Abstract

A novel virus-like particle was isolated from wild coarse fish during an investigation of mass mortalities of perch (Perca fluviatilis) and roach (Rutilus rutilus) in the Erne river catchment, Ireland. The culture and electron microscopic characteristics of this isolate appear to be different from those of viruses previously isolated from fish in Ireland and also from those of viruses isolated elsewhere from perch or roach.

Introduction

About 75% of the earth’s surface is covered in water. Only 3% of this is fresh water, the remainder is in the marine environment. Three quarters of the fresh water is found in the frozen form at the earth’s poles and in glaciers. The remainder constitutes our rivers and lakes.

Although only a small part of the planet’s total water, the rivers and lakes are a vital constituent of the circulatory system in the purifying water cycle; from sea to cloud to land to sea. Fresh water rivers and lakes are an important natural resource and provide us with an essential fresh water supply network throughout major land masses. The vital position of rivers and lakes within a major natural cycle makes the health of these freshwater bodies of paramount importance. Knowledge about health factors in these ecosystems may also provide us with key indicators of general environmental health which stretch beyond the geographic boundaries of lake shores and riverbanks.

Anyone who has set out to investigate disease problems in rivers or lakes soon comes to realize the vastness of this environment, despite it representing such a small percentage of the earth’s waters. Epidemiologic disease information is generally more readily available on land dwelling animals and more basic work is required for many species of fish.

In recent years veterinary investigations of wild fish diseases have been carried out to enable the development of a National Fish Health Management Plan with a view to assisting the rational and sustainable use of Ireland’s natural fisheries resources.¹⁴ As with any endeavor where background information is scarce, the initial findings often raise more questions than they provide answers.

Ireland has approximately 140,000 hectares of freshwater lakes and 14,000 kilometers of main channel rivers. There are strong recreational fisheries interests in these fresh waters. From a wildlife vet’s point of view this proves a major benefit. Fish inhabit an environment where it is difficult to oversee their well-being and they would often go unnoticed but for anglers. It is common for wildfish “disease” problems to be first noticed and brought to the forefront by anglers. Such problems include mass mortalities, decreased availability of fish, skin diseases noticed on caught fish, odd-shaped fish or fish behaving strangely.

Report

Investigations of wild fish diseases, in common with any wildlife disease investigation, generally lead to the identification of a number of disease agents. Some of these may be intimately associated with the problem under investigation, some may be “normal background” and others may be unknown entities requiring further study. This communication reports preliminary findings of the isolation by culture and the detection by electron microscopy of a novel virus-like particle from wild coarse fish during an investigation of mass mortalities of perch and roach in the Erne river catchment, Ireland. The complete findings from this investigation are currently being analyzed.

The River Erne catchment covers approximately 4340 square kilometers. It is the fourth largest river in Ireland. The catchment covers areas in both the Republic of Ireland and Northern Ireland. The river rises in Beaghy Lough in County Cavan in the Republic of Ireland. It flows 64 miles through Loughs Gowna and Oughter and Upper and Lower Lough Erne before entering the sea at Ballyshannon, County Donegal. For 30 miles between Crossdoney in County Cavan to the town of Enniskillen, County Fermanagh it is difficult to distinguish the river as it winds its way through hundreds of interconnected loughs nestling among the drumlin hills of Counties Cavan and Fermanagh.

Many of the waters in the Erne catchment are used for sport fishing. Sport fishing is of economic importance to this predominantly rural area since it attracts tourists who use local facilities. Many tourists visit Ireland primarily to fish. Sport fishing consists of game or coarse fishing. The vast majority of fishing sites within the Erne catchment are for coarse fish (bream Abramis abrama, roach Rutilus rutilus, perch Perca fluviatilis, pike Esox lucius and tench Tinca tinca) but game fish (salmon Salmo salar and trout Salmo trutta) are also a relatively popular angler’s quarry on the river system.

A major incident of coarse fish mortalities had occurred in the lakes of the Erne. Initial reports were of tens of thousands of dead and dying fish fry in Loughs Gowna and Oughter in the summer months. Perch were the predominant species affected in Lough Gowna but the majority of later reports involved both perch and roach. Later again, smaller numbers of perch up to 5-years-old were reported dead or dying. By mid-August, 14 other lakes within the Erne system were experiencing similar problems. By the end of August it was estimated that over one million fish had died (A. Ní Shuilleabhain, Northern Regional Fisheries Board, personal communication).

Samples of perch and roach fry from Loughs Gowna and Oughter and Annaghmakerrig Lake were submitted to virology testing procedures similar to those laid down under EC Decision 92/532/EEC.⁴

Inoculated cultures were incubated at 15°C and 22°C for seven days and inspected daily for the presence of cytopathogenic effect (CPE) at 40x magnification. Flasks and trays with no CPE after 1 week were subcultivated using the following procedures. Aliquots of medium from all wells constituting the primary culture were pooled following one freeze-thaw cycle of the cultures and 0.1 ml of this inoculated at 1:1 and 1:100 dilutions. These were then incubated and examined as for the primary cultures.

CPE consisting of a slow thinning of cell monolayers was noted initially from perch tissues on bluegill fry (BF-2) cell lines at 15°C after seven days. CPE also developed from perch tissues on the chinook salmon embryo (CHSE) and epithelioma papulosum cyprini (EPC) cell lines and from roach tissues on BF-2 cell lines after serial passage. The CPE resultant from inoculae of tissues from both perch and roach occurred earlier (within three days in some instances) after serial passage. Overall, however, despite attempts to improve methodology, the extent of CPE in repeat wells and flasks and between species was somewhat variable suggesting culture conditions may not be ideal.

The CPE was not affected by neutralizing antibodies against IPN (specific divalent antiserum to reference strains Sp and Ab), infectious hematopoietic necrosis (IHN) (group specific antibody) or viral hemorrhagic septicaemia (VHS) (group specific antibody).

Suspensions for electron microscopic (EM) examinations were from cultures in which CPE had developed. These were put through a freeze-thaw cycle and centrifuged at 3,000 rpm for 20 min to sediment tissue culture cells and debris. The resultant supernatant was ultracentrifuged at 60,000 rpm for 2 hours. The sediment was stained with 1% phosphotungstic acid, pH 6.5 for 1 minute and examined with a Philips Model EM 201 at 30,000x magnification.

Attempts to identify viral particles by EM examination proved negative initially suggesting that viral titres may be low, again possibly due to the lack of ideal culture conditions. It has not been possible to achieve sufficient titers from cultures of roach tissues to detect virus by EM. However, icosahedral particles approximately 70 nm in diameter with an electron dense core and with an apparent less dense capsid were found in tissue cultures of samples of perch tissues.

Discussion

Although, worldwide, there are numerous diseases in fish that are associated with viruses,^13,15 there are reports of only three viruses, capable of causing fish disease, which have been cultured in Ireland. These are infectious pancreatic necrosis (IPN) virus,¹ pike fry rhabdovirus¹² and salmon pancreas disease virus (SPDV)¹².

The described culture and EM characteristics of this isolate appear to be different from those of viruses previously isolated from fish in Ireland and also from those of viruses isolated elsewhere from perch or roach.^6,7,13,15 The relationship of this isolate to other known viruses deserves further study.

No viruses were isolated during investigations of diseases affecting coarse fish in England, Scotland and Ireland during the 1960s and 1970s, despite attempts by some investigators.^3,5,16 More recently, the effects of pollution have been attributed as the principal cause of wild fish deaths in Ireland.^8-11

There is insufficient information at present to determine the role of this isolate in the fish deaths in the Erne and further work is necessary in regard to this area.

Acknowledgments

Thanks are due to the staff of the Fish Health Unit (FHU), Department of the Marine and the Northern Regional Fisheries Board (NRFB) in particular C. Hickey, Virology Technician, FHU and A. Ni Shuilleabhain, Senior Fisheries Environmental Officer, NRFB. Also, thanks to P. Dillon of the Department of Agriculture, Veterinary Research Laboratory for electron microscopy technical assistance and to M.G. Scullion.

Literature Cited

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2.  Adair, B.M. and M. Mc Loughlin. 1986. Isolation of pike fry rhabdovirus from brown trout (Salmo trutta). Bulletin of the European Association of Fish Pathologists 6 (3): 85.

3.  Ajmal, M. and B.C. Hobbs. 1967. Columnaris disease in roach and perch from English waters. Nature, London 215: 141–142.

4.  Anon. 1992. Laying down the sampling plans and diagnostic methods for the detection and confirmation of certain fish diseases. Commission Decision 92/532/EEC. No L 337/18.

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7.  Langdon, J.S., J.D. Humphrey, L.M. Williams, A.D. Hyatt, and H.A. Westbury. 1986. First virus isolation from Australian fish: an iridovirus-like pathogen from redfin perch, Perca fluviatilis L. J. of Fish Dis. 9, 263–268.

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11.  Moriarty, C. 1993. Fish kills in Ireland 1991–1992. Fishery Leaflet 157. Department of the Marine, Dublin.

12.  Nelson, R.T., M.F. Mc Loughlin, H.M. Rowley, M.A. Platten, and J.I. Mc Cormick. 1995. Isolation of a toga-like virus from farmed Atlantic salmon Salmo salar with pancreas disease. Diseases of Aquatic Organisms 22, 25–32.

13.  Roberts, R.J. 1989. Fish Pathology. 2nd ed. London, W.B. Saunders.

14.  Scullion, F.T. 1995. Veterinary health management in the rehabilitation of wild fish stocks in Ireland. XXV World Veterinary Association Scientific Programme, World Veterinary Congress, Yokohama, Japan (Abstract), p 23.

15.  Stoskopf, M.K. 1993. Fish Medicine. London, W.B. Saunders.

16.  Willoughby, L.G. 1970. Mycological aspects of a disease of young perch in Windermere. Journal of Fish Biology 2, 113–116.