PRESENCE OF HERPESVIRUS ANGUILLAE (ANGHV1) DNA IN THE NATIVE ICHTHYOFAUNA OF NORTH-WESTERN POLAND

Nguyen T. Tuan¹, Jolanta Kempter², Remigiusz Panicz^3
1 Faculty of Agriculture-Forestry-Fisheries, Vinh University, Vietnam
² Department of Aquaculture, Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
³ Department of Meat Technology, Faculty of Food Technology and Fisheries,
West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

Anguillid herpesvirus 1 (AngHV1), an enveloped double-stranded DNA virus, occurs in eel and poses a threat to the farmed and wild eel species. With a prevalence of up to 48%, the virus causes severe losses in aquaculture and seriously hampers the recovery of the severely depleted eel stocks. To date, there have been no reports on non-eel species that may act as vectors of AngHV1 transmission. Therefore, the aim of this study was to screen marine and freshwater fish species as potential vectors. Totally, 332 fish samples representing 20 families were collected, and DNA from the gills as well as internal organs (liver, kidney, spleen, intestine and heart) was extracted. Detection of the gene fragment encoding the AngHV1 DNA polymerase was positive in 17 fish samples obtained from the Prussian carp Carassius gibelio (n = 2), European perch Perca fluviatilis (n = 2), pikeperch Stizostedion lucioperca (n = 2), sterlet Acipenser ruthenus (n = 5), and round goby Neogobius melanostomus (n = 6). This is the first study in which non-eel species have been reported as possible AngHV1 carriers. Cohabitation studies should be conducted to test whether these possible vectors may transmit AngHV1 to eel and thereby cause mortality of eel.

Key words: transmission, Anguilla anguilla, AngHV1, vectors, herpesvirus.

INTRODUCTION

Herpesvirus anguillae (HVA) or scientifically anguillid herpesvirus 1 (AngHV1) is a species of the Cyprinivirus genus within the family Alloherpesviridae [9]. It is an enveloped double-stranded DNA virus that belongs to the largest group of DNA viruses that occur in eel and poses a threat to the farmed and wild eel species. The size of the complete genome of AngHV1 comprises 249 kb, including an 11-kb terminal direct repeat that contains 7 out of the 136 predicted protein-coding open reading frames [16]. AngHV1 was isolated from diseased Japanese eel and European eel in 1985 in Japan [13]. AngHV1 was detected in eel from eel farms and from natural habitats, rivers and lakes, in the Netherlands [4] and Germany [8, 14,15], with a prevalence of up to 48% in eel from some Bavarian water bodies [15]. The mortality caused by AngHV1 in eel ranges from 1 to 10%, but with a stress response it can increase up to 100% in aquaculture. The virus has caused severe losses in aquaculture as well as in wild eel in German rivers and lakes [14, 15]. Some authors [3, 8] consider AngHV1 as the most significant viral threat to the European eel because of its high virulence. The virus has also been isolated from cultured eel in several countries worldwide, including Japan, Taiwan and the Netherlands [3, 4, 13]. A virological analysis conducted in the wild eel from Lake Albufera (Spain) in 2004 and 2008 (83% of the analysed eel resulted positive for the presence of the virus) showed that AngHV1 was the most frequently detected viral agent, followed by aquabirnavirus. As noted by the authors, the diversity of viral agents and the high frequency of virus detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought [1]. According to Nguyen et al. [10], an average of 30% of the investigated eel in western Poland carried the virus and the risk of mass mortality of the European eel in domestic waters after the introduction of the herpesvirus AngHV1 from imported eel was high. The European eel (Anguilla anguilla) is a fish species of special economic importance, but its natural stocks decline rapidly. Therefore, the IUCN status of the European eel is CR (critically endangered). As a result of the steep decline in the European eel population, the European Commission has proposed a Community Action Plan for the protection and recovery of the severely depleted eel stocks [2]. In recent years, many countries have implemented the Eel Management Plan, including restocking of the European eel in Europe. Therefore, it was necessary to monitor the health status of the European eel, particularly in terms of transmission of possible hazardous and virulent viruses.

Van Beurden et al. [17] sought other fish hosts of AngHV1. Up to 2012, there was no detection of AngHV1 infection in other species than the Japanese eel and the American eel [7]. AngHV1 is widely distributed and infects the wild population of eel with a high incidence. This is one of the reasons to hypothesize the existence of some natural vectors of AngHV1 in addition to the eel, as investigated in the current study.

MATERIAL AND METHODS

Freshwater and marine fish were collected by anglers or fishermen in 2015. A total of 332 individuals representing 20 species, obtained from 6 locations were analysed (Tab. 1). Each fish batch was transported to the laboratory at 4°C and the subsequent DNA extraction was conducted within 24 hours. Organ material was collected per fish under sterile conditions by dissection, in accordance with standard procedures of veterinary practice. The study was not meant to verify pathological changes in the sampled fish. From each fish, fragments of the gills and internal organs, such as liver, kidneys, spleen, intestine and heart, were taken. In view of the large number of samples and the aim of the study, the organ samples from each fish were pooled (combined at an equal weight ratio and treated as a single sample). DNA extraction from 664 samples was conducted using the peqGOLD Tissue DNA Mini Kit (PeqLab, Germany), according to the manufactures instructions attached to the kit. The qualitative and quantitative assessment of the obtained DNA was carried out by electrophoresis of DNA isolates in 1.5% agarose gel, followed by absorbance measurements using the NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific). Detection of the gene fragment encoding the AngHV1 DNA polymerase was conducted using the primers HVAPOLVPSD and HVAPOLOOSN (Tab. 2), according to the procedure developed by Rijsewijk et al. [12]. Briefly, PCR reaction mix was prepared based on the GoTaq®G2 Hot Start polymerase (Promega), Green Master Mix 12.5 μL, F-primer 0.5 μL (10 pmol·μL^-1), R-primer 0.5 μL (10 pmol·μL^-1), DEPC-treated H2O 10.5 μL, DNA 1 μL. The PCR program included: 5 min at 94°C; 40 cycles of 30 s at 94°C, 45 s at 65°C and 60 s at 72°C; 7 min at 72°C, followed by 4°C until the samples were removed. PCR was conducted on a GeneAmp PCR system 9700 (Perkin-Elmer Applied Biosytems) and the results were evaluated by amplicon electrophoresis in 2% agarose gel.

RESULTS

Detection of a fragment of AngHV1 genome after amplification and electrophoretic separation in agarose gel showed the presence of AngHV1 genome in 17 samples. The size of the obtained amplicons of 394 bp was consistent with the products described by Rijsewijk et al. [12]. The positive results indicate fish species that may act as reservoirs for the transmission of AngHV1 in natural waters. The species (and no. of positives) include: Prussian carp (2), perch (2), pikeperch (2), sterlet (5) and round goby (6). In other fish species, the described PCR did not confirm the presence of AngHv1 genome.

DISCUSSION

Viral eel diseases are a factor limiting the effectiveness of eel stocking programmes as well as the eel aquaculture. Among the primary problems are the absence of specific signs of viral infections and the limited diagnostic capabilities of various fish diagnostic laboratories. To date, among the viral diseases considered potentially dangerous for eel are the aquabirnavirus eel virus European (EVE), anguillid herpesvirus-1 (AngHV1) and the eel virus European X (EVEX). Taking into account the management of eel stocks in Poland [11], and particularly the fear of spontaneous reintroduction of known viral eel diseases by restocking eel, the authors of this study attempted to confirm or exclude vector species among the native ichthyofauna. The first confirmed detection of AngHV1 in Poland [7] concerned eel caught in Dąbie Lake and the Szczecin Lagoon. In the list reported by Haenen et al. [5] and Lehmann et al. [8], herpesvirus anguillae was confirmed in wild eel in Germany and in the Netherlands. As for farmed eel, the virus was confirmed in Germany, Japan, Taiwan and the Netherlands [8, 13, 17]. The prevalence of infection and spread are unfortunately difficult to verify as they are closely linked with the intensity of diagnostic surveillance conducted in the area, as well as the nature of any herpesviral infection known to date: latency or persistence. As demonstrated by Nguyen et al. [2016], five of the six investigated sampling sites of the European eel (Anguilla anguilla) in north-western Poland proved to be positive for AngHV1. Particularly dangerous in terms of epizootics is the presence of the virus genome on the gills of juvenile eel imported directly from Denmark and intended for pre-rearing and use in reintroduction programmes. Additionally, AngHV1 was also detected in elvers directly from Atlantic Ocean (Bergmann, pers. observation). From this study, it seems that transmission of the virus is possible not only through the introduction of infected eel, but also through the presence of vector fish species for which the virus is not pathogenic. As demonstrated in this study, the transfer of AngHV1 in the aquatic environment might also occur through such species as: Prussian carp, European perch, zander, sterlet or round goby. Taking into account that among 332 organ samples obtained from 20 fish species, 17 tests representing 5 fish species were positive, steps should be taken to assess the potential for transferring the virus by the mentioned fish species to healthy eel. Among the species with AngHV1 detected, particularly sterlet may be a potential threat due to the transmission of the virus, as sterlet rearing centres are often involved in the pre-rearing of stocking material or for the purpose of sales on the secondary market, not necessarily directly to the consumer. Such a system enables the potential transmission of AngHV1 between centres, resulting in the introduction of infected fish into open water. Of additional concern is the presence of AngHV1 in species such as the European perch or zander. These species are not subject to veterinary checks in aquaculture, however, they often pass into rearing centres during the flooding of ponds or basins with surface waters, and thereby they might spread the virus. On the other hand, the used conventional PCR is very specific but has unknown sensitivity. Due to this fact, it may also happen that more species are found as healthy carriers if, e.g., real-time (q)PCR or nested PCR are used.

CONCLUSION

The possibility of fish vectors potentially enabling the horizontal transmission of AngHV1 indicates a potentially dangerous situation. Future studies should include cohabitation experiments to investigate the possible viral transmission of AngHV1 to eels. Understanding the routes and dynamics of the spread of AngHV1 in aquatic ecosystems is the key element for the development of a systematic monitoring programme aiming to exclude infected fish from the reintroduction fish stock marketed in Poland. Our study indicated that AngHV1 transmission might occur in freshwater as well in marine environment, which additionally indicates that the eel population might be under serious threat. Monitoring programmes should be carefully developed as well as the diagnostic tools used for the detection of the disease-causing agent.

REFERENCES

Accepted for print: 22.11.2016

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