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The prevention and cure of an infectious disease in cultivated juvenile Dover sole, Solea solea (L.)
Aquaculture, 26 (1981/1982) 213-222 Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
213
THE PREVENTION AND CURE OF AN INFECTIOUS DISEASE IN CULTIVATED JUVENILE DOVER SOLE, SOLEA SOLEA (L.)
A.H. McVICAR and P.G. WHITE?* Marine Laboratory,
Victoria Road, Aberdeen
+ WFA Marine Cultivation Unit, Hunterston,
(Great Britain) Ayrshire
(Great Brifain)
*Present address: Hellenic Fish Farm, Varda, Ilias (Greece). (Accepted
5 May 1981)
ABSTRACT McVicar, A.H. and White, P.G., 1982. The prevention and cure of an infectious disease of cultivated juvenile Dover sole, Solea solea (L.). Aquaculture, 26: 213-222. The disease condition “Black Patch Necrosis” which causes serious mortalities of cultivated Dover sole through the development of areas of fin and skin necrosis, was shown to be infectious by co-habitation of diseased and healthy stocks. Spread of the disease throughout an affected tank was rapid and high pathogenicity was evident, with most affected sole dying l-2 days after the first appearance of clinical signs. Addition of a sand substrate to tanks both prevented occurrence of the disease and cured an established disease situation. No aetiological agent has yet been identified.
INTRODUCTION
The culture of Dover sole has been under investigation at the White Fish Authority’s (WFA) Marine Farming Unit at Hunterston for several years. Serious mortalities associated with the development of fin and skin lesions were first noted in “0” group stocks in 1976 and the disease condition, now colloqually known as “Black Patch Necrosis” (BPN) has since recurred each year in “0” group and “I” group stocks and has also affected stocks on a commercial farm. In 1977 an 83% loss of the total metamorphosed stock of over 51000 sole was largely attributed to the disease. McVicar and White (1979) described the clinical signs and histopathology of the disease and gave some circumstantial evidence of the involvement of an unidentified infective agent. Control of the disease’by standard methods proved difficult. A wide range of antibiotics administered orally and as a bath had little effect. Continuous formalin treatment held the disease in check although did not cure it, and toxic effects of the treatment to the sole were found. However, it was demonstrated that the addition of a sand substrate to affected tanks would rapidly cure the disease and
0044-8486/82/0000-0000/$02.75
0
1982 Elsevier Scientific Publishing Company
214
improved hatchery survivals of 51% in 1978 and 81% in 1979 may be largely attributed to the more general use of sand, although other changes in husbandry methods probably also contributed. The objectives of the study reported in this paper were to investigate the possible infective nature of the disease and to determine the prophylactic benefits from using sand continuously in rearing tanks. MATERIALS
AND METHODS
The study was carried out on sole hatched and reared at Hunterston and three main groups of fish were used: (1) Experimental tanks (Hunterston) Experiments were performed at Hunterston in conditions which matched as closely as possible those used routinely and successfully by the WFA in juvenile sole rearing. A single stock of approximately 2000 juvenile sole, derived from one production hatching, was subdivided randomly at 70 days post-metamorphosis (16 August) into six approximately equal groups and held in 1 m diamter (75 1) glass reinforced plastic tanks each with an outflow through a central bottom screen (28 cm diameter with 3 mm holes). Raised rims formed from 3 cm diameter polythene tubing were placed round the outlets of all tanks to prevent sand substrate washing out and in tanks without sand to maintain standard conditions between tanks. Fine grained pre-washed quarry sand, as used in building construction, was placed and maintained in selected tanks at a depth of approximately 20 mm over the whole tank bottom. All of the tanks (numbered l-6) were situated in a single row on a common water supply (mixed warm power station discharge and sub-sand filtered seawater at a flow rate of 11 l/h to each tank). Temperatures, monitored twice daily, were in the range 14.7 to 21.2”C and lighting was provided continuously at a level of 200 lux by four 100 Watt bulbs. Individual hand nets were used for each tank and precautions were taken to avoid contact of water or fish between tanks. For most of the experimental period the fish were fed only a live diet of redworm, Lumbricillus riualis Levinsen aug. Ditlevsen, given twice daily to satiation to avoid complications associated with weaning. However, because of supply problems with Lumbricillus, it was necessary to start a weaning programme towards the end of the period. The following procedures were followed in the sequence (a)-(d) (note Fig. 1) (a) Sand was introduced into two tanks (Nos 2,4) at the start of the experiment to determine any prophylactic effects.
215
r
+ -
SAND
0 -
DISEASE
ADDEG
TO TANK AND
APPEAREO
400-
MAINTAINED
THEREAFTER
SPONTANEOUSLY
-
-
WEANING
PERIOD
0. TANK 1
2004 '....._._._
O-
4*r
t_
-----
-'
-'I
TANK 2 ...-_.__
4
200
t 0 f&o-._-_ cn 200. .s .1 2 i?l O-
4
_--.__t,
TANK 3 ....,
-__ ... I
-'
4
z 4OOr + 2'jt 2
1
-1
-------.,
TANK 4 ._.-_
200
4--_.
400
TANK 5
200 t
400-
0
---
TANK 6
200OL
'.._ 20
40
60
-80
-100
1 110
days Fig. 1. Survival of juvenile Dover sole in trial tanks l-6. Day 0 = 70 days post metamorphosis. Broken lines indicate one or more mortalities.
(b) The disease was allowed to occur naturally in any two tanks (it appeared in two without sand Nos. 1 and 6) Sand was then added to one of them (No. 6) in an attempt to cure the disease.
216
(c) To investigate the infectivity of the disease, all tanks (i.e. three then with sand and three without) were challenged with BPN by introducing one sole showing overt disease signs into each. (d) On the outbreak of induced disease in two tanks (Nos. 3 and 5, both without substrate) sand was added to one and the other allowed to continue without interference. All dead fish were removed from the tanks and recorded daily and an accurate census carried out at the end of the experiment. (2) Production tanks (Hunterston) Because of the dramatic effect of sand in curing BPN, demonstrated by McVicar and White (1979), sand substrate was introduced into the ten 1.5 m3 glass reinforced concrete production tanks holding 15000 fish on day 80 before the onset of disease and maintained at a depth of approximately 10 mm thereafter. All mortalities were recorded daily. (3) Aquarium tanks (Marine Laboratory) Approximately 50 “0” group sole were transferred from WFA Hunterston to the Marine Laboratory aquarium in Aberdeen in April before any appearance of BPN at Hunterston and were held in glass fibre tanks without sand with seawater recirculated through filters individual to each tank., WFA composite diet was used throughout. Using these non diseased stocks the following three experiments on transmission were carried out: (a) co-habitation with BPN diseased sole from Hunterston; (b) intraperitioneal injection of bacteria cultured from diseased sole at Hunterston; (c) removal of dorsal scales and damage to fins while in contact with bacterial isolates from Hunterston. (II) Bat teriology Standard loop samples were taken from the centre of lesions, subdermally close to lesions, from liver and from blood and were streaked on to Zobell marine agar and Furunculosis agar with and without 4% NaCl. Sole showing as wide a range of disease stages as possible were selected. RESULTS
(1) Experimental
tanks (Fig. 1) (Table I)
(I) Continuous sand substrate (tanks 2, 4) Few mortalities were recorded in the two populations of sole held continuously on a sand substrate (tanks 2 and 4) (even after introduction of a diseased sole)_ No signs of BPN lesions were ever observed in these tanks but the small numbers of mortalities, common to both 5-10 days after introducing diseased sole many have been associated with a low level occurrence of the disease.
I
2 65 1 40 92 17 3 1 1 2 1 0
350 348 283 143 51 34 31 30 29 27 26
0 1 2 3 4 5 6 7 8 9 10
0.6 19.1 62.7 85.4 SO.3 91.1 91.4 91.7 92.3 92.6 ’
% 273 269 269 268 268 268 266 266 266 266 266
No
aBPN induced on Day 61, by adding one diseased fish. bSand substrate added. ‘No change in % has occurred.
Mort.
NO
45
Days from first BPN associated mortality
Day of first BPN associated mortality
Tank 1
Pattern of sole mortality associated with BPN
TABLE
1.5 c
4 0 1 0 0 2 0 0 0 0 0 2.6 c
1.8 c c
%
Mart.
66a
Tank2
261 261 58 t 249 246 241 230 225 221 220
No
3
9 3 5 11 5 4 1 0
5 0
Mort.
66a
3.0 6.4 7.5 9.4 13.5 15.4 16.9 17.3 c
1.9 c
%
Tank 3
295 294 294 294 293 293 293 292 292 292 292
No.
0 1 0 0 1 0 0 0 0
1 0
Mort.
67a
= 0.7 c c 1.0 c c c c
0.3 c
%
Tank 4
318 316 274 151 51 24 15 11 9
322 318
No
2 42 123 100 27 9 4 2 0
4 0
Mart.
66a
Tank5
C
1.9 14.9 53.1 84.2 92.6 95.3 96.6 97.2
1.2 c
%
42 38 32a 26 26 26 26 24
311 05 2 78
NO
106 127 36 4 7 6 0 0 0 2 0
Mart.
57
34.1 75.0 86.5 87.8 90.0 92.0 c = c 92.6 c
‘70
Tank 6
218
(II) Tanks without sand (a) Natural disease outbreaks (tanks 1, 6). Spontaneous outbreaks of BPN with typical clinical signs appeared in tank 1 at day 44 and in tank 6 at day 56. Heavy mortalities rapidly ensued in both groups of sole, and although sand was added to tank 6 three days after the disease was first recognised, the population of both was reduced to very low levels (<30) in approximately a week (Table I). When a fish showed overt signs of the disease they normally survived less than 24 h. After the initial period of heavy mortality most of the residual fish survived until the end of the experiment. (b) Induced disease (tanks 3, 5). Overt signs of BPN appeared in the two remaining tanks without sand 2-3 days after introduction of diseased sole and significant mortalities were recorded after 4 days. In the population allowed to continue without sand (tank 5) a similar mortality pattern to that observed in natural outbreaks was found, the number surviving being reduced to 9 within 10 days. Although significant mortalities (17.3%) were also recorded in tank 3 in the lo-day period after the disease appeared (Table I) no mortalities attributable to BPN were observed from 6 days after adding sand to that tank. All tanks showed some mortalities towards the end of the experiment, without clinical signs of BPN, and these may have been associated with inadequacies of the Lumbricillus diet for sole of that age, or to the start of the weaning programme. (2) Production tanks With the routine use of sand in production tanks, total mortalities of juveniles were reduced to a low level (19%) in comparison to previous years figures of 49% in 1978 and 85% in 19’77 (WFA, unpublished report). However, other conditions, particularly weaning and composition of prepared diets, were not identical in the different years and as many of these stocks were under different trials throughout the periods it is probable other factors had some influence on the relative mortalities. (3) Aquarium experiments The sole transferred from Hunterston to the Marine Laboratory Aberdeen did not develop BPN although they were reared in the absence of sand. Similarly no BPN signs or mortalities resulted from the transmission experiments. (4) Bacteriology of lesions Plating of samples from open ulcers normally produced prolific growth of a range of morphological types of bacteria predominately gram-negative rods. Samples taken from liver and blood of affected sole gave variable
219
results, many fish being negative and others producing pure cultures, but not always of one bacterial colony morphology. Because of the failure to regularly isolate one bacterial type in association with the disease, detailed identification of the bacteria was not made. The morphology and antibiotic sensitivity of pure isolates from blood were not inconsistent with those belonging to the Vibrio/Aeromonas group. DISCUSSION
The above results demonstrate that BPN is an infectious disease which may easily be transmitted between Dover sole by co-habitation. Although a specific disease agent has not yet been isolated evidence was strong for the existence of one. A high degree of virulence was indicated by the ease which the disease could be induced in tank populations without sand, and the potential risk in commercial conditions of disease dissemination through movement of affected stock well illustrated. This confirms the suggestion made by McVicar and White (1979) that an outbreak of BPN in the Institute of Marine Biochemistry (IMB) Aberdeen was associated with the introduction of diseased stock a short time previously from Hunterston. The natural occurrence of the disease both within the WFA Hunterston farm and in other sole rearing units was typically sporadic. During the present study in both the experimental and production tanks, and in earlier WFA stocks (noted by McVicar and White (1979)), there was no synchronism in the outbreaks of disease in different groups of apparently identical fish. Some tanks were severely affected while neighbouring tanks on the same water supply escaped disease for long periods, or even totally. Signs of a BPN-like disease have also been noted in the sole reared at the CNEXO laboratory in Brest, France (Girin and Harache, 1979) and a serious outbreak occurred in a commercial farm adjacent to the WFA Hunterston unit. However, no disease signs have been found in a west coast site of the same company, although stocks and husbandry techniques were similar in both sites (A. Brown, personal communication, 1980). The disease was also absent from the IMB aquarium in Aberdeen until the introduction of affected stock from Hunterston (McVicar and White, 1979), and sole reared in the MAFF Lowestoft Laboratory have not shown any signs of BPN (B. Howell, personal communication, 1980). This suggests the disease may have a restricted geographical distribution. Failure to experimentally transmit BPN in the aquarium and the efficiency of sand in preventing and curing the disease suggests that an outbreak of disease may be dependent on particular conditions. There was some evidence from the few numbers of mortalities in sand-bottomed tanks immediately following experimental challenge with BPN that infection was being contracted by at least some fish in these, but without the widespread, serious effects observed in tanks without substrate; it is not known if sand prevents infection or the onset of clinical disease. It has been shown that a variety
220
of factors alone or in combination may influence fish disease resistance or susceptibility (Wedemeyer et al., 1976), but apart from sand, those having significant roles in the BPN/sole relationship have not been identified. Two possible roles for sand have been identified; sand cover in tanks can greatly reduce stress as measured by heart beat and respiration rates (Peyraud and Labat, 1962), and preliminary studies with the light and electron microscope have indicated that sand may be beneficial in assisting sloughing of moribund and dead cells from the skin surface (unpublished results). When conditions were suitable, a rapid spread of disease was evident from the explosive mortality rate within very few days of BPN first appearing in a tank of sole. Both production and experimental tanks (with natural and induced disease) showed the same pattern. In this aspect BPN has many of the features of an acute form of disease, although, as was also noted by McVicar and White (1979), in residual populations surviving the initial outbreak, a chronic phase may be shown with mortalities persisting at a lower level for long periods. Pathogenicity of the disease was high during the present study, most sole dying within 24-48 h after the first appearance of clinical signs. The pathology of the disease was as described by McVicar and White (1979): localised darkening of skin especially between fin rays, the development of a large black area and finally sloughing of epithelium forming open ulcers. Initial blistering of the skin surface occurred in larger fish (McVicar and White, 1979), but was seldom observed in “0” sole, possibly because of the rate of progression of the lesion in them. Fish probably died because of extensive loss of body fluids through the ulcers or secondary infection of the lesion, and septicaemia. No protozoan or metazoan parasites were associated with the disease but bacteria were typically found in advanced lesions, often at high concentrations. However, the evidence at present suggests that these were probably secondary opportunistic invaders. The types of bacterium were variable in different diseased fish, largely reflecting the groups commonly found on the surface of healthy sole and in the tank water, and were .neither uniquely associated with BPN outbreaks nor showed the sporadic pattern of occurrence of the disease. The absence of systemic infection, even in severely damaged fish, and the failure to induce disease by experimentally challenging sole with culture isolates, also suggested these bacteria were not the primary cause of the disease. A skin disease of cod in Danish waters, the ulcus syndrome, with similar pathology and transmission characteristics was considered by Larsen and Jensen (1979) not to be primarily due to bacterial infection, although various species including Vibrio and Aeromonas were implicated in the advanced development of ulcers. One of two viruses isolated from diseased cod by Jensen et al. (1979) and Jensen and Larsen (1981) caused pathogenesis typical of the initial stage of the ulcus-syndrome, but no virus associated with BPN lesions has been detected by tissue culture and electron microscope studies.
221
The beneficial effects of sand in preventing and curing BPN were so spectacular that, as a prophylactic measure in a farm site where there is a risk of the disease being contracted, it is recommended that young sole be reared through high disease risk periods on a sand substrate. High mortalities could occur if sand was only used to cure established disease because of its high rate of spread, the very rapid pathogenic effect, the 4-5 day delay in the effect of sand and the difficulty in recognising very early lesions. Larger sole (“I” group and above) were less seriously affected and succumbed more slowly to the disease so that it may be possible to pursue a sand treatment policy with such stocks. Although the use of sand causes husbandry difficulties resulting in some increased effort in tank maintenance (estimated at about 15% greater than in tanks without substrate), sole reared on sand have shown a greatly increased growth rate of 0.87% per day over a trial period of 84 days, compared with an identical stock held in tanks without sand substrate with a growth rate of 0.53% per day (WFA, unpublished report). Enhanced food conversion ratios were also observed. It is therefore a matter of deciding if the advantages of sand outweigh the disadvantages. However, recent experience with the use of a raised sump to retain sand in production tanks at Hunterston has shown that a self-cleaning sanded tank requiring minimum additional maintenance is possible providing certain husbandry parameters are correct. Water flow must be sufficient to flush uneatenfood away but not too great to remove sand; a raised rim around the sump must be introduced to prevent sand loss; stocking density must be sufficient for the fish to agitate the sand frequently to keep particulate waste in suspension and prevent anaerobism; and overfeeding of the stock must be avoided. Extra maintenance of properly adjusted tanks was reduced to twice daily flushing of sumps and periodic replenishment or replacement of sand. ACKNOWLEDGEMENTS
The authors would like to express their thanks to Marine Laboratory and White Fish Authority staff who made helpful suggestions during the preparation of this manuscript.
REFERENCES Girin, M. and Harache, Y., 1979. L’elevage des poissons en eau de mer; nouveaux resultats francais en mat&e de recherche et de developpement. In: T.V.R. Pillay and W.A. Dill (Editors), Advances in Aquaculture. Fishing News Book Ltd, Farnham, Surrey, pp. 173-179. Jensen, N.J. and Larsen, J.L., 1981. The ulcus-syndrome in cod (Gadus morhua). IV. Transmission and inoculation experiments. Implication of an icoschedral virus. Nord. Veterinaermed., in press.
222 Jensen, N.J., Bloch, B. and Larsen, J.L., 1979. The ulcus-syndrome in cod (Gadus morhua). III. A preliminary virological report. Nord. Veterinaermed., 31: 436-442. Larsen, J.L. and Jensen, N.J., 1979. The ulcus-syndrome in cod (Gadus morhua). II. A bacteriological investigation. Nord. Veterinaermed., 31: 289-296. McVicar, A.H. and White, P.G., 1979. Fin and skin necrosis of cultivated Dover sole (Soieo solea (L.)). J. Fish Dis., 2: 557-562. Peyraud, C. and Labat, R., 1962. Reactions cardio respiratoires observees chez la sole au cows de l’ensablement. Hydrobiologia, 19: 351-356 Wedemeyer, G.A., Meyer, F.P. and Smith, L., 1976. Environmental Stress and Fish Diseases. T.F.H. Publications Inc. Ltd., Neptune City, NJ, 192 pp.
Printed in The Netherlands
213
THE PREVENTION AND CURE OF AN INFECTIOUS DISEASE IN CULTIVATED JUVENILE DOVER SOLE, SOLEA SOLEA (L.)
A.H. McVICAR and P.G. WHITE?* Marine Laboratory,
Victoria Road, Aberdeen
+ WFA Marine Cultivation Unit, Hunterston,
(Great Britain) Ayrshire
(Great Brifain)
*Present address: Hellenic Fish Farm, Varda, Ilias (Greece). (Accepted
5 May 1981)
ABSTRACT McVicar, A.H. and White, P.G., 1982. The prevention and cure of an infectious disease of cultivated juvenile Dover sole, Solea solea (L.). Aquaculture, 26: 213-222. The disease condition “Black Patch Necrosis” which causes serious mortalities of cultivated Dover sole through the development of areas of fin and skin necrosis, was shown to be infectious by co-habitation of diseased and healthy stocks. Spread of the disease throughout an affected tank was rapid and high pathogenicity was evident, with most affected sole dying l-2 days after the first appearance of clinical signs. Addition of a sand substrate to tanks both prevented occurrence of the disease and cured an established disease situation. No aetiological agent has yet been identified.
INTRODUCTION
The culture of Dover sole has been under investigation at the White Fish Authority’s (WFA) Marine Farming Unit at Hunterston for several years. Serious mortalities associated with the development of fin and skin lesions were first noted in “0” group stocks in 1976 and the disease condition, now colloqually known as “Black Patch Necrosis” (BPN) has since recurred each year in “0” group and “I” group stocks and has also affected stocks on a commercial farm. In 1977 an 83% loss of the total metamorphosed stock of over 51000 sole was largely attributed to the disease. McVicar and White (1979) described the clinical signs and histopathology of the disease and gave some circumstantial evidence of the involvement of an unidentified infective agent. Control of the disease’by standard methods proved difficult. A wide range of antibiotics administered orally and as a bath had little effect. Continuous formalin treatment held the disease in check although did not cure it, and toxic effects of the treatment to the sole were found. However, it was demonstrated that the addition of a sand substrate to affected tanks would rapidly cure the disease and
0044-8486/82/0000-0000/$02.75
0
1982 Elsevier Scientific Publishing Company
214
improved hatchery survivals of 51% in 1978 and 81% in 1979 may be largely attributed to the more general use of sand, although other changes in husbandry methods probably also contributed. The objectives of the study reported in this paper were to investigate the possible infective nature of the disease and to determine the prophylactic benefits from using sand continuously in rearing tanks. MATERIALS
AND METHODS
The study was carried out on sole hatched and reared at Hunterston and three main groups of fish were used: (1) Experimental tanks (Hunterston) Experiments were performed at Hunterston in conditions which matched as closely as possible those used routinely and successfully by the WFA in juvenile sole rearing. A single stock of approximately 2000 juvenile sole, derived from one production hatching, was subdivided randomly at 70 days post-metamorphosis (16 August) into six approximately equal groups and held in 1 m diamter (75 1) glass reinforced plastic tanks each with an outflow through a central bottom screen (28 cm diameter with 3 mm holes). Raised rims formed from 3 cm diameter polythene tubing were placed round the outlets of all tanks to prevent sand substrate washing out and in tanks without sand to maintain standard conditions between tanks. Fine grained pre-washed quarry sand, as used in building construction, was placed and maintained in selected tanks at a depth of approximately 20 mm over the whole tank bottom. All of the tanks (numbered l-6) were situated in a single row on a common water supply (mixed warm power station discharge and sub-sand filtered seawater at a flow rate of 11 l/h to each tank). Temperatures, monitored twice daily, were in the range 14.7 to 21.2”C and lighting was provided continuously at a level of 200 lux by four 100 Watt bulbs. Individual hand nets were used for each tank and precautions were taken to avoid contact of water or fish between tanks. For most of the experimental period the fish were fed only a live diet of redworm, Lumbricillus riualis Levinsen aug. Ditlevsen, given twice daily to satiation to avoid complications associated with weaning. However, because of supply problems with Lumbricillus, it was necessary to start a weaning programme towards the end of the period. The following procedures were followed in the sequence (a)-(d) (note Fig. 1) (a) Sand was introduced into two tanks (Nos 2,4) at the start of the experiment to determine any prophylactic effects.
215
r
+ -
SAND
0 -
DISEASE
ADDEG
TO TANK AND
APPEAREO
400-
MAINTAINED
THEREAFTER
SPONTANEOUSLY
-
-
WEANING
PERIOD
0. TANK 1
2004 '....._._._
O-
4*r
t_
-----
-'
-'I
TANK 2 ...-_.__
4
200
t 0 f&o-._-_ cn 200. .s .1 2 i?l O-
4
_--.__t,
TANK 3 ....,
-__ ... I
-'
4
z 4OOr + 2'jt 2
1
-1
-------.,
TANK 4 ._.-_
200
4--_.
400
TANK 5
200 t
400-
0
---
TANK 6
200OL
'.._ 20
40
60
-80
-100
1 110
days Fig. 1. Survival of juvenile Dover sole in trial tanks l-6. Day 0 = 70 days post metamorphosis. Broken lines indicate one or more mortalities.
(b) The disease was allowed to occur naturally in any two tanks (it appeared in two without sand Nos. 1 and 6) Sand was then added to one of them (No. 6) in an attempt to cure the disease.
216
(c) To investigate the infectivity of the disease, all tanks (i.e. three then with sand and three without) were challenged with BPN by introducing one sole showing overt disease signs into each. (d) On the outbreak of induced disease in two tanks (Nos. 3 and 5, both without substrate) sand was added to one and the other allowed to continue without interference. All dead fish were removed from the tanks and recorded daily and an accurate census carried out at the end of the experiment. (2) Production tanks (Hunterston) Because of the dramatic effect of sand in curing BPN, demonstrated by McVicar and White (1979), sand substrate was introduced into the ten 1.5 m3 glass reinforced concrete production tanks holding 15000 fish on day 80 before the onset of disease and maintained at a depth of approximately 10 mm thereafter. All mortalities were recorded daily. (3) Aquarium tanks (Marine Laboratory) Approximately 50 “0” group sole were transferred from WFA Hunterston to the Marine Laboratory aquarium in Aberdeen in April before any appearance of BPN at Hunterston and were held in glass fibre tanks without sand with seawater recirculated through filters individual to each tank., WFA composite diet was used throughout. Using these non diseased stocks the following three experiments on transmission were carried out: (a) co-habitation with BPN diseased sole from Hunterston; (b) intraperitioneal injection of bacteria cultured from diseased sole at Hunterston; (c) removal of dorsal scales and damage to fins while in contact with bacterial isolates from Hunterston. (II) Bat teriology Standard loop samples were taken from the centre of lesions, subdermally close to lesions, from liver and from blood and were streaked on to Zobell marine agar and Furunculosis agar with and without 4% NaCl. Sole showing as wide a range of disease stages as possible were selected. RESULTS
(1) Experimental
tanks (Fig. 1) (Table I)
(I) Continuous sand substrate (tanks 2, 4) Few mortalities were recorded in the two populations of sole held continuously on a sand substrate (tanks 2 and 4) (even after introduction of a diseased sole)_ No signs of BPN lesions were ever observed in these tanks but the small numbers of mortalities, common to both 5-10 days after introducing diseased sole many have been associated with a low level occurrence of the disease.
I
2 65 1 40 92 17 3 1 1 2 1 0
350 348 283 143 51 34 31 30 29 27 26
0 1 2 3 4 5 6 7 8 9 10
0.6 19.1 62.7 85.4 SO.3 91.1 91.4 91.7 92.3 92.6 ’
% 273 269 269 268 268 268 266 266 266 266 266
No
aBPN induced on Day 61, by adding one diseased fish. bSand substrate added. ‘No change in % has occurred.
Mort.
NO
45
Days from first BPN associated mortality
Day of first BPN associated mortality
Tank 1
Pattern of sole mortality associated with BPN
TABLE
1.5 c
4 0 1 0 0 2 0 0 0 0 0 2.6 c
1.8 c c
%
Mart.
66a
Tank2
261 261 58 t 249 246 241 230 225 221 220
No
3
9 3 5 11 5 4 1 0
5 0
Mort.
66a
3.0 6.4 7.5 9.4 13.5 15.4 16.9 17.3 c
1.9 c
%
Tank 3
295 294 294 294 293 293 293 292 292 292 292
No.
0 1 0 0 1 0 0 0 0
1 0
Mort.
67a
= 0.7 c c 1.0 c c c c
0.3 c
%
Tank 4
318 316 274 151 51 24 15 11 9
322 318
No
2 42 123 100 27 9 4 2 0
4 0
Mart.
66a
Tank5
C
1.9 14.9 53.1 84.2 92.6 95.3 96.6 97.2
1.2 c
%
42 38 32a 26 26 26 26 24
311 05 2 78
NO
106 127 36 4 7 6 0 0 0 2 0
Mart.
57
34.1 75.0 86.5 87.8 90.0 92.0 c = c 92.6 c
‘70
Tank 6
218
(II) Tanks without sand (a) Natural disease outbreaks (tanks 1, 6). Spontaneous outbreaks of BPN with typical clinical signs appeared in tank 1 at day 44 and in tank 6 at day 56. Heavy mortalities rapidly ensued in both groups of sole, and although sand was added to tank 6 three days after the disease was first recognised, the population of both was reduced to very low levels (<30) in approximately a week (Table I). When a fish showed overt signs of the disease they normally survived less than 24 h. After the initial period of heavy mortality most of the residual fish survived until the end of the experiment. (b) Induced disease (tanks 3, 5). Overt signs of BPN appeared in the two remaining tanks without sand 2-3 days after introduction of diseased sole and significant mortalities were recorded after 4 days. In the population allowed to continue without sand (tank 5) a similar mortality pattern to that observed in natural outbreaks was found, the number surviving being reduced to 9 within 10 days. Although significant mortalities (17.3%) were also recorded in tank 3 in the lo-day period after the disease appeared (Table I) no mortalities attributable to BPN were observed from 6 days after adding sand to that tank. All tanks showed some mortalities towards the end of the experiment, without clinical signs of BPN, and these may have been associated with inadequacies of the Lumbricillus diet for sole of that age, or to the start of the weaning programme. (2) Production tanks With the routine use of sand in production tanks, total mortalities of juveniles were reduced to a low level (19%) in comparison to previous years figures of 49% in 1978 and 85% in 19’77 (WFA, unpublished report). However, other conditions, particularly weaning and composition of prepared diets, were not identical in the different years and as many of these stocks were under different trials throughout the periods it is probable other factors had some influence on the relative mortalities. (3) Aquarium experiments The sole transferred from Hunterston to the Marine Laboratory Aberdeen did not develop BPN although they were reared in the absence of sand. Similarly no BPN signs or mortalities resulted from the transmission experiments. (4) Bacteriology of lesions Plating of samples from open ulcers normally produced prolific growth of a range of morphological types of bacteria predominately gram-negative rods. Samples taken from liver and blood of affected sole gave variable
219
results, many fish being negative and others producing pure cultures, but not always of one bacterial colony morphology. Because of the failure to regularly isolate one bacterial type in association with the disease, detailed identification of the bacteria was not made. The morphology and antibiotic sensitivity of pure isolates from blood were not inconsistent with those belonging to the Vibrio/Aeromonas group. DISCUSSION
The above results demonstrate that BPN is an infectious disease which may easily be transmitted between Dover sole by co-habitation. Although a specific disease agent has not yet been isolated evidence was strong for the existence of one. A high degree of virulence was indicated by the ease which the disease could be induced in tank populations without sand, and the potential risk in commercial conditions of disease dissemination through movement of affected stock well illustrated. This confirms the suggestion made by McVicar and White (1979) that an outbreak of BPN in the Institute of Marine Biochemistry (IMB) Aberdeen was associated with the introduction of diseased stock a short time previously from Hunterston. The natural occurrence of the disease both within the WFA Hunterston farm and in other sole rearing units was typically sporadic. During the present study in both the experimental and production tanks, and in earlier WFA stocks (noted by McVicar and White (1979)), there was no synchronism in the outbreaks of disease in different groups of apparently identical fish. Some tanks were severely affected while neighbouring tanks on the same water supply escaped disease for long periods, or even totally. Signs of a BPN-like disease have also been noted in the sole reared at the CNEXO laboratory in Brest, France (Girin and Harache, 1979) and a serious outbreak occurred in a commercial farm adjacent to the WFA Hunterston unit. However, no disease signs have been found in a west coast site of the same company, although stocks and husbandry techniques were similar in both sites (A. Brown, personal communication, 1980). The disease was also absent from the IMB aquarium in Aberdeen until the introduction of affected stock from Hunterston (McVicar and White, 1979), and sole reared in the MAFF Lowestoft Laboratory have not shown any signs of BPN (B. Howell, personal communication, 1980). This suggests the disease may have a restricted geographical distribution. Failure to experimentally transmit BPN in the aquarium and the efficiency of sand in preventing and curing the disease suggests that an outbreak of disease may be dependent on particular conditions. There was some evidence from the few numbers of mortalities in sand-bottomed tanks immediately following experimental challenge with BPN that infection was being contracted by at least some fish in these, but without the widespread, serious effects observed in tanks without substrate; it is not known if sand prevents infection or the onset of clinical disease. It has been shown that a variety
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of factors alone or in combination may influence fish disease resistance or susceptibility (Wedemeyer et al., 1976), but apart from sand, those having significant roles in the BPN/sole relationship have not been identified. Two possible roles for sand have been identified; sand cover in tanks can greatly reduce stress as measured by heart beat and respiration rates (Peyraud and Labat, 1962), and preliminary studies with the light and electron microscope have indicated that sand may be beneficial in assisting sloughing of moribund and dead cells from the skin surface (unpublished results). When conditions were suitable, a rapid spread of disease was evident from the explosive mortality rate within very few days of BPN first appearing in a tank of sole. Both production and experimental tanks (with natural and induced disease) showed the same pattern. In this aspect BPN has many of the features of an acute form of disease, although, as was also noted by McVicar and White (1979), in residual populations surviving the initial outbreak, a chronic phase may be shown with mortalities persisting at a lower level for long periods. Pathogenicity of the disease was high during the present study, most sole dying within 24-48 h after the first appearance of clinical signs. The pathology of the disease was as described by McVicar and White (1979): localised darkening of skin especially between fin rays, the development of a large black area and finally sloughing of epithelium forming open ulcers. Initial blistering of the skin surface occurred in larger fish (McVicar and White, 1979), but was seldom observed in “0” sole, possibly because of the rate of progression of the lesion in them. Fish probably died because of extensive loss of body fluids through the ulcers or secondary infection of the lesion, and septicaemia. No protozoan or metazoan parasites were associated with the disease but bacteria were typically found in advanced lesions, often at high concentrations. However, the evidence at present suggests that these were probably secondary opportunistic invaders. The types of bacterium were variable in different diseased fish, largely reflecting the groups commonly found on the surface of healthy sole and in the tank water, and were .neither uniquely associated with BPN outbreaks nor showed the sporadic pattern of occurrence of the disease. The absence of systemic infection, even in severely damaged fish, and the failure to induce disease by experimentally challenging sole with culture isolates, also suggested these bacteria were not the primary cause of the disease. A skin disease of cod in Danish waters, the ulcus syndrome, with similar pathology and transmission characteristics was considered by Larsen and Jensen (1979) not to be primarily due to bacterial infection, although various species including Vibrio and Aeromonas were implicated in the advanced development of ulcers. One of two viruses isolated from diseased cod by Jensen et al. (1979) and Jensen and Larsen (1981) caused pathogenesis typical of the initial stage of the ulcus-syndrome, but no virus associated with BPN lesions has been detected by tissue culture and electron microscope studies.
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The beneficial effects of sand in preventing and curing BPN were so spectacular that, as a prophylactic measure in a farm site where there is a risk of the disease being contracted, it is recommended that young sole be reared through high disease risk periods on a sand substrate. High mortalities could occur if sand was only used to cure established disease because of its high rate of spread, the very rapid pathogenic effect, the 4-5 day delay in the effect of sand and the difficulty in recognising very early lesions. Larger sole (“I” group and above) were less seriously affected and succumbed more slowly to the disease so that it may be possible to pursue a sand treatment policy with such stocks. Although the use of sand causes husbandry difficulties resulting in some increased effort in tank maintenance (estimated at about 15% greater than in tanks without substrate), sole reared on sand have shown a greatly increased growth rate of 0.87% per day over a trial period of 84 days, compared with an identical stock held in tanks without sand substrate with a growth rate of 0.53% per day (WFA, unpublished report). Enhanced food conversion ratios were also observed. It is therefore a matter of deciding if the advantages of sand outweigh the disadvantages. However, recent experience with the use of a raised sump to retain sand in production tanks at Hunterston has shown that a self-cleaning sanded tank requiring minimum additional maintenance is possible providing certain husbandry parameters are correct. Water flow must be sufficient to flush uneatenfood away but not too great to remove sand; a raised rim around the sump must be introduced to prevent sand loss; stocking density must be sufficient for the fish to agitate the sand frequently to keep particulate waste in suspension and prevent anaerobism; and overfeeding of the stock must be avoided. Extra maintenance of properly adjusted tanks was reduced to twice daily flushing of sumps and periodic replenishment or replacement of sand. ACKNOWLEDGEMENTS
The authors would like to express their thanks to Marine Laboratory and White Fish Authority staff who made helpful suggestions during the preparation of this manuscript.
REFERENCES Girin, M. and Harache, Y., 1979. L’elevage des poissons en eau de mer; nouveaux resultats francais en mat&e de recherche et de developpement. In: T.V.R. Pillay and W.A. Dill (Editors), Advances in Aquaculture. Fishing News Book Ltd, Farnham, Surrey, pp. 173-179. Jensen, N.J. and Larsen, J.L., 1981. The ulcus-syndrome in cod (Gadus morhua). IV. Transmission and inoculation experiments. Implication of an icoschedral virus. Nord. Veterinaermed., in press.
222 Jensen, N.J., Bloch, B. and Larsen, J.L., 1979. The ulcus-syndrome in cod (Gadus morhua). III. A preliminary virological report. Nord. Veterinaermed., 31: 436-442. Larsen, J.L. and Jensen, N.J., 1979. The ulcus-syndrome in cod (Gadus morhua). II. A bacteriological investigation. Nord. Veterinaermed., 31: 289-296. McVicar, A.H. and White, P.G., 1979. Fin and skin necrosis of cultivated Dover sole (Soieo solea (L.)). J. Fish Dis., 2: 557-562. Peyraud, C. and Labat, R., 1962. Reactions cardio respiratoires observees chez la sole au cows de l’ensablement. Hydrobiologia, 19: 351-356 Wedemeyer, G.A., Meyer, F.P. and Smith, L., 1976. Environmental Stress and Fish Diseases. T.F.H. Publications Inc. Ltd., Neptune City, NJ, 192 pp.