² = 0.45, N = 72) correlated with the time of sunset, with the majority (72%) recorded during the hours of darkness. The results of this investigation have implications for the design/timing of surveys of yellow eel populations and their habitat associations and suggest that successfully determining the microhabitat preferences of active resident eels will require remote monitoring throughout the 24‐h period.     

Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

Growth differences of Japanese eels Anguilla japonica between fresh and brackish water habitats in relation to annual food consumption in the Kojima Bay‐Asahi River system,Japan

To learn about the relationships between feeding and growth of temperate eels in freshwater and brackish water habitats, we analysed 533 yellow‐phase Japanese eels Anguilla japonica collected in both types of habitats in southeastern Japan. Because male eels were very rare in each habitat (FW, N = 1; BW, N = 20), characteristics of female eels were compared between the different habitats. Annual food consumption was evaluated with the consideration of instantaneous food consumption and annual activity period. Stomach fullness index (stomach content weight/body weight) was used as an indicator of instantaneous food consumption. The ratios of number of months with eel catch to those when eel sampling was conducted were used as an indicator of activity period. Female yellow eels tended to be older and slower growing in fresh water (N = 78; age, mean ± SD = 7.9 ± 2.4 years; growth rate, 59.8 ± 14.0 mm year^?1) than in brackish water (N = 229; age, 5.5 ± 1.8 years; growth rate, 90.1 ± 24.4 mm year^?1). Irrespective of sex, yellow eels in brackish water had a higher stomach fullness index and a greater ratio of months with eel catches, indicating greater annual food consumption by brackish water eels. These results indicate that greater annual food consumption contributes to the greater growth rates of Japanese eels in brackish water habitats.