Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla

Abstract

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. KEY WORDS: Herpesvirus · Eel · Latency · Dexamethasone Resale or republication not permitted without written consent of the publisherDis Aquat Org 45: [103][104][105][106][107] 2001 MATERIALS AND METHODS Cells, virus, antiserum. EK-1 cells (Chen et al. 1982) were used in the isolation and propagation of HVA throughout this study. Cells were routinely cultured in sterile plastic flasks in Leibovitz-L15 medium (Life Technologies BV, Breda, The Netherlands) supplemented with 5% foetal bovine serum, antibiotics and 0.075 to 0.15% (w/v) bicarbonate. Cultures were incubated at 26°C in a CO 2 incubator. The Dutch isolate 500138 of HVA was propagated in EK-1 cells, and virus was concentrated and purified from cell culture medium by centrifugation for 4 h at 53 000 × g through 25% (w/w) sucrose in TEN buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA; pH 7.4). The virus pellet was resuspended in TE buffer (10 mM Tris. HCl, 1 mM EDTA, pH 7.4) and virus protein concentration was determined using the BCA (bicinchoninic acid) protein assay with bovine serum albumin as a standard (Pierce, Rockford, IL, USA). To obtain specific antiserum, rabbits were immunised 3 times both intramuscularly and subcutaneously with a dose of 1 mg virus protein in complete or incomplete Freund adjuvans. The rabbit immune serum had a titre of 1:1060 in a virus neutralisation test using 150 TCID 50 of HVA. The rabbit anti-HVA serum was used to type virus isolates, which showed a cytopathic effect characteristic of HVA.Fish. Eighty outwardly healthy eels ranging in weight from 150 to 200 g were purchased from an eel farm. Fishes were kept at 23°C in six 200 l glass aquariums half filled with tap water, which was aerated continuously and changed for fresh water each day. Water temperature was recorded each day. Eels were offered a hiding place in these aquariums and were subjected to a 12/12 h light/dark cycle. They were fed at a maintenance level with commercial dry pellet feed. For individual identification of fishes from which blood samples were collected weekly, 26 fishes in 2 aquariums were tagged by scratching their skin.Experimental design. Eels were placed in 6 aquariums (A to F) in groups of 13 or 14 eels. First blood samples were collected from the caudal vein from all fishes. The experiment was started on Day 0, which was 4 d after eels arrived at our institute. Fishes in Aquariums A, B, and C were untreated contr...

Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands

“…AngHV1 was subsequently isolated from wild A. anguilla from the Netherlands (van Ginneken et al 2004 and Germany (only PCR-positive) (Jakob et al 2009), and from farmed A. anguilla from the Netherlands (Haenen et al 2002, van Ginneken et al 2004) and Greece (Varvarigos et al 2011). The Dutch AngHV1 isolates were shown to be antigenically and genetically related to the Japanese AngHV1 isolate (Davidse et al 1999, van Nieuwstadt et al 2001, Rijsewijk et al 2005). Overall, AngHV1 has been reported in A. japonica and A. anguilla in Japan and Taiwan, A. anguilla in the Netherlands, Germany and Greece, and A. rostrata in Taiwan.…”

Herpesviruses of Fish, Amphibians and Invertebrates

“…It has been shown in vitro that CyHV-3 is able to persist in cultured cells at a nonpermissive temperature, with viral propagation and viral gene transcription being turned off, and reactivated upon return to the permissive temperature (Dishon et al, 2007). Reactivation of fish herpesviruses in vivo has been demonstrated for AngHV-1 following dexamethasone treatment (van Nieuwstadt et al, 2001), and for CyHV-3 following temperature stress (Eide et al, 2011b;St-Hilaire et al, 2005).…”

Pathogenesis of Herpesvirus anguillae (HVA) in juvenile European eel Anguilla anguilla after infection by bath immersion

“…The fact that diseased eels were not obtained in their infection experiment while disease was induced in the present study might be explained by the use of smaller sized and HVA-naïve eels in our study. Additionally, the European eels in the study of Van Nieuwstadt et al (2001) were sourced from southwest Europe, and probably had been pre-exposed to HVA.In the present study we used a virus isolate from diseased eels to expose, by immersion, healthy glass eels which developed disease and from which the virus HVA could be re-isolated, therefore fulfilling Koch's postulates (Evans 1976) for HVA in European eel. HVA infection did not cause mortality, which supports the non-lethal nature of this virus as reported by Kobayashi & Miyazaki (1997) in Japanese eel Anguilla japonica.…”

“…A4503) for 10 min in phosphate-buffered saline (PBS; pH 7.4). The slides were blotted dry and incubated with the primary rabbit anti-HVA serum (Van Nieuwstadt et al 2001) at a dilution of 1:1000 in 5% (v/v) normal horse serum in PBS for 60 min. After 2 × 5 min washing in PBS with 0.05% Tween-20, the secondary antibody, goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase (HRP, DAKO A/S, Cat.…”