Identification of virus infections of European eels intended for stocking measures.

The spread of viral diseases in eels is suggested to severely affect the European eel (Anguilla anguilla) panmictic population. The European Commission has initiated the Eel Recovery Plan (Council Regulation No. 1100/2007) to try to return the European eel stock to more sustainable levels within that measures eel restocking. However, scientific evidence evaluating the efficacy of stocking remains scarce. In addition, knowledge about the impact and contribution of eel stocking on the distribution of infectious diseases is insufficient. In this study, we aimed to investigate virus infections in batches of eels intended for restocking. We analysed samples of glass eels from certified fisheries and farmed European eels from different aquaculture farms. All analysed eels were purchased within a North Rhine Westphalian conservation program. Via a combination of cell culture and qPCR-based techniques, we detected infections of glass eels with the rhabdovirus Eel Virus European X and anguillid herpesvirus 1 infections in farmed eels (10-15 cm).

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.

Is humane slaughtering of rainbow trout achieved in conventional production chains in Germany? Results of a pilot field and laboratory study.

BACKGROUND: Rainbow trout, Oncorhynchus mykiss, is an important fish in European freshwater aquaculture. This industry sector is dominated by small family-owned enterprises located in rural areas. A large percentage of rainbow trout produced by these small enterprises is marketed directly and killed on demand and not processed in commercial processing plants. EU and national regulations stipulate that fish shall be stunned prior to killing and slaughter. The overall objective of this study was to monitor how stunning interventions were integrated into the production chains of German conventional trout aquaculture in order to safeguard animal welfare during stunning and killing. For this, the stunning and slaughtering processes were monitored on 18 rainbow trout farms in various German federal states. During the on-farm research, (i) the stunning success, (ii) injuries related to the stunning procedure, (iii) duration between stunning and killing, and (iv) visible responses at the time of slaughtering were assessed as welfare indicators. In addition, haematological and biochemical blood parameters were measured as indicators for physiological stress. Due to the fact that stunning interventions should induce a loss of consciousness in fish, in a laboratory study, it was examined whether the absence of the brainstem/ behavioural responses, opercular movements (OM) or eye-rolling reflex (vestibulo-ocular reflex, VOR) was correlated with the stage of insensibility. RESULTS: The majority of rainbow trout farms applied manual percussion (38%) or electrical stunning (48%), while on 14% of the farms, the fish were stunned by electrical stunning which was immediately followed by manual percussion. After percussive stunning, about 92.3% of the rainbow trout displayed no OM or VOR as brainstem/ behavioural indicators of consciousness. This percentage varied on farms which applied electrical stunning. While on the majority of farms, 95 to 100% of the fish were unconscious according to the observation of brainstem/ behavioural indicators, the stunning intervention was less effective on farms where rainbow trout were stunned at current densities below 0.1 A dm2 or for a few seconds only. The laboratory study confirmed that the absence of brainstem/ behavioural indicators correlated with the absence of visually evoked responses (VER) of the brain to light stimuli as a neuronal indicator of insensibility. Therefore, the brainstem/ behavioural signs can be used to interpret the stage of insensibility in rainbow trout. A stage of insensibility could safely be induced by exposing portion-sized rainbow trout to an electric current density above 0.1 A dm2. This was not influenced by the orientation of the electric field. CONCLUSIONS: In conventional aquaculture, rainbow trout can effectively be stunned by manual percussion or electrical stunning. Consciousness can be monitored by the absence of opercular movements or the eye-rolling reflex, which are lost approximately at the same time as neurological responses like VER. For safeguarding animal welfare during stunning and killing of rainbow trout in conventional production processes, the stunning process requires careful attention and the operating personnel need to be trained in using the stunning devices and recognising indicators of consciousness.

Stunning of common carp: Results from a field and a laboratory study.

BACKGROUND: Common carp Cyprinus carpio is an important food fish in Central Europe, which in some regions is consumed as part of local tradition. The majority of carp are sold by small retailers and not processed in commercial processing plants. The overall objective of this study was to monitor how animal welfare is safeguarded during the stunning and slaughtering of carp for retail sale. For this, the stunning and slaughtering process was monitored on 12 carp farms. Four welfare-related parameters were assessed: (i) stunning success, (ii) injuries related to the applied stunning method, (iii) time between stunning and slaughter, and (iv) visible responses of carp during slaughtering. In addition, indicators of physiological stress were measured. In order to analyse whether the absence of behavioural indicators of consciousness after electrical stunning was correlated with unconsciousness a complementary laboratory study was performed. Here, carp were exposed to electrical current densities between 0.09 and 0.41 A/dm2. The presence of behavioural responses and visually-evoked responses (VER) in the electro-encephalogram in response to light flashes as indicators for an absence of consciousness was recorded. RESULTS: The carp farms applied manual percussive (18%) or electrical (23%) stunning methods, while the majority of farms used a combination of electrical stunning immediately followed by manual percussive stunning (59%). In the latter condition, 92.6% of stunned carp displayed no behavioural indicators of consciousness and significantly fewer injuries related to mishits compared to sole percussive stunning. In the laboratory study, behavioural indicators of consciousness recovered in carp between 1 and 9 min following removal of the electrical current. However, VER could be recorded already at 30 ± 8 s post stunning. This indicates a fast recovery of carp from electrical stunning when exposed to current densities in the range of those generated by commercially available stunning instruments for fish. CONCLUSIONS: Under field conditions, percussion (applied manually) and electrical stunning might be poor inducers of unconsciousness before slaughter, while a combination was most effective. In order to undertake improvements in electrical stunning, further investigations into the current density, required for inducing prolonged insensibility in carp during electrical stunning, are needed.

Concentration of carp edema virus (CEV) DNA in koi tissues affected by koi sleepy disease (KSD).

Carp edema virus (CEV), the causative agent of 'koi sleepy disease' (KSD), appears to be spreading worldwide and to be responsible for losses in koi, ornamental varieties of the common carp Cyprinus carpio. Clinical signs of KSD include lethargic behaviour, swollen gills, sunken eyes and skin alterations and can easily be mistaken for other diseases, such as infection with cyprinid herpesvirus 3 (CyHV-3). To improve the future diagnosis of CEV infection and to provide a tool to better explore the relationship between viral load and clinical disease, we developed a specific quantitative PCR (qPCR) for strains of the virus known to infect koi carp. In samples from several clinically affected koi, CEV-specific DNA was present in a range from 1 to 2,046,000 copies, with a mean of 129,982 copies and a median of 45 copies per 250 ng of isolated DNA, but virus DNA could not be detected in all clinically affected koi. A comparison of the newly developed qPCR, which is based on a dual-labelled probe, to an existing end-point PCR procedure revealed higher specificity and sensitivity of the qPCR and demonstrated that the new protocol could improve CEV detection in koi. In addition to improved diagnosis, the newly developed qPCR test would be a useful research tool. For example, studies on the pathobiology of CEV could employ controlled infection experiments in which the development of clinical signs could be examined in parallel with a quantitative determination of virus load.

Another potential carp killer?: Carp Edema Virus disease in Germany.

BACKGROUND: Infections with carp edema virus, a pox virus, are known from Japanese koi populations since 1974. A characteristic clinical sign associated with this infection is lethargy and therefore the disease is called "koi sleepy disease". Diseased koi also show swollen gills, enophthalmus, and skin lesions. Mortality rates up to 80 % are described. For a long period of time, disease outbreaks seemed to be restricted to Japan. However, during the last years clinical outbreaks of koi sleepy disease also occurred in the UK and in the Netherlands. CASE PRESENTATION: In spring 2014 koi from different ponds showing lethargic behavior, skin ulcers, inflammation of the anus, enophthalmus, and gill necrosis were presented to the laboratory for diagnosis. In all cases, new koi had been purchased earlier that spring from the same retailer and introduced into existing populations. Eleven koi from six ponds were examined for ectoparasites and for bacterial and viral infections (cyprinid herpesviruses in general and especially koi herpesvirus (KHV) known formally as Cyprinid herpesvirus 3 (CyHV-3); and Carp Edema Virus). In most of the cases parasites were not detected from skin and gills. Only opportunistic freshwater bacteria were isolated from skin ulcers. In cell cultures no cytopathic effect was observed, and none of the samples gave positive results in PCR tests for cyprinid herpesviruses. By analyzing gill tissues for CEV in seven out of eleven samples by a nested PCR, PCR products of 547 bp and 180 bp (by using nested primers) could be amplified. An outbreak of Koi Sleepy Disease was confirmed by sequencing of the PCR products. These results confirm the presence of CEV in German koi populations. CONCLUSION: A clinical outbreak of "koi sleepy disease" due to an infection with Carp Edema Virus was confirmed for the first time in Germany. To avoid transmission of CEV to common carp testing of CEV should become part of fish disease surveillance programs.

Detection of piscine orthoreoviruses (PRV-1 and PRV-3) in Atlantic salmon and rainbow trout farmed in Germany.

Piscine orthoreoviruses (PRVs) are emerging pathogens causing circulatory disorders in salmonids. PRV-1 is the etiological cause of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), characterized by epicarditis, inflammation and necrosis of the myocardium, myositis and necrosis of red skeletal muscle. In 2017, two German breeding farms for Atlantic salmon and rainbow trout (Oncorhynchus mykiss) experienced disease outbreaks with mortalities of 10% and 20% respectively. The main clinical signs were exhaustion and lethargic behaviour. During examinations, PRV-1 in salmon and PRV-3 in trout were detected for the first time in Germany. Further analyses also indicated the presence of Aeromonas salmonicida in internal tissues of both species. While PRV-1 could be putatively linked with the disease in Atlantic salmon, most of the rainbow trout suffered from an infection with A. salmonicida and not with PRV-3. Interestingly, the sequence analysis suggests that the German PRV-3 isolate is more similar to a Chilean PRV-3 isolate from Coho salmon (Oncorhynchus kisutch) than to PRV-3 from rainbow trout from Norway. This indicates a wide geographic distribution of this virus or dispersal by global trade. These findings indicate that infections with PRVs should be considered when investigating disease outbreaks in salmonids.