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Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea
Virus Research 63 (1999) 95 – 106 www.elsevier.com/locate/virusres
Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea Helle Frank Mortensen *, Ole Eske Heuer, Niels Lorenzen, Lars Otte, Niels Jørgen Olesen Danish Veterinary Laboratory, Department of Poultry, Fish and Fur Animals, Hangø6ej 2, DK-8200, A, rhus N, Denmark
Abstract In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine environment surrounding Denmark, fish tissue samples were collected on four cruises with the research vessel H/S Dana in 1996 and 1997. The sampling comprised 923 samples totalling 7344 fish representing 29 different species. VHSV was isolated from 24 fish samples from the Baltic Sea, four samples from Skagerrak and three samples from the North Sea. The virus-positive host species included herring Clupea harengus (11 isolates), sprat Sprattus sprattus (eight isolates), cod Gadus morhua (six isolates), rockling Rhinonemus cimbrius (one isolate), Norway pout Trisopterus esmarkii (one isolate), blue whiting Micromesistius poutassou (one isolate), whiting Merlangius merlangus (two isolates) and lesser argentine Argentina sphyraena (one isolate). VHSV has previously been reported from cod and herring, but not from the other five species. A virus belonging to serogroup II of the aquatic birnaviruses was isolated from three samples of flounder Platichthys flesus and three samples of dab Limanda limanda and a virus preliminary identified as iridovirus (lymphocystis virus) was isolated from seven samples of long rough dab Hippoglossoides platessoides. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Viral haemorrhagic septicaemia virus; Marine fish; Birnavirus serogroup II
1. Introduction Viral haemorrhagic septicaemia virus (VHSV) is the most serious viral pathogen affecting farmed rainbow trout (Oncorhynchus mykiss) in * Corresponding author. Tel.: +45-35-300100; fax: + 4589-372470. E-mail address: [email protected] (H.F. Mortensen)
Europe. Until the first isolation of VHSV from fish in the marine environment in 1979 (Jensen et al., 1979; Jørgensen and Olesen, 1987), it was reported only from fish in fresh or brackish waters. Castric and de Kinkelin (1980) reported outbreaks of VHS in sea farmed rainbow trout in France, and Hørlyck et al. (1984) reported several similar outbreaks in Denmark. In most cases, the origin of these outbreaks could be traced back to
0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 6 2 - 3
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the fish farms supplying trout to the seawater farms. Since 1986, Danish marine fish farms have experienced numerous outbreaks of VHS, of which some were due to the above-mentioned transfers, and some to waterborne spread between farms in seawater (Jørgensen, 1992). The VHS outbreaks in farmed turbot (Scophthalmus maximus) in Germany in 1991 (Schlotfeldt et al., 1991) and in Scotland in 1994 (Ross et al., 1994) and the isolation of VHSV from haddock (Melanogrammus aeglefinus) and cod (Gadus morhua) caught close to the Shetland Islands in 1993 and 1995, (Smail, 1995 and Smail, personal communication), were indications of the presence of VHSV in a wider range of fish species from the marine environment. Until 1989, VHSV had only been isolated in Europe, but following the isolation in 1989 of VHSV from returning Chinook salmon (Oncorhyncus tshawytscha) (Hopper, 1989), and coho salmon (Oncorhyncus kisutch) (Brunson et al., 1989) at two different sites in Washington State, USA, the virus has now been reported to be enzootic in the North Eastern Pacific Ocean among Pacific herring stocks, with Pacific cod (Gadus macrocephalus) serving as a secondary reservoir (Meyers and Winton, 1995). Genomic analysis has shown that these American isolates can be distinguished from the European VHSV isolates (Batts et al., 1993; Oshima et al., 1993; Einer-Jensen et al., 1995; Benmansour et al., 1997; Stone et al., 1997). The findings described above caused the Danish Veterinary Laboratory (DVL) to participate in a research cruise with the Danish research vessel H/S Dana in the Baltic Sea in June/July 1996. During that cruise, DVL staff collected tissue specimens from marine fish for virological examination. The objective was to determine whether VHSV could be isolated from marine fish species living in areas close to Denmark. The results of that cruise prompted the laboratory to continue sampling in other areas and at other seasons. The present paper describes the sampling and the results of four research cruises with H/S Dana in the Baltic Sea, the North Sea, Skagerrak and Kattegat in 1996 and 1997.
2. Materials and methods
2.1. Cruises 1 – 4 Samples for virological examination were obtained from catches onboard the research vessel H/S Dana during four cruises in 1996 and 1997. The first cruise took place in the Baltic Sea in June/July 1996 (Fig. 1) and samples were collected from 33 haul stations. The second cruise took place in the North Sea, Skagerrak and Kattegat in November 1996 (Fig. 1) and samples were collected from 34 haul stations. The third cruise took place in the North Sea in February 1997 (Fig. 2) and samples were collected from 24 haul stations in the North Sea, Skagerrak and Kattegat. The last cruise took place in July 1997 (Fig. 2) and samples were collected from 34 haul stations. For cruises 1, 3 and 4, the tissue samples were taken by DVL staff onboard the vessel; whereas on cruise 2 the samples were collected by the crew onboard Dana and delivered to the laboratory as whole, frozen fish. Three types of specimens were sampled, consisting of either internal organs (for cruise 1: brain; heart; liver; spleen; kidney and gonads (if present); for the other three cruises: heart; kidney and spleen), skin with lesions or skin without lesions. The fishing was performed as demersal or pelagic trawling, with a trawling time of either 30 min or 1 h for cruise 1, 30 min for cruise 2 and 3 and 1 h for cruise 4. The hauls took place at daytime for the first three cruises whereas for cruise 4 the hauls were conducted both at daytime and in the night and besides demersal and pelagic trawling, surface trawling was also done. Immediately after landing, the catch was inspected and fish selected for sampling. Species, constitution and clinical health status were noted before processing the samples. Samples of tissue specimens from single fish were transferred to 10 ml plastic vials with 2 ml of sterile Eagles minimal essential medium (MEM) with 10% new-born calf serum, TRIS-buffer and antibiotics (penicillin 90.9 IU/ml and streptomycin 90.9 mg/ml) (transport medium). When tissues from more than one fish were sampled, the material was transferred to
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
10 ml plastic vials with 4 ml of transport medium. One pair of sterile scissors was used for every individual or pooled sample. Gloves were washed thoroughly in hot freshwater and soap followed by disinfection in Virkon®S 2% in between every sampling procedure. On cruise 4, both disposable gloves and gloves washed and scrubbed in disinfecting soap instead of Virkon®S, were used. While sampling, the test tubes were placed on ice, and not more than 60 min after processing of the first sample, all the collected material from the haul station were frozen at − 80°C. For cruise 2, plastic bags with whole fish were noted with the haul number and frozen at − 30°C within 1 h after fishing. For cruise 4, fish caught in the last night haul were placed on ice in a cool room until next morning before processing took place and, due to high sampling intensity, processing of selected fish took up to three h after the catch was taken onboard.
97
2.2. Sampling strategy The sampling strategy for cruise 1 had two main objectives. The prime objective was to collect one pool of tissue from 10 randomly selected fish from each species caught, at each haul. The secondary objective was to collect samples from fish with and without lesions or external signs of disease to examine if presence of virus was correlated with skin lesions or other pathological changes. From selected fish that had either gross pathological changes or looked healthy, pieces of skin were cut out and transferred to a vial with transport medium. In fish with skin lesions, an area with lesion was included in the sample. The skin samples were either processed as pools of 2–10 fish or as individual fish. In most cases the internal organs and skin specimens were collected in separate vials. For cruise 2, the crew onboard H/S Dana were asked to collect 10 herring, 10 sprat and, if available, 10 other species of small
Fig. 1. Area of fishing, shown by ICES squares, and geographical positions of VHSV isolates for cruise 1 and 2.
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H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
Fig. 2. Area of fishing, shown by ICES squares, and geographical positions of VHSV isolates for cruises 3 and 4.
fish (B30 cm) at each haul in plastic bags. The sampling strategy for cruise 3 was the same as for cruise 1, except that instead of one pool of 10 fish the prime objective was to take three pools of 10 fish from each of the species cod, haddock (Melanogrammus aeglefinus), herring and sprat, at each haul, if possible. The sampling strategy for cruise 4 was primarily to test as many fish and species as feasible, so whenever possible three pools of 10 fish each per species caught at each haul were sampled. All samples were transported frozen to the laboratory after arrival of the ship in port, and kept at either −30 or − 80°C until further processing.
2.3. Virological examination At the laboratory, the samples were thawed in cold tap water (cruise 1, 3 and 4) and homogenised by use of mortar, pestle and sterile sand. The samples were diluted to contain one
part tissue material and nine part transport medium and centrifuged for 15 min at 4000× g at 4°C. After centrifugation gentamicin was added to the supernatant (final concentration of 1 mg/ ml) which was then incubated overnight at 4°C. The supernatant was inoculated into 24 h old BF-2 cell cultures (Wolf et al., 1966) in 24-well trays, resulting in final dilutions of tissue material in cell culture medium of 1:100, 1:1000 and 1:10 000, respectively. Seven samples (3 wells per sample) were inoculated on each tray and three wells served as negative controls. The inoculated cell cultures were incubated at 15°C, and inspected three times over 7 days. If no cytopathic effect (CPE) had occurred after 7 days, the samples were sub-cultivated and observed for another 7 days, before the test was terminated. If CPE occurred, VHSV was verified by ELISA as described by Olesen and Jørgensen (1991) with the modification that the plates were incubated for 1 h at 37°C instead of the described 30 min and
Table 1 The number of samples and fish and the result of the virological examination for all four research cruises Species
Dana cruise 1 Number of samples
Number of fish processed
VHSV positive
Number of samples
Dana cruise 3 Number of fish processed
VHSV positive
Number of samples
Dana cruise 4 Number of fish processed
VHSV positive
Number of samples
All four Dana cruises Number of fish processed
VHSV positive
Number of samples
Number of fish processed
VHSV positive
350
10
24
225
0
66
655
0
87
870
1
212
2100
11
350
8
15
143
0
41
405
0
3
30
0
94
928
8
2
0
28
235
0
31
286
0
33
328
2
93
851
2
351
5
13
93
0
59
158
0
22
218
1
198
820
6
1
10
0
39
390
0
40
400
0
20
166
0
21
210
0
46
382
0
3
20
0
35
346
1
38
366
1
14
141
1
21
204
0
35
351
1
12
114
0d
44
339
0
14
129
0
22
133
0
18
122
0c
18
122
0
11
115
0
11
115
0
9
90
0
10
100
0
3
23
0
5
34
0
5
32
8
1
35
10
12
225
0
0a
0
0
2
11
0
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106 99
Herring (Clu35 pea harengus) Sprat (Sprat35 tus sprattus) Whiting 1 (Merlangius merlangus) Cod (Gadus 104 morhua) Mackerel (Scomber scombrus) Haddock (Melanogra mmus aeglefinus) Blue whiting (Micromesistius poutassou) Norway pout (Trisopterus esmarki ) Dab (Limanda limanda) Saithe (Pollachius 6irens) Long Rough Dab (Hippoglossoides platessoides) Pearlsides (Maurolicus muelleri ) Scad (Trachurus trachurus) Grey gurnard (Eutrigla gurnardus)
Dana cruise 2
8
3
4
4
2
4
1109
11
7
198
30
6
24
0
0
0
1
0
129
1 1
Number of samples
VHSV positive
Number of samples
Number of fish processed
Dana cruise 2
Dana cruise 1
1091
1 12
Number of fish processed
1
0 0
VHSV positive
b
One sample positive for Birna II virus. Three samples positive for Birna II virus. c Seven samples positive for virus, preliminarily identified as Iridovirus (lymphocystis virus). d Two samples positive for Birna II virus.
a
Total
Lesser Argentine (Argentina sphyraena) Greater Sandeel (Hyperoplus lanceolatus) Sandeel (Ammodytes lancea) Roundnosed Grenadier (Coryphaenoide s rupestris) Vahl =s eelpout (Lycodes 6ahli ) Garfish (Belone belone) Isopodes (Sadurion enthomon) Rockling (Rhinonemus cimbrius) Rockling spp. Poor cod (Trisopterus minutus) Plaice (Pleuronectes platessa) Three spined stickleback (Gasterosteus aculeatus) Flounder (Platichthys flesus) Lumpsucker (Cyclopterus lumpus) European hake (Merluccius merluccius) Smear dab (Microstomus kitt)
Species
Table 1 (Continued)
1
2
1775
1
1
251
3
1
4
9
2
8
5
Number of fish processed
1
Number of samples
Dana cruise 3
0
0
0
0
0b
0
0
VHSV positive
30
30
30
30
3
3
3
3
3369
30
3
346
30
Number of fish processed
3
Number of samples
Dana cruise 4
6
0
0
0
0
0
1
VHSV positive
924
1
1
5
10
4
2
2 1
7
6
3
3
3
3
3
3
Number of samples
7344
1
1
7
7
8
9
6 12
11
30
30
30
30
30
30
30
Number of fish processed
All four Dana cruises
31
0
0
0
0
0
0
0 0
1
0
0
0
0
0
0
1
VHSV positive
100 H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
instead of horseradish-peroxydase (HRP) conjugated rabbit anti-VHSV the monoclonal antibody IP5B11 (Lorenzen et al., 1988) was used with a subsequent layer of HRP conjugated rabbit antimouse immunoglobulins (P0260, DAKO A/S, Denmark). If the cause of CPE was not identified by ELISA the virus was examined by immunofluorescence as previously described (Olesen et al., 1988; Jørgensen et al., 1993) using antisera against VHSV, IPNV (serotype Sp) and birnavirus (serogroup II). For fish sampled on cruise 2, the fish were thawed in cold tap water and processed for virological examination within 1 h after thawing. New disposable gloves were used for each sample.
3. Results
3.1. Cruise 1 The first group of samples taken by DVL staff during the June/July 1996 research cruise with H/S Dana in the Baltic Sea, comprised a total of 198 pooled and single samples from 1109 fish (representing nine species) from which 24 isolations of VHSV were made. The distribution of the VHSV positive samples in the different species, as well as the number of samples and the number of collected fish for each species appears in Table 1. Most VHSV isolates were from herring (10 from 35 pooled samples) and from sprat (8 from 35 pooled samples). Five VHSV isolates were from cod and one from rockling. All isolates revealed strong reactions in the ELISA using the monoclonal antibody IP5B11 (Lorenzen et al., 1988). The geographic positions of VHSV positive samples are given in Fig. 1. The VHSV positive samples of cod, herring and sprat are evenly distributed over the fishing areas. The relatively high concentration of positive samples in an area just east of Bornholm is considered to be due to the fact that the fishing was more intense there. Except for cod with minor ulcers (the highest prevalence found on the stations close to Bornholm) and a few rocklings with ulcerative changes, no visible signs of disease in either of the
101
other VHSV-positive species represented in the catches were detected. Except for a few sticklebacks (Gasterosteus aculeatus), flounders, rocklings and isopodes (Sadurion enthomon), all predominant in the eastern part of the Baltic Sea, no other species than cod, herring and sprat were caught. The latter was the only species represented on every station. Therefore the majority of the pooled samples (pool size varying from 2–10 single fish) consisted of one of the three most prevalent species (cod, herring, sprat) and fish with no visible sign of disease. Consistent with the secondary objective of the sampling strategy for the first cruise, samples were taken from cod with lesions and cod without lesions. The aim of this part of the examination was to verify earlier reports of correlation between lesions in cod and the presence of VHSV (Smail, 1995). The type of lesions recorded were ulcers and haemorrhages. The numbers, as they appear from Table 2, have been examined by a Fisher Exact test which revealed a P-value of 1.000 (Siegel, 1956). It was not possible to establish a clear correlation between lesions and virus isolation from the data in this survey: only 3 of 50 samples of cod with lesions were VHSV positive; while 2 of 37 samples of cod without lesions were VHSV positive. VHSV was isolated from cod skin specimens with lesions (two isolates) as well as pieces of normal skin specimen (one isolate). Two cod VHSV isolates were from internal organs.
Table 2 Samples from cod with lesions and from cod without lesions, from the June/July 1996 research cruise with H/S Danaa Cod
Lesions
No lesions
Total
VHSV positive VHSV negative
3 (2.87b) 47 (47.1)
2 (2.13) 35 (34.9)
5 82
Total
50
37
87
a
The data contains single samples and pooled samples. Different samples consisting of specimens from the same fish have been regarded as one sample. b Expected frequencies under the assumption that there is no correlation between ulcers and infection with VHSV.
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H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
3.2. Cruise 2 The second group of samples originated from the North Sea, Skagerrak and Kattegat. The fish were sampled by the crew on H/S Dana during a cruise in October/November 1996 and transferred to the laboratory as whole frozen fish. Organs and skin samples for virological examination were taken from the frozen fish after thawing. As shown in Table 1, a total of 129 samples consisting of 1091 fish, representing 10 species, yielded one VHSV positive sample. The VHSV positive sample was from Norway pout caught in Skagerrak just north of Jutland (Fig. 1).
3.3. Cruise 3 The third group of samples originated from the North Sea and was sampled by DVL staff during February 1997. A total of 251 pooled and single samples comprising a total of 1775 fish from 13 different fish species were collected (Table 1). Except for a few flatfish [dab, smear dab (Microstomus kitt) and flounder] with ulcers, no visible signs of disease in any of the other species represented in the catches were detected. VHSV was not isolated, but a birnavirus belonging to serogroup II was isolated from three samples of flounder consisting of two samples of organs and one sample of skin originating from two fish with ulcers. The last skin sample was negative. A birnavirus belonging to serogroup II was also isolated from one pooled sample of dab.
sample of organs was negative. Virus was also isolated from two pooled samples of dab. These seven virus isolates were not VHS virus or birnavirus, but were preliminarily identified as an iridovirus (lymphocystis virus) based on the fact that the fish showed signs of lymphocystis and electron microscopic examination of one of the isolates showed iridovirus-like particles. A birnavirus belonging to serogroup II was isolated from two pooled samples of dab. Except for long rough dab and dab with lesions comparable to lymphocystis, a few mackerel (Scomber scombrus) and a garfish (Belone belone) with processes in the internal organs and a saithe (Pollachius 6irens) with exophthalmia, no visible signs of disease in either of the other species represented in the catches were observed.
3.5. All 4 Dana cruises A total of 924 samples from 7344 fish and isopodes representing 29 species yielded a total of 31 VHSV isolates (Table 3) from eight species Birnavirus isolates belonging to serogroup II were isolated from six samples of dab, originating from two single fish and three pooled samples, and iridovirus (presumably) was isolated from three skin samples and four organ samples originating from three single fish and two pooled samples. Virus was identified in all tissue cultures showing CPE.
4. Discussion
3.4. Cruise 4 The fourth group of samples originated from Kattegat, Skagerrak and the North Sea in July 1997. A total of 346 pooled and single fish samples from 3369 fish, representing 20 species, yielded six isolates of VHSV (Table 1). The geographic positions of these isolates are shown in Fig. 2. The VHSV isolates were obtained from specimens of cod, herring, whiting, blue whiting and lesser argentine. In addition, virus was isolated from five organ/tissue samples taken from three long rough dab comprising of three samples of skin and two samples of organs. The third
Since 1989, a number of reports have described isolation of VHSV from fish in the marine environment (Brunson et al., 1989; Hopper, 1989; Smail, 1995; Dixon et al., 1997). The data from the Baltic Sea survey indicates that VHSV is present in at least four different marine species, i.e. cod, herring, sprat and rockling, in the southeastern part of the Baltic Sea. It seems likely to assume that VHSV is widespread in the Baltic Sea, while the results from the North Sea, Skagerrak and Kattegat indicates a low prevalence there of VHSV infected fish among the species tested (Tables 1 and 3). However, the comparatively low
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
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Table 3 VHSV and birnavirus II isolates from Dana cruise 1, 2, 3 and 4 Isolate
Date of sampling
Host species
Number of fish in sample
Material
Water
ICES square
Virus
1e62 1p3 1p8 1p12 1p40 1p49 1p50 1p52 1p53 1p54 1p55 1p64 1p85 1p86 1p93 1p109 1p110 1p111 1p116 1p120 1p121 1p124 1p125 1p128 2p51 4p37 4p51
02.07.1996 26.06.1996 27.06.1996 27.06.1996 28.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 30.06.1996 01.07.1996 01.07.1996 01.07.1996 02.07.1996 02.07.1996 02.07.1996 02.07.1996 03.07.1996 03.07.1996 03.07.1996 03.07.1996 03.07.1996 28.10.1996 04.07.1997 04.07.1997
1 10 10 10 3 10 10 10 10 5 10 10 10 10 10 10 10 10 5 10 10 10 10 10 10 10 10
Skin (vesicles) Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Skin (normal) Organs Organs Skin (vesicles) Organs Organs Organs Organs Organs Organs Organs Organs
Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Skagerrak North Sea North Sea
39G5 39G3 40G7 40G8 43G9 42G9 42G9 41G9 41G9 41G9 41G9 40G9 38G9 38G9 39G9 39G5 39G5 39G5 39G5 39G5 39G5 39G5 39G5 39G5 44G0 45F5 44F5
VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV
4p59 4p100 4p101 4p168 3e3 (bvII) 3e4 (bvII) 3e5 (bvII) 3p37 (bvII) 4p76 (bvII) 4p78 (bvII)
04.07.1997 06.07.1997 06.07.1997 08.07.1997 12.02.1997 12.02.1997 12.02.1997 13.02.1997 04.07.1997 04.07.1997
Cod Herring Herring Herring Rockling Herring Sprat Sprat Herring Cod Sprat Herring Herring Sprat Cod Cod Herring Sprat Cod Herring Sprat Sprat Herring Sprat Norway pout Blue whiting Lesser argentine Cod Whiting Whiting Herring Flounder Flounder Flounder Dab Dab Dab
8 10 10 10 1 1 1 10 10 10
Organs Organs Organs Organs Organs Skin (ulcer) Organs Organs Organs Organs
North Sea Skagerrak Skagerrak Skagerrak North Sea North Sea North Sea North Sea North Sea North Sea
44F5 43F7 43F7 44G0 39F5 39F5 39F5 40F3 44F5 44F5
VHSV VHSV VHSV VHSV Birnavirus Birnavirus Birnavirus Birnavirus Birnavirus Birnavirus
number of isolates from cruise 2 (one isolate), can be attributed to other factors than the prevalence of infected fish. The second group of samples was taken from whole frozen fish after thawing. Rapid degradation of the organs while thawing may have influenced the outcome of the virological examination. However, cruise 3 samples, also from the North Sea, did not result in any isolations of VHSV, even though the samples were
II II II II II II
taken from the fish shortly after capture. The samples from cruise 4 resulted in six VHSV isolations, three from the North Sea close to Skagerrak and three from Skagerrak. It is not known yet if there is a seasonal variation in VHSV-positive fish in the sampled sea areas. The cruises that resulted in VHSV isolations took place in the summer or in the autumn whereas the winter cruise did not result in any
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isolation. Still, except for the part of the North Sea in proximity to Skagerrak, no isolations were made from the North Sea either in autumn or in winter. Colleagues at the Marine Laboratory, Aberdeen, Scotland, conducted cruises in the North Sea in April and August 1997 sampling fish for virological examination and did not succeed in isolating VHSV (Rob Raynard, personal communication). Unfortunately no cruise has, so far, been conducted in the Baltic Sea in the winter. A relatively high proportion of the samples taken from herring and sprat sampled from the Baltic Sea, were VHSV-positive, compared to a lower proportion of VHSV positive cod (Table 1). Due to a large difference in number of samples, the number of VHSV isolations from cod, herring and sprat are regarded not comparable to the number of positives for the other species in that cruise. As the fish were not selected randomly, the proportions of positive samples in the different cruises are not directly comparable and the results must not be interpreted as demonstration of the prevalence rate. As can be seen in Table 1, in most cases it was necessary to examine a large number of fish to find one VHSV-positive sample. Perhaps a higher proportion of fish are infected than those detected but the number of infective virus particles are so few as to be below the detection limit. BF-2 cells are now considered to be the most susceptible cell line for the detection of VHSV (Olesen and Jørgensen, 1992). The use of fewer fish in each pooled sample or the inoculation of samples on larger cell areas might have enhanced the test sensitivity but would have had the practical impact that fewer fish would have been processed. From the results of the virological examination of samples from cod with lesions and cod without lesions, no correlation between lesions and the presence of VHSV could be established (Table 2). The result of the Fisher Exact test should be viewed with caution, since it is was applied to material where a different number of fish were included in the different samples. Since the cod without lesions were sampled randomly, whereas the cod with lesions were selected, the proportion of cod with lesions in the samples is higher than the proportion of cod with lesions in the catch,
resulting in the relationship between lesions and VHSV will be enhanced. Furthermore, in some of the samples counted as cod with lesions that were VHSV positive not all of the fish in the sample had lesions. If it was one of the lesion-free fish that contained VHSV this also would enhance the apparent relationship between lesions and VHSV. Thus it was not possible to conclude a relationship exists. The four cruises have added six new species to the list of known VHSV-susceptible European fish species (Table 4), which now comprises 10 marine fish species. This number of species will probably rise as more fish are examined in the future. The general conclusions appear to be that VHSV occurs considerably more often in marine fish than originally thought. This raises several important questions: (a) are the marine VHSV isolates pathogenic to wild or farmed fish?; (b) are the marine VHSV isolates antigenically and/or genetically distinguishable from freshwater isolates? (c) how should the marine VHSV isolates be regarded in terms of veterinary regulations for prevention and control of VHS disease? Table 4 European marine fish species from which VHS virus has been isolated Species
Reference
Cod
Gadus morhua
Turbot
Scophthalmus maximus Clupea harengus Melanogrammus aeglefinus Sprattus sprattus Rhinonemus cimbrius Merlangius merlangus Trisopterus esmarki Micromesistius poutassou Argentina sphyraena
Jensen et al., (1979) and Jørgensen and Olesen (1987) Schlotfeldt et al., 1991
Herring Haddock Sprat Rockling Whiting Norway pout Blue whiting Lesser argentine
Dixon et al., 1997 Smail (unpublished) This study This study This study This study This study This study
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
Challenge experiments conducted by Kocan et al. (1997), using North American VHSV isolates, on Pacific herrings (Clupea pallasi ) showed that the external signs of disease were usually limited to 1 –2 mm haemorrhagic areas on the lower jaw and isthmus and around the eye and that few fish exhibited extensive cutaneous haemorrhaging. It has not been possible to detect these signs on herrings caught on the Dana cruises since the herrings got squeezed in the trawl and subsequently developed cutaneous and sometimes internal haemorrhages such that it was not possible to distinguish between these bleedings and pathological haemorrhages. The challenge experiments in the USA with herring also demonstrated that VHSV could be the reason for the declines in the North American Pacific herring stocks in 1993 and 1994. It is not known if the European marine VHSV isolates are able to induce morbidity and/ or mortality in Atlantic herring, but there is evidence that morbidity caused by marine VHSV can be induced in turbot (Ross et al., 1994 and McArdle, Fisheries Research Centre, Dublin, Ireland, personal communication). Preliminary infection trials have indicated that the isolates from marine fish species are nonpathogenic or have low pathogenicity to rainbow trout (Mortensen et al., manuscript in preparation). Jørgensen (1992) reported that the Danish M. rhabdovirus isolate from cod in 1979 was avirulent for rainbow trout by bath immersion challenge. Dixon et al. (1997) obtained no mortality when rainbow trout were challenged by bath immersion with an Atlantic herring VHSV isolate and the Danish M. rhabdo virus isolate. United Kingdom, Norway, Sweden, Finland and a large part of Denmark are classified as approved VHS-free zones according to Directives of the European Union. The approvals concern both inland and coastal zones. The implication of the above findings might be the cancellation of approval of coastal zones in Northern Europe if no methods for discriminating between freshwater pathogenic isolates and marine, apparently nonpathogenic, isolates are developed. Beside VHSV, a birnavirus belonging to serogroup II [first described by Underwood et al. (1977)] was isolated from flatfish in the North Sea
105
and Skagerrak. The findings are not considered to be correlated with the pathological changes found in a few of the fish. There is, however, one report of a birnavirus belonging to serogroup II causing disease and mortality in brook trout (Sal6elinus fontinalis, Ahne et al., 1989). The impact of the presence of this virus in dab is still unknown, but is considered to be low as is the case for the presence of infectious pancreatic necrosis (IPN) virus (birnavirus serogroup I) in a large range of aquatic animals.
Acknowledgements Stig Mellergaard, Danish Institute for Fisheries and Marine Research, is acknowledged for helping to organise all four cruises. We thank the cruise leaders Jonna Tomkiewicz, Niels Jørgen Pihl and Jens Pedersen, for letting us participate in the cruises with H/S Dana and Niels Jørgen Pihl for collecting fish on the second research cruise. The crew onboard Dana are also thanked. The technicians at DVL are acknowledged for all their help with the processing of the samples and a special thank to Mette Storgaard Lorentzen for help with the sampling onboard the ship on the fourth research cruise. Biologist Kim Michelsen is thanked for helping with sampling onboard the ship in the middle of the night, at cruise 4, when we were tired. Part of this work has been done under the EEC-project Rhabdoviruses in Wild Marine Fish in European Coastal Waters: Characterisation and Significance for Aquaculture. CT 96-1594%%.
References Ahne, W., Jørgensen, P.E.V., Olesen, N.J., Fischer-Scherl, T., Hoffmann, R., 1989. Aquatic birnaviruses: Virus of the serogroup II isolated from an IP7N outbreak in brook trout (Sal6elinus fontinalis). Bull. Eur. Ass. Fish Pathol. 9 (1), 14 – 16. Batts, W.N., Arakawa, C.K., Bernard, J., Winton, J.R., 1993. Isolates of viral hemorrhagic septicemia virus from North America and Europe can be detected and distinguished by DNA probes. Dis. Aquat. Org. 17, 67 – 71.
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Benmansour, A., Basurco, B., Monnier, A.F., Vende, P., Winton, J.R., de Kinkelin, P., 1997. Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus. J. Gen. Virol. 78, 2837–2846. Brunson, R., True, K., Yancey, J., 1989. VHS virus isolated at Makah National Fish Hatchery. Fish Health Sect. Am. Fish Soc. Newslett. 17, 3–4. Castric, J., de Kinkelin, P., 1980. Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea-water. J. Fish Dis. 3, 21–27. Dixon, P.F., Feist, S., Kehoe, E., Parry, L., Stone, D.M., Way, K., 1997. Isolation of viral haemorrhagic septicaemia virus from Atlantic herring Clupea harengus from the English Channel. Dis. Aquat. Org. 30, 81–89. Einer-Jensen, K., Olesen, N.J., Lorenzen, N., Jørgensen, P.E.V., 1995. Use of the polymerase chain reaction (PCR) to differentiate serologically similar viral haemorrhagic septicaemia (VHS) virus isolates from Europe and America. Vet. Res. 26, 464–469. Hopper, K., 1989. The isolation of VHSV from chinook salmon at Glenwood Springs, Orcas Island, Washington. Fish Health Sect. Am. Fish Soc. Newslett. 17, 1. Hørlyck, V., Mellerga˚rd, S., Dalsgaard, I., Jørgensen, P.E.V., 1984. Occurrence of VHS in danish maricultured rainbow trout. Bull. Eur. Ass. Fish Pathol. 4 (1), 11–13. Jensen, N.J., Bloch, B., Larsen, J.L., 1979. The Ulcus syndrome in cod (Gadus morhua) III. A preliminary virological report. Nord. Vet.-Med. 31, 436–442. Jørgensen, P.E.V., 1992. Recent advances in surveillance and control of viral haemorrhagic septicaemia (VHS) of trout. In: Proceedingsof The OJI International Symposium on Salmonid Diseases. Hokkaido University Press, Sapporo, Japan, pp. 60 – 71. Jørgensen, P.E.V., Olesen, N.J., 1987. Cod ulcus syndrome rhabdovirus is indistinguishable from the egtved (VHS) virus. Bull. Eur. Ass. Fish Pathol. 7 (3), 73–74. Jørgensen, P.E.V., Olesen, N.J., Ahne, W., Wahli, T., Meier, W., 1993. Isolation of a previously undescribed rhabdovirus from pike Esox lucius. Dis. Aquat. Org. 16, 171– 179. Kocan, R., Bradley, M., Elder, N., Meyers, T., Batts, W., Winton, J., 1997. North American strain of viral hemorrhagic septicemia virus is highly pathogenic for laboratoryreared pacific herring. J. Aquat. Anim. Health 9, 279–290. Lorenzen, N., Olesen, N.J., Jørgensen, P.E.V., 1988. Production and characterization of monoclonal antibodies to four Egtved virus structural proteins. Dis. Aquat. Org. 4, 35– 42.
.
Meyers, T.R., Winton, J.R., 1995. Viral hemorrhagic septicemia virus in North America. Ann. Rev. Fish Dis. 5, 3 – 24. Olesen, N.J., Jørgensen, P.E.V., 1991. Rapid detection of viral haemorrhagic septicaemia virus in fish by ELISA. J. Appl. Ichthyol. 7, 183 – 186. Olesen, N.J., Jørgensen, P.E.V., 1992. Comparative susceptibility of three fish cell lines to Egtved virus, the virus of viral haemorrhagic septicaemia (VHS). Dis. Aquat. Org. 12, 235 – 237. Olesen, N.J., Jørgensen, P.E.V., Bloch, B., Mellergaard, S., 1988. Isolation of an IPN-like virus belonging to the serogroup II of the aquatic birnaviruses from dab, Limanda limanda L. J. Fish Dis. 11, 449 – 451. Oshima, K.H., Higman, K.H., Arakawa, C.K., de Kinkelin, P., Jørgensen, P.E.V., Meyers, T.R., Winton, J.R., 1993. Genetic comparison of viral hemorrhagic septicemia virus isolates from North America and Europe. Dis. Aquat. Org. 17, 73 – 80. Ross, K., McCarthy, U., Huntly, P.J., Wood, B.P., Stuart, D., Rough, E.I., Smail, D.A., Bruno, D.W., 1994. An outbreak of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bull. Eur. Ass. Fish Pathol. 14 (6), 213 – 214. Schlotfeldt, H.-J., Ahne, W., Jørgensen, P.E.V., Glende, W., 1991. Occurrence of viral haemorrhagic septicaemia in turbot (Scophthalmus maximus) — a natural outbreak. Bull. Eur. Ass. Fish Pathol. 11 (3), 105 – 107. Siegel, S., 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, New York. Smail, D.A., 1995. Isolation and identification of viral haemorrhagic septicaemia (VHS) virus from North Sea cod (Gadus morhua L.). In: ICES International Council of Explo. Sea CM 1995/F:15. Stone, D.M., Way, K., Dixon, P.F., 1997. Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol. 78, 1319 – 1326. Underwood, B.O., Smale, C.J., Brown, F., 1977. Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus. J. Gen. Virol. 36, 93 – 109. Wolf, K., Gravell, M., Malsberger, R.G., 1966. Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151, 1004 – 1005.
Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea Helle Frank Mortensen *, Ole Eske Heuer, Niels Lorenzen, Lars Otte, Niels Jørgen Olesen Danish Veterinary Laboratory, Department of Poultry, Fish and Fur Animals, Hangø6ej 2, DK-8200, A, rhus N, Denmark
Abstract In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine environment surrounding Denmark, fish tissue samples were collected on four cruises with the research vessel H/S Dana in 1996 and 1997. The sampling comprised 923 samples totalling 7344 fish representing 29 different species. VHSV was isolated from 24 fish samples from the Baltic Sea, four samples from Skagerrak and three samples from the North Sea. The virus-positive host species included herring Clupea harengus (11 isolates), sprat Sprattus sprattus (eight isolates), cod Gadus morhua (six isolates), rockling Rhinonemus cimbrius (one isolate), Norway pout Trisopterus esmarkii (one isolate), blue whiting Micromesistius poutassou (one isolate), whiting Merlangius merlangus (two isolates) and lesser argentine Argentina sphyraena (one isolate). VHSV has previously been reported from cod and herring, but not from the other five species. A virus belonging to serogroup II of the aquatic birnaviruses was isolated from three samples of flounder Platichthys flesus and three samples of dab Limanda limanda and a virus preliminary identified as iridovirus (lymphocystis virus) was isolated from seven samples of long rough dab Hippoglossoides platessoides. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Viral haemorrhagic septicaemia virus; Marine fish; Birnavirus serogroup II
1. Introduction Viral haemorrhagic septicaemia virus (VHSV) is the most serious viral pathogen affecting farmed rainbow trout (Oncorhynchus mykiss) in * Corresponding author. Tel.: +45-35-300100; fax: + 4589-372470. E-mail address: [email protected] (H.F. Mortensen)
Europe. Until the first isolation of VHSV from fish in the marine environment in 1979 (Jensen et al., 1979; Jørgensen and Olesen, 1987), it was reported only from fish in fresh or brackish waters. Castric and de Kinkelin (1980) reported outbreaks of VHS in sea farmed rainbow trout in France, and Hørlyck et al. (1984) reported several similar outbreaks in Denmark. In most cases, the origin of these outbreaks could be traced back to
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the fish farms supplying trout to the seawater farms. Since 1986, Danish marine fish farms have experienced numerous outbreaks of VHS, of which some were due to the above-mentioned transfers, and some to waterborne spread between farms in seawater (Jørgensen, 1992). The VHS outbreaks in farmed turbot (Scophthalmus maximus) in Germany in 1991 (Schlotfeldt et al., 1991) and in Scotland in 1994 (Ross et al., 1994) and the isolation of VHSV from haddock (Melanogrammus aeglefinus) and cod (Gadus morhua) caught close to the Shetland Islands in 1993 and 1995, (Smail, 1995 and Smail, personal communication), were indications of the presence of VHSV in a wider range of fish species from the marine environment. Until 1989, VHSV had only been isolated in Europe, but following the isolation in 1989 of VHSV from returning Chinook salmon (Oncorhyncus tshawytscha) (Hopper, 1989), and coho salmon (Oncorhyncus kisutch) (Brunson et al., 1989) at two different sites in Washington State, USA, the virus has now been reported to be enzootic in the North Eastern Pacific Ocean among Pacific herring stocks, with Pacific cod (Gadus macrocephalus) serving as a secondary reservoir (Meyers and Winton, 1995). Genomic analysis has shown that these American isolates can be distinguished from the European VHSV isolates (Batts et al., 1993; Oshima et al., 1993; Einer-Jensen et al., 1995; Benmansour et al., 1997; Stone et al., 1997). The findings described above caused the Danish Veterinary Laboratory (DVL) to participate in a research cruise with the Danish research vessel H/S Dana in the Baltic Sea in June/July 1996. During that cruise, DVL staff collected tissue specimens from marine fish for virological examination. The objective was to determine whether VHSV could be isolated from marine fish species living in areas close to Denmark. The results of that cruise prompted the laboratory to continue sampling in other areas and at other seasons. The present paper describes the sampling and the results of four research cruises with H/S Dana in the Baltic Sea, the North Sea, Skagerrak and Kattegat in 1996 and 1997.
2. Materials and methods
2.1. Cruises 1 – 4 Samples for virological examination were obtained from catches onboard the research vessel H/S Dana during four cruises in 1996 and 1997. The first cruise took place in the Baltic Sea in June/July 1996 (Fig. 1) and samples were collected from 33 haul stations. The second cruise took place in the North Sea, Skagerrak and Kattegat in November 1996 (Fig. 1) and samples were collected from 34 haul stations. The third cruise took place in the North Sea in February 1997 (Fig. 2) and samples were collected from 24 haul stations in the North Sea, Skagerrak and Kattegat. The last cruise took place in July 1997 (Fig. 2) and samples were collected from 34 haul stations. For cruises 1, 3 and 4, the tissue samples were taken by DVL staff onboard the vessel; whereas on cruise 2 the samples were collected by the crew onboard Dana and delivered to the laboratory as whole, frozen fish. Three types of specimens were sampled, consisting of either internal organs (for cruise 1: brain; heart; liver; spleen; kidney and gonads (if present); for the other three cruises: heart; kidney and spleen), skin with lesions or skin without lesions. The fishing was performed as demersal or pelagic trawling, with a trawling time of either 30 min or 1 h for cruise 1, 30 min for cruise 2 and 3 and 1 h for cruise 4. The hauls took place at daytime for the first three cruises whereas for cruise 4 the hauls were conducted both at daytime and in the night and besides demersal and pelagic trawling, surface trawling was also done. Immediately after landing, the catch was inspected and fish selected for sampling. Species, constitution and clinical health status were noted before processing the samples. Samples of tissue specimens from single fish were transferred to 10 ml plastic vials with 2 ml of sterile Eagles minimal essential medium (MEM) with 10% new-born calf serum, TRIS-buffer and antibiotics (penicillin 90.9 IU/ml and streptomycin 90.9 mg/ml) (transport medium). When tissues from more than one fish were sampled, the material was transferred to
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
10 ml plastic vials with 4 ml of transport medium. One pair of sterile scissors was used for every individual or pooled sample. Gloves were washed thoroughly in hot freshwater and soap followed by disinfection in Virkon®S 2% in between every sampling procedure. On cruise 4, both disposable gloves and gloves washed and scrubbed in disinfecting soap instead of Virkon®S, were used. While sampling, the test tubes were placed on ice, and not more than 60 min after processing of the first sample, all the collected material from the haul station were frozen at − 80°C. For cruise 2, plastic bags with whole fish were noted with the haul number and frozen at − 30°C within 1 h after fishing. For cruise 4, fish caught in the last night haul were placed on ice in a cool room until next morning before processing took place and, due to high sampling intensity, processing of selected fish took up to three h after the catch was taken onboard.
97
2.2. Sampling strategy The sampling strategy for cruise 1 had two main objectives. The prime objective was to collect one pool of tissue from 10 randomly selected fish from each species caught, at each haul. The secondary objective was to collect samples from fish with and without lesions or external signs of disease to examine if presence of virus was correlated with skin lesions or other pathological changes. From selected fish that had either gross pathological changes or looked healthy, pieces of skin were cut out and transferred to a vial with transport medium. In fish with skin lesions, an area with lesion was included in the sample. The skin samples were either processed as pools of 2–10 fish or as individual fish. In most cases the internal organs and skin specimens were collected in separate vials. For cruise 2, the crew onboard H/S Dana were asked to collect 10 herring, 10 sprat and, if available, 10 other species of small
Fig. 1. Area of fishing, shown by ICES squares, and geographical positions of VHSV isolates for cruise 1 and 2.
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H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
Fig. 2. Area of fishing, shown by ICES squares, and geographical positions of VHSV isolates for cruises 3 and 4.
fish (B30 cm) at each haul in plastic bags. The sampling strategy for cruise 3 was the same as for cruise 1, except that instead of one pool of 10 fish the prime objective was to take three pools of 10 fish from each of the species cod, haddock (Melanogrammus aeglefinus), herring and sprat, at each haul, if possible. The sampling strategy for cruise 4 was primarily to test as many fish and species as feasible, so whenever possible three pools of 10 fish each per species caught at each haul were sampled. All samples were transported frozen to the laboratory after arrival of the ship in port, and kept at either −30 or − 80°C until further processing.
2.3. Virological examination At the laboratory, the samples were thawed in cold tap water (cruise 1, 3 and 4) and homogenised by use of mortar, pestle and sterile sand. The samples were diluted to contain one
part tissue material and nine part transport medium and centrifuged for 15 min at 4000× g at 4°C. After centrifugation gentamicin was added to the supernatant (final concentration of 1 mg/ ml) which was then incubated overnight at 4°C. The supernatant was inoculated into 24 h old BF-2 cell cultures (Wolf et al., 1966) in 24-well trays, resulting in final dilutions of tissue material in cell culture medium of 1:100, 1:1000 and 1:10 000, respectively. Seven samples (3 wells per sample) were inoculated on each tray and three wells served as negative controls. The inoculated cell cultures were incubated at 15°C, and inspected three times over 7 days. If no cytopathic effect (CPE) had occurred after 7 days, the samples were sub-cultivated and observed for another 7 days, before the test was terminated. If CPE occurred, VHSV was verified by ELISA as described by Olesen and Jørgensen (1991) with the modification that the plates were incubated for 1 h at 37°C instead of the described 30 min and
Table 1 The number of samples and fish and the result of the virological examination for all four research cruises Species
Dana cruise 1 Number of samples
Number of fish processed
VHSV positive
Number of samples
Dana cruise 3 Number of fish processed
VHSV positive
Number of samples
Dana cruise 4 Number of fish processed
VHSV positive
Number of samples
All four Dana cruises Number of fish processed
VHSV positive
Number of samples
Number of fish processed
VHSV positive
350
10
24
225
0
66
655
0
87
870
1
212
2100
11
350
8
15
143
0
41
405
0
3
30
0
94
928
8
2
0
28
235
0
31
286
0
33
328
2
93
851
2
351
5
13
93
0
59
158
0
22
218
1
198
820
6
1
10
0
39
390
0
40
400
0
20
166
0
21
210
0
46
382
0
3
20
0
35
346
1
38
366
1
14
141
1
21
204
0
35
351
1
12
114
0d
44
339
0
14
129
0
22
133
0
18
122
0c
18
122
0
11
115
0
11
115
0
9
90
0
10
100
0
3
23
0
5
34
0
5
32
8
1
35
10
12
225
0
0a
0
0
2
11
0
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106 99
Herring (Clu35 pea harengus) Sprat (Sprat35 tus sprattus) Whiting 1 (Merlangius merlangus) Cod (Gadus 104 morhua) Mackerel (Scomber scombrus) Haddock (Melanogra mmus aeglefinus) Blue whiting (Micromesistius poutassou) Norway pout (Trisopterus esmarki ) Dab (Limanda limanda) Saithe (Pollachius 6irens) Long Rough Dab (Hippoglossoides platessoides) Pearlsides (Maurolicus muelleri ) Scad (Trachurus trachurus) Grey gurnard (Eutrigla gurnardus)
Dana cruise 2
8
3
4
4
2
4
1109
11
7
198
30
6
24
0
0
0
1
0
129
1 1
Number of samples
VHSV positive
Number of samples
Number of fish processed
Dana cruise 2
Dana cruise 1
1091
1 12
Number of fish processed
1
0 0
VHSV positive
b
One sample positive for Birna II virus. Three samples positive for Birna II virus. c Seven samples positive for virus, preliminarily identified as Iridovirus (lymphocystis virus). d Two samples positive for Birna II virus.
a
Total
Lesser Argentine (Argentina sphyraena) Greater Sandeel (Hyperoplus lanceolatus) Sandeel (Ammodytes lancea) Roundnosed Grenadier (Coryphaenoide s rupestris) Vahl =s eelpout (Lycodes 6ahli ) Garfish (Belone belone) Isopodes (Sadurion enthomon) Rockling (Rhinonemus cimbrius) Rockling spp. Poor cod (Trisopterus minutus) Plaice (Pleuronectes platessa) Three spined stickleback (Gasterosteus aculeatus) Flounder (Platichthys flesus) Lumpsucker (Cyclopterus lumpus) European hake (Merluccius merluccius) Smear dab (Microstomus kitt)
Species
Table 1 (Continued)
1
2
1775
1
1
251
3
1
4
9
2
8
5
Number of fish processed
1
Number of samples
Dana cruise 3
0
0
0
0
0b
0
0
VHSV positive
30
30
30
30
3
3
3
3
3369
30
3
346
30
Number of fish processed
3
Number of samples
Dana cruise 4
6
0
0
0
0
0
1
VHSV positive
924
1
1
5
10
4
2
2 1
7
6
3
3
3
3
3
3
Number of samples
7344
1
1
7
7
8
9
6 12
11
30
30
30
30
30
30
30
Number of fish processed
All four Dana cruises
31
0
0
0
0
0
0
0 0
1
0
0
0
0
0
0
1
VHSV positive
100 H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
H.F. Mortensen et al. / Virus Research 63 (1999) 95–106
instead of horseradish-peroxydase (HRP) conjugated rabbit anti-VHSV the monoclonal antibody IP5B11 (Lorenzen et al., 1988) was used with a subsequent layer of HRP conjugated rabbit antimouse immunoglobulins (P0260, DAKO A/S, Denmark). If the cause of CPE was not identified by ELISA the virus was examined by immunofluorescence as previously described (Olesen et al., 1988; Jørgensen et al., 1993) using antisera against VHSV, IPNV (serotype Sp) and birnavirus (serogroup II). For fish sampled on cruise 2, the fish were thawed in cold tap water and processed for virological examination within 1 h after thawing. New disposable gloves were used for each sample.
3. Results
3.1. Cruise 1 The first group of samples taken by DVL staff during the June/July 1996 research cruise with H/S Dana in the Baltic Sea, comprised a total of 198 pooled and single samples from 1109 fish (representing nine species) from which 24 isolations of VHSV were made. The distribution of the VHSV positive samples in the different species, as well as the number of samples and the number of collected fish for each species appears in Table 1. Most VHSV isolates were from herring (10 from 35 pooled samples) and from sprat (8 from 35 pooled samples). Five VHSV isolates were from cod and one from rockling. All isolates revealed strong reactions in the ELISA using the monoclonal antibody IP5B11 (Lorenzen et al., 1988). The geographic positions of VHSV positive samples are given in Fig. 1. The VHSV positive samples of cod, herring and sprat are evenly distributed over the fishing areas. The relatively high concentration of positive samples in an area just east of Bornholm is considered to be due to the fact that the fishing was more intense there. Except for cod with minor ulcers (the highest prevalence found on the stations close to Bornholm) and a few rocklings with ulcerative changes, no visible signs of disease in either of the
101
other VHSV-positive species represented in the catches were detected. Except for a few sticklebacks (Gasterosteus aculeatus), flounders, rocklings and isopodes (Sadurion enthomon), all predominant in the eastern part of the Baltic Sea, no other species than cod, herring and sprat were caught. The latter was the only species represented on every station. Therefore the majority of the pooled samples (pool size varying from 2–10 single fish) consisted of one of the three most prevalent species (cod, herring, sprat) and fish with no visible sign of disease. Consistent with the secondary objective of the sampling strategy for the first cruise, samples were taken from cod with lesions and cod without lesions. The aim of this part of the examination was to verify earlier reports of correlation between lesions in cod and the presence of VHSV (Smail, 1995). The type of lesions recorded were ulcers and haemorrhages. The numbers, as they appear from Table 2, have been examined by a Fisher Exact test which revealed a P-value of 1.000 (Siegel, 1956). It was not possible to establish a clear correlation between lesions and virus isolation from the data in this survey: only 3 of 50 samples of cod with lesions were VHSV positive; while 2 of 37 samples of cod without lesions were VHSV positive. VHSV was isolated from cod skin specimens with lesions (two isolates) as well as pieces of normal skin specimen (one isolate). Two cod VHSV isolates were from internal organs.
Table 2 Samples from cod with lesions and from cod without lesions, from the June/July 1996 research cruise with H/S Danaa Cod
Lesions
No lesions
Total
VHSV positive VHSV negative
3 (2.87b) 47 (47.1)
2 (2.13) 35 (34.9)
5 82
Total
50
37
87
a
The data contains single samples and pooled samples. Different samples consisting of specimens from the same fish have been regarded as one sample. b Expected frequencies under the assumption that there is no correlation between ulcers and infection with VHSV.
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3.2. Cruise 2 The second group of samples originated from the North Sea, Skagerrak and Kattegat. The fish were sampled by the crew on H/S Dana during a cruise in October/November 1996 and transferred to the laboratory as whole frozen fish. Organs and skin samples for virological examination were taken from the frozen fish after thawing. As shown in Table 1, a total of 129 samples consisting of 1091 fish, representing 10 species, yielded one VHSV positive sample. The VHSV positive sample was from Norway pout caught in Skagerrak just north of Jutland (Fig. 1).
3.3. Cruise 3 The third group of samples originated from the North Sea and was sampled by DVL staff during February 1997. A total of 251 pooled and single samples comprising a total of 1775 fish from 13 different fish species were collected (Table 1). Except for a few flatfish [dab, smear dab (Microstomus kitt) and flounder] with ulcers, no visible signs of disease in any of the other species represented in the catches were detected. VHSV was not isolated, but a birnavirus belonging to serogroup II was isolated from three samples of flounder consisting of two samples of organs and one sample of skin originating from two fish with ulcers. The last skin sample was negative. A birnavirus belonging to serogroup II was also isolated from one pooled sample of dab.
sample of organs was negative. Virus was also isolated from two pooled samples of dab. These seven virus isolates were not VHS virus or birnavirus, but were preliminarily identified as an iridovirus (lymphocystis virus) based on the fact that the fish showed signs of lymphocystis and electron microscopic examination of one of the isolates showed iridovirus-like particles. A birnavirus belonging to serogroup II was isolated from two pooled samples of dab. Except for long rough dab and dab with lesions comparable to lymphocystis, a few mackerel (Scomber scombrus) and a garfish (Belone belone) with processes in the internal organs and a saithe (Pollachius 6irens) with exophthalmia, no visible signs of disease in either of the other species represented in the catches were observed.
3.5. All 4 Dana cruises A total of 924 samples from 7344 fish and isopodes representing 29 species yielded a total of 31 VHSV isolates (Table 3) from eight species Birnavirus isolates belonging to serogroup II were isolated from six samples of dab, originating from two single fish and three pooled samples, and iridovirus (presumably) was isolated from three skin samples and four organ samples originating from three single fish and two pooled samples. Virus was identified in all tissue cultures showing CPE.
4. Discussion
3.4. Cruise 4 The fourth group of samples originated from Kattegat, Skagerrak and the North Sea in July 1997. A total of 346 pooled and single fish samples from 3369 fish, representing 20 species, yielded six isolates of VHSV (Table 1). The geographic positions of these isolates are shown in Fig. 2. The VHSV isolates were obtained from specimens of cod, herring, whiting, blue whiting and lesser argentine. In addition, virus was isolated from five organ/tissue samples taken from three long rough dab comprising of three samples of skin and two samples of organs. The third
Since 1989, a number of reports have described isolation of VHSV from fish in the marine environment (Brunson et al., 1989; Hopper, 1989; Smail, 1995; Dixon et al., 1997). The data from the Baltic Sea survey indicates that VHSV is present in at least four different marine species, i.e. cod, herring, sprat and rockling, in the southeastern part of the Baltic Sea. It seems likely to assume that VHSV is widespread in the Baltic Sea, while the results from the North Sea, Skagerrak and Kattegat indicates a low prevalence there of VHSV infected fish among the species tested (Tables 1 and 3). However, the comparatively low
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Table 3 VHSV and birnavirus II isolates from Dana cruise 1, 2, 3 and 4 Isolate
Date of sampling
Host species
Number of fish in sample
Material
Water
ICES square
Virus
1e62 1p3 1p8 1p12 1p40 1p49 1p50 1p52 1p53 1p54 1p55 1p64 1p85 1p86 1p93 1p109 1p110 1p111 1p116 1p120 1p121 1p124 1p125 1p128 2p51 4p37 4p51
02.07.1996 26.06.1996 27.06.1996 27.06.1996 28.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 29.06.1996 30.06.1996 01.07.1996 01.07.1996 01.07.1996 02.07.1996 02.07.1996 02.07.1996 02.07.1996 03.07.1996 03.07.1996 03.07.1996 03.07.1996 03.07.1996 28.10.1996 04.07.1997 04.07.1997
1 10 10 10 3 10 10 10 10 5 10 10 10 10 10 10 10 10 5 10 10 10 10 10 10 10 10
Skin (vesicles) Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Organs Skin (normal) Organs Organs Skin (vesicles) Organs Organs Organs Organs Organs Organs Organs Organs
Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Baltic Sea Skagerrak North Sea North Sea
39G5 39G3 40G7 40G8 43G9 42G9 42G9 41G9 41G9 41G9 41G9 40G9 38G9 38G9 39G9 39G5 39G5 39G5 39G5 39G5 39G5 39G5 39G5 39G5 44G0 45F5 44F5
VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV VHSV
4p59 4p100 4p101 4p168 3e3 (bvII) 3e4 (bvII) 3e5 (bvII) 3p37 (bvII) 4p76 (bvII) 4p78 (bvII)
04.07.1997 06.07.1997 06.07.1997 08.07.1997 12.02.1997 12.02.1997 12.02.1997 13.02.1997 04.07.1997 04.07.1997
Cod Herring Herring Herring Rockling Herring Sprat Sprat Herring Cod Sprat Herring Herring Sprat Cod Cod Herring Sprat Cod Herring Sprat Sprat Herring Sprat Norway pout Blue whiting Lesser argentine Cod Whiting Whiting Herring Flounder Flounder Flounder Dab Dab Dab
8 10 10 10 1 1 1 10 10 10
Organs Organs Organs Organs Organs Skin (ulcer) Organs Organs Organs Organs
North Sea Skagerrak Skagerrak Skagerrak North Sea North Sea North Sea North Sea North Sea North Sea
44F5 43F7 43F7 44G0 39F5 39F5 39F5 40F3 44F5 44F5
VHSV VHSV VHSV VHSV Birnavirus Birnavirus Birnavirus Birnavirus Birnavirus Birnavirus
number of isolates from cruise 2 (one isolate), can be attributed to other factors than the prevalence of infected fish. The second group of samples was taken from whole frozen fish after thawing. Rapid degradation of the organs while thawing may have influenced the outcome of the virological examination. However, cruise 3 samples, also from the North Sea, did not result in any isolations of VHSV, even though the samples were
II II II II II II
taken from the fish shortly after capture. The samples from cruise 4 resulted in six VHSV isolations, three from the North Sea close to Skagerrak and three from Skagerrak. It is not known yet if there is a seasonal variation in VHSV-positive fish in the sampled sea areas. The cruises that resulted in VHSV isolations took place in the summer or in the autumn whereas the winter cruise did not result in any
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isolation. Still, except for the part of the North Sea in proximity to Skagerrak, no isolations were made from the North Sea either in autumn or in winter. Colleagues at the Marine Laboratory, Aberdeen, Scotland, conducted cruises in the North Sea in April and August 1997 sampling fish for virological examination and did not succeed in isolating VHSV (Rob Raynard, personal communication). Unfortunately no cruise has, so far, been conducted in the Baltic Sea in the winter. A relatively high proportion of the samples taken from herring and sprat sampled from the Baltic Sea, were VHSV-positive, compared to a lower proportion of VHSV positive cod (Table 1). Due to a large difference in number of samples, the number of VHSV isolations from cod, herring and sprat are regarded not comparable to the number of positives for the other species in that cruise. As the fish were not selected randomly, the proportions of positive samples in the different cruises are not directly comparable and the results must not be interpreted as demonstration of the prevalence rate. As can be seen in Table 1, in most cases it was necessary to examine a large number of fish to find one VHSV-positive sample. Perhaps a higher proportion of fish are infected than those detected but the number of infective virus particles are so few as to be below the detection limit. BF-2 cells are now considered to be the most susceptible cell line for the detection of VHSV (Olesen and Jørgensen, 1992). The use of fewer fish in each pooled sample or the inoculation of samples on larger cell areas might have enhanced the test sensitivity but would have had the practical impact that fewer fish would have been processed. From the results of the virological examination of samples from cod with lesions and cod without lesions, no correlation between lesions and the presence of VHSV could be established (Table 2). The result of the Fisher Exact test should be viewed with caution, since it is was applied to material where a different number of fish were included in the different samples. Since the cod without lesions were sampled randomly, whereas the cod with lesions were selected, the proportion of cod with lesions in the samples is higher than the proportion of cod with lesions in the catch,
resulting in the relationship between lesions and VHSV will be enhanced. Furthermore, in some of the samples counted as cod with lesions that were VHSV positive not all of the fish in the sample had lesions. If it was one of the lesion-free fish that contained VHSV this also would enhance the apparent relationship between lesions and VHSV. Thus it was not possible to conclude a relationship exists. The four cruises have added six new species to the list of known VHSV-susceptible European fish species (Table 4), which now comprises 10 marine fish species. This number of species will probably rise as more fish are examined in the future. The general conclusions appear to be that VHSV occurs considerably more often in marine fish than originally thought. This raises several important questions: (a) are the marine VHSV isolates pathogenic to wild or farmed fish?; (b) are the marine VHSV isolates antigenically and/or genetically distinguishable from freshwater isolates? (c) how should the marine VHSV isolates be regarded in terms of veterinary regulations for prevention and control of VHS disease? Table 4 European marine fish species from which VHS virus has been isolated Species
Reference
Cod
Gadus morhua
Turbot
Scophthalmus maximus Clupea harengus Melanogrammus aeglefinus Sprattus sprattus Rhinonemus cimbrius Merlangius merlangus Trisopterus esmarki Micromesistius poutassou Argentina sphyraena
Jensen et al., (1979) and Jørgensen and Olesen (1987) Schlotfeldt et al., 1991
Herring Haddock Sprat Rockling Whiting Norway pout Blue whiting Lesser argentine
Dixon et al., 1997 Smail (unpublished) This study This study This study This study This study This study
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Challenge experiments conducted by Kocan et al. (1997), using North American VHSV isolates, on Pacific herrings (Clupea pallasi ) showed that the external signs of disease were usually limited to 1 –2 mm haemorrhagic areas on the lower jaw and isthmus and around the eye and that few fish exhibited extensive cutaneous haemorrhaging. It has not been possible to detect these signs on herrings caught on the Dana cruises since the herrings got squeezed in the trawl and subsequently developed cutaneous and sometimes internal haemorrhages such that it was not possible to distinguish between these bleedings and pathological haemorrhages. The challenge experiments in the USA with herring also demonstrated that VHSV could be the reason for the declines in the North American Pacific herring stocks in 1993 and 1994. It is not known if the European marine VHSV isolates are able to induce morbidity and/ or mortality in Atlantic herring, but there is evidence that morbidity caused by marine VHSV can be induced in turbot (Ross et al., 1994 and McArdle, Fisheries Research Centre, Dublin, Ireland, personal communication). Preliminary infection trials have indicated that the isolates from marine fish species are nonpathogenic or have low pathogenicity to rainbow trout (Mortensen et al., manuscript in preparation). Jørgensen (1992) reported that the Danish M. rhabdovirus isolate from cod in 1979 was avirulent for rainbow trout by bath immersion challenge. Dixon et al. (1997) obtained no mortality when rainbow trout were challenged by bath immersion with an Atlantic herring VHSV isolate and the Danish M. rhabdo virus isolate. United Kingdom, Norway, Sweden, Finland and a large part of Denmark are classified as approved VHS-free zones according to Directives of the European Union. The approvals concern both inland and coastal zones. The implication of the above findings might be the cancellation of approval of coastal zones in Northern Europe if no methods for discriminating between freshwater pathogenic isolates and marine, apparently nonpathogenic, isolates are developed. Beside VHSV, a birnavirus belonging to serogroup II [first described by Underwood et al. (1977)] was isolated from flatfish in the North Sea
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and Skagerrak. The findings are not considered to be correlated with the pathological changes found in a few of the fish. There is, however, one report of a birnavirus belonging to serogroup II causing disease and mortality in brook trout (Sal6elinus fontinalis, Ahne et al., 1989). The impact of the presence of this virus in dab is still unknown, but is considered to be low as is the case for the presence of infectious pancreatic necrosis (IPN) virus (birnavirus serogroup I) in a large range of aquatic animals.
Acknowledgements Stig Mellergaard, Danish Institute for Fisheries and Marine Research, is acknowledged for helping to organise all four cruises. We thank the cruise leaders Jonna Tomkiewicz, Niels Jørgen Pihl and Jens Pedersen, for letting us participate in the cruises with H/S Dana and Niels Jørgen Pihl for collecting fish on the second research cruise. The crew onboard Dana are also thanked. The technicians at DVL are acknowledged for all their help with the processing of the samples and a special thank to Mette Storgaard Lorentzen for help with the sampling onboard the ship on the fourth research cruise. Biologist Kim Michelsen is thanked for helping with sampling onboard the ship in the middle of the night, at cruise 4, when we were tired. Part of this work has been done under the EEC-project Rhabdoviruses in Wild Marine Fish in European Coastal Waters: Characterisation and Significance for Aquaculture. CT 96-1594%%.
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Meyers, T.R., Winton, J.R., 1995. Viral hemorrhagic septicemia virus in North America. Ann. Rev. Fish Dis. 5, 3 – 24. Olesen, N.J., Jørgensen, P.E.V., 1991. Rapid detection of viral haemorrhagic septicaemia virus in fish by ELISA. J. Appl. Ichthyol. 7, 183 – 186. Olesen, N.J., Jørgensen, P.E.V., 1992. Comparative susceptibility of three fish cell lines to Egtved virus, the virus of viral haemorrhagic septicaemia (VHS). Dis. Aquat. Org. 12, 235 – 237. Olesen, N.J., Jørgensen, P.E.V., Bloch, B., Mellergaard, S., 1988. Isolation of an IPN-like virus belonging to the serogroup II of the aquatic birnaviruses from dab, Limanda limanda L. J. Fish Dis. 11, 449 – 451. Oshima, K.H., Higman, K.H., Arakawa, C.K., de Kinkelin, P., Jørgensen, P.E.V., Meyers, T.R., Winton, J.R., 1993. Genetic comparison of viral hemorrhagic septicemia virus isolates from North America and Europe. Dis. Aquat. Org. 17, 73 – 80. Ross, K., McCarthy, U., Huntly, P.J., Wood, B.P., Stuart, D., Rough, E.I., Smail, D.A., Bruno, D.W., 1994. An outbreak of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bull. Eur. Ass. Fish Pathol. 14 (6), 213 – 214. Schlotfeldt, H.-J., Ahne, W., Jørgensen, P.E.V., Glende, W., 1991. Occurrence of viral haemorrhagic septicaemia in turbot (Scophthalmus maximus) — a natural outbreak. Bull. Eur. Ass. Fish Pathol. 11 (3), 105 – 107. Siegel, S., 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, New York. Smail, D.A., 1995. Isolation and identification of viral haemorrhagic septicaemia (VHS) virus from North Sea cod (Gadus morhua L.). In: ICES International Council of Explo. Sea CM 1995/F:15. Stone, D.M., Way, K., Dixon, P.F., 1997. Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol. 78, 1319 – 1326. Underwood, B.O., Smale, C.J., Brown, F., 1977. Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus. J. Gen. Virol. 36, 93 – 109. Wolf, K., Gravell, M., Malsberger, R.G., 1966. Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science 151, 1004 – 1005.