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Bacterial disease of sea bass (Dicentrarchus labrax (L.)) reared in the laboratory: An approach to treatment
Aquaculture, 10 (1977) 317-322 317 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BACTERIAL DISEASE OF SEA BASS (DIClW?‘RARCHUS LABRAX (L.)) REARED IN THE LABORATORY: AN APPROACH TO TREATMENT
MARIA HELENA BARAHONA-FERNANDES Faculdade
de Ci.&zcias, Departamento
de Zoofogia,
Lisbon
(Portugal)
Present address: Centre Oceanologique de Bretagne, P.B. 337, 29273 Brest (France) Contribution no. 524 Departement Scientifique du Centre Oceanologique de Bretagne (Received 21 December 1976)
ABSTRACT Barahona-Fernandes, M.H., 1977. Bacterial disease of sea bass (Dicentrarchus labrax (L.)) reared in the laboratory: an approach to treatment. Aquaculture, 10: 317-322. Two nitrofuran derivatives and one antiseptic were tested on the control of a bacterial disease in laboratory reared sea bass. Best results were obtained with 20-min bath treatments of Furanace at 12 mg/l.
INTRODUCTION
For the last three years there has been a high mortality between May and September in sea bass juveniles already well adapted to pellets and reared at the Centre Oceanologique de Bretagne. Clinical symptoms include progressive haemorrhaging in cervical, jugular, opercular and ventral regions, white stripes on the middle body and fin rot in the last stages of the disease. There is a consequent mortality of 30-40% affecting only fish from 70 to 120 days old (300-l 900 mg). In contrast, it has never occurred on 1-2-year-old fish kept in the laboratory for growth experiments. The gross pathology and the epizootic pattern of the disease suggested bacteria were the causative agents, as the disease was similar to those described by Oppenheimer (1962), Akazawa (1968), Amend (1970), Bullock and McLaughlin (1970), Pacha and Ordal (1970), Sindermann (1970) and Van Duijn (1972) for other species of fish. At the Laboratoire National de Pathologie des Animaux Aquatiques at the Centre Oceanologique de Bretagne the presence of both Pseudomonas sp. and Vibrio sp. as well as myxobacteria was demonstrated; however, the primary aetiological agent could not be confirmed. Recent developments in the use of nitrofuran for treatment of bacterial infection are described by Shimizu and Takase (1967), Takase et al. (1968), Amend (1970), Amend and Ross (1970), Ross (1972), Pearse et al. (1974)
and Holt et al. (1975); they note its absorption by the skin and gills and its low price compared to other antibiotic agents. Therefore, two nitrofuran derivatives, Furanace (from R. Bellon Laboratory) and Furoxone (= Furazolidone from Hess and Clark C.P.), were compared to a mixture of zinc-free malachite green (E. Merck Co.) and formalin adapted for salt water at Station Marine d’Endoume from the work of Leteux and Meyer (1972). MATERIAL
AND METHODS
All fish used were grown from larvae hatched in the laboratory and had been reared in half-recirculating units following the technique described by Girin et al. (1975) and Barahona-Fernandes and Girin (1976). After the pellet adaptation period, all juveniles were fed on “Aqualim” dry pellets from Grandes Semouleries de l’Ouest, in 400-2 000 1 PVC square tanks with a 14-h photoperiod. Samples of ten fish were taken every 10 days and preserved in 5% neutralized formalin for measuring and weighing. Dead fish were removed and counted daily. The three drugs, Furanace, Furoxone and malachite green-formalin, were administered in a bath: at the start of treatment the water inlet was closed, to be reopened 20 min later, allowing slow removal of the drug. The duration and concentration (12 mg/l) of the bath treatment used for Furanace were similar to those used by Takase et al. (1968) and Pearse et al. (1974) for other species of marine fish. The Furoxone concentration was also 12 mg/l, that is, ten times lower than the one used by Shimizu and Takase (1967) for in vitro studies. For the mixture of malachite green and formalin, the concentration was 0.04 cc/l (3 g zinc-free malachite green in 1 1 of 40% of formalin). Experiment 1 was carried out to test the relative effect of these drugs. Five batches of 500 fish from the same spawn were taken from a 2-m3 tank containing 5 010 fish, 87 days old, at a biomass of 6 g/l. The temperature was 23°C and the flow rate 400 l/h. There was 12.9% mortality due to the disease in the 5 days preceding the experiment. Each batch of 500 fish was transferred into a 400-l tank at 18”C, with an initial biomass of 1.2 g/l and a flow rate of 80 l/h. One received a Furanace treatment, one a Furoxone treatment, one a malachite green-formalin treatment, one no treatment (control tank) and the last one no treatment but the “Aqualim” pellets were supplemented with 4% of a mixture of vitamins and oligoelements (Metailler and Girin, 1976). All tanks received the same Furanace treatment 8 days later. Experiment 2 compared a Furanace treatment in the original tank with no treatment on part of the population transferred to a clean tank. The original Z-m3 tank A contained 4 167 fish, 93 days old and from the same spawn. The mortality in the last 5 days before the experiment was only 3.6% but a large percentage of sick fish were noted. Water temperature was 23”C, flow rate of 400 l/h and the biomass I.3 g/l. A batch of 1 360 fish was moved into
319
a 400-l tank at 18”C, with a flow rate of 40 l/h and a biomass of 2.3 g/l, and received no treatment (tank A’). The remaining fish in tank A received a Furanace treatment. Both tanks received the same Furanace treatment 9 days later.. Experiment 3 was carried out to test a Furoxone treatment on handled and unhandled fish. It was started in 2-m3 tanks with 96-day-old (fish from the same spawn; tank B contained 3 274 fish, water at lS”C, had a flow rate of 400 l/h and a biomass 1.7 g/l. Tank C contained 1 265 fish in water at 24°C had a flow rate of 400 l/h and a biomass 0.6 g/l. In the last 5 days before the experiment, tank B had a disease mortality of 15.2% and tank C 15.9%. A batch of 1 064 fish was transferred from tank B to a similar one (tank B’) and then both were treated with Furoxone. Tank C received no treatment. Initial biomass in tank B was 1.2 g/l; that in tank B’ and in tank C, 0.6 g/l. For technical reasons, in experiments 2 and 3 it was not possible to work at the same temperature. The evolution of the biomass in each tank was calculated from the growth curve of weight and the daily number of fish in the tank. RESULTS
The results of experiment 1 are plotted in Fig. 1. The mortality after 8 days was 28% in the control tank, 30.4% in the batch whose pellets were supplemented with vitamins and oligoelements, 20% in the malachite green-formalin treated tank, 9.2% in the Furoxone treated tank and 6.4% in the Furanace treated one. % MORTALITY
T
Fig. 1. Mortality curves of experiment 1: control tank ooo ; vitamins and oligoelements supplemented tank - * a;malachite green-formalin in treated tank -W-V--; Furoxone treated tank -; Furanace treated tank - - - -. l
320
For experiment 2, the mortality curves are plotted in Fig. 2 a and the changes in the biomass in each tank are shown in Fig. 2 b. Eight days after the beginning of the experiment, the mortality was 5% in the treated original tank A and 35% in the transferred untreated batch (A’). For experiment 3, the mortality curves are plotted in Fig. 3 a and the changes in the biomass in Fig. 3 b. Six days after the beginning of the experiment, the mortality was 8% in the untreated tank C; in the Furoxone treated tank B it was 11.6%. a little higher than in the transferred and Furoxone treated batch B’, where it was5.4%. q/t
% MORTALITY T
,
3.0
42_
I I l,‘e
35 -
)c--
28 2.5
34_
2,4
/!
30_ /
I
2,2
26 22_
ti;
Ia_ _ 14 _ IO 1
iI $1 =I I I I
I’ I I
11111,11111,,,,,1,,11,,,,
93
95
97
99
IO1 103 105 107 109 Ill
85
57
59
91
93
95
(days)
97
99
IO1 103 I05 107 109 (days 1
Fig, 2. (a) Mortality curves of experiment 2; treated tank -; untreated tank ----. (b) Changes in the biomass in experiment 2: original tank ***a; Furanace treated tank A p; untreated tank A’ - - - .
%MORTALITY
pr/l
,,I-
T
Fl 96
I
97
98
I
I
I
I)
99 100 IO1 IO2
.I
lllll’lr,,,,, AGE (days)
91
93
95
97
99
IO1
)AGE (days1
Fig. 3. (a) Mortality curves of experiment 3: untreated tank C .*.*; unhandled Furoxone treated tank B-I-*-* , handled Furoxone treated tank B’ - - - . (b) Changes in the biomass in experiment 3 : original tank B -. original tank C .*.*; unhandled Furoxone treated tank B -+-*; handled Puroxone treaded tank B’ ---.
321
DISCUSSION
In the first experiment, both nitrofuran derivatives showed similarly good efficiencies in the control of disease. The antiseptic solution (malachite green-formalin) showed about half of their efficiency. The complement to the pellets of 4% more vitamins and oligoelements had no effect. These results can be compared to the cost of the different treatments: for a cubic metre of treated water, it costs roughly 0.54 F for the malachite greenformalin, 0.90 F for the Furoxone and 18.00 F for the Furanace. In the second experiment, the Furanace confirmed its good efficiency in the control of disease in the original tank, while a transfer to a new tank did not prove to be useful. In the third experiment, the results disagree with those of the first experiment; they seem to indicate that the Furoxone is less effective than the Furanace at the same concentration, Furthermore, the effect of the biomass might be more important in the development of the disease than the effect of handling the fish; of both Furoxone treated tanks, tank B, with the higher biomass and unhandled fish, had higher mortality than handled fish in tank B’ with the lower biomass. In the untreated tank C, with a low biomass, the mortality remained low. Furoxone should be tested a little more before being used as a routine method in the treatment of bacterial disease. These experiments lead us to the conclusion that the bacterial disease in reared sea bass can be efficiently controlled by a 20-min bath treatment of Furanace at 12 mg/l. A higher concentration of Furoxone might be needed to always obtain the same efficiency. One could put forward the hypothesis that malachite green-formalin treatments might control the disease before, or at very early stages of, its external symptoms. The causes of stress in our laboratory are not established, but crowding and imbalance of the pellet formulation could be important factors in the development of disease. Research on these factors must be continued, as well as studies on prophylaxis. ACKNOWLEDGEMENTS
The author acknowledges the fellowship received from the French Government through the Cultural Services of the French Embassy in Lisbon. Also, F.L. Baudin, G. Tixerant and J. Thery for their advice on pathology, L. Laubier, M. Girin, I. Paperna, F.L. Baudin and M. Francheteau for their critical reading of the manuscript, and G. Nedelec for his technical help.
REFERENCES Amend, D.F., 1970. Myxobacterial infections of salmonids: prevention and treatment. In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 258-264.
322
Amend, D.F. and Ross, A.J., 1970. Experimental control of columnaris disease with a new nitrofuran drug, P-7138. Prog. Fish Cult., 32(l): 19-25. Akazawa, H., 1968. Bacterial disease of marine fish. In: Symposium on Bacterial Fish Diseases. Bull. Jpn. Sot. Sci. Fish., 34(3): 271-272. Barahona-Fernandes, M.H. and Girin, M., 1976. Preliminary tests on the optimal pelletadaptation age for sea bass larvae (Pisces, Dicentrarchus labrax L. 1758). Aquaculture, 8: 283-290. Bullock, G.L. and McLaughlin, J.J.A., 1970. Advances in knowledge concerning bacteria pathogenic to fishes (1954-1968). In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 231-242. Girin, M., Barahona-Fernandes, M.H. and Le Roux, A., 1975. Larval Rearing of Sea Bass (Dicentrarchus labrax (L.)) with a High Survival. Presented at Int. Count. Explor. Sea, Comm. Meet. 1975/G: 14,8 pp. Holt, R.A., Conrad, J.F. and Fryer, J.L., 1975. Furanace for control of Cytophaga psychrophilu, the causitive agent of cold water disease in coho salmon. Prog. Fish Cult., 37(3): 137-139. Leteux, F. and Meyer, F.P., 1972. Mixtures of malachite green and formalin for controlling Zchthyophthirius and other protozoan parasites of fish. Prog. Fish Cult., 34(l): 21-26. Metailler, R. and Girin, M., 1976. Croissance de jeunes soles (Solea solea) nees en laboratoire et conditionnees B l’aliment compos& Int. Count. Explor. Sea, 2nd Meet. Work. Group Maricult., Hamburg, May 1976,12 pp. Oppenheimer, C.H., 1962. On marine fish diseases. In: G. Borgston (Editor), Fish as Food, vol. 2. Academic Press, New York, N.Y., 541 pp. Pacha, R.E. and Ordal, E.Y., 1970. Myxobacterial diseases of salmonids. In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 243-255. Pearse, L., Pullin, R.S.V., Conroy, D.A. and McGregor, D., 1974. Observations on the use of furanace for the control of Vibrio disease in marine flatfish. Aquaculture, 3: 295302. Sindermann, C.J., 1970. Principal Diseases of Marine Fish and Shellfish. Academic Press, New York, N.Y., London, 369 pp. Shimizu, M. and Takase, Y., 1967. A potent chemotherapeutic agent against fish diseases: 6-Hydroxymethyl-2-[ 2-(5-nitro-2-furyl) vinyl] pyridine (P-7138). Bull. Jpn. Sot. Sci. Fish., 33(6): 544-554. Takase, Y., Shimizu, M. and Kubota, S.S., 1968. The absorption and distribution of a chemotherapeutic agent, P-7138, in fishes. Bull. Jpn. Sot. Sci. Fish., 34(2): 1118-1123 Van Duijn Jr, C., 1972. Diseases of Fishes. Iliffe Books, London, 3rd edn, 372 pp.
BACTERIAL DISEASE OF SEA BASS (DIClW?‘RARCHUS LABRAX (L.)) REARED IN THE LABORATORY: AN APPROACH TO TREATMENT
MARIA HELENA BARAHONA-FERNANDES Faculdade
de Ci.&zcias, Departamento
de Zoofogia,
Lisbon
(Portugal)
Present address: Centre Oceanologique de Bretagne, P.B. 337, 29273 Brest (France) Contribution no. 524 Departement Scientifique du Centre Oceanologique de Bretagne (Received 21 December 1976)
ABSTRACT Barahona-Fernandes, M.H., 1977. Bacterial disease of sea bass (Dicentrarchus labrax (L.)) reared in the laboratory: an approach to treatment. Aquaculture, 10: 317-322. Two nitrofuran derivatives and one antiseptic were tested on the control of a bacterial disease in laboratory reared sea bass. Best results were obtained with 20-min bath treatments of Furanace at 12 mg/l.
INTRODUCTION
For the last three years there has been a high mortality between May and September in sea bass juveniles already well adapted to pellets and reared at the Centre Oceanologique de Bretagne. Clinical symptoms include progressive haemorrhaging in cervical, jugular, opercular and ventral regions, white stripes on the middle body and fin rot in the last stages of the disease. There is a consequent mortality of 30-40% affecting only fish from 70 to 120 days old (300-l 900 mg). In contrast, it has never occurred on 1-2-year-old fish kept in the laboratory for growth experiments. The gross pathology and the epizootic pattern of the disease suggested bacteria were the causative agents, as the disease was similar to those described by Oppenheimer (1962), Akazawa (1968), Amend (1970), Bullock and McLaughlin (1970), Pacha and Ordal (1970), Sindermann (1970) and Van Duijn (1972) for other species of fish. At the Laboratoire National de Pathologie des Animaux Aquatiques at the Centre Oceanologique de Bretagne the presence of both Pseudomonas sp. and Vibrio sp. as well as myxobacteria was demonstrated; however, the primary aetiological agent could not be confirmed. Recent developments in the use of nitrofuran for treatment of bacterial infection are described by Shimizu and Takase (1967), Takase et al. (1968), Amend (1970), Amend and Ross (1970), Ross (1972), Pearse et al. (1974)
and Holt et al. (1975); they note its absorption by the skin and gills and its low price compared to other antibiotic agents. Therefore, two nitrofuran derivatives, Furanace (from R. Bellon Laboratory) and Furoxone (= Furazolidone from Hess and Clark C.P.), were compared to a mixture of zinc-free malachite green (E. Merck Co.) and formalin adapted for salt water at Station Marine d’Endoume from the work of Leteux and Meyer (1972). MATERIAL
AND METHODS
All fish used were grown from larvae hatched in the laboratory and had been reared in half-recirculating units following the technique described by Girin et al. (1975) and Barahona-Fernandes and Girin (1976). After the pellet adaptation period, all juveniles were fed on “Aqualim” dry pellets from Grandes Semouleries de l’Ouest, in 400-2 000 1 PVC square tanks with a 14-h photoperiod. Samples of ten fish were taken every 10 days and preserved in 5% neutralized formalin for measuring and weighing. Dead fish were removed and counted daily. The three drugs, Furanace, Furoxone and malachite green-formalin, were administered in a bath: at the start of treatment the water inlet was closed, to be reopened 20 min later, allowing slow removal of the drug. The duration and concentration (12 mg/l) of the bath treatment used for Furanace were similar to those used by Takase et al. (1968) and Pearse et al. (1974) for other species of marine fish. The Furoxone concentration was also 12 mg/l, that is, ten times lower than the one used by Shimizu and Takase (1967) for in vitro studies. For the mixture of malachite green and formalin, the concentration was 0.04 cc/l (3 g zinc-free malachite green in 1 1 of 40% of formalin). Experiment 1 was carried out to test the relative effect of these drugs. Five batches of 500 fish from the same spawn were taken from a 2-m3 tank containing 5 010 fish, 87 days old, at a biomass of 6 g/l. The temperature was 23°C and the flow rate 400 l/h. There was 12.9% mortality due to the disease in the 5 days preceding the experiment. Each batch of 500 fish was transferred into a 400-l tank at 18”C, with an initial biomass of 1.2 g/l and a flow rate of 80 l/h. One received a Furanace treatment, one a Furoxone treatment, one a malachite green-formalin treatment, one no treatment (control tank) and the last one no treatment but the “Aqualim” pellets were supplemented with 4% of a mixture of vitamins and oligoelements (Metailler and Girin, 1976). All tanks received the same Furanace treatment 8 days later. Experiment 2 compared a Furanace treatment in the original tank with no treatment on part of the population transferred to a clean tank. The original Z-m3 tank A contained 4 167 fish, 93 days old and from the same spawn. The mortality in the last 5 days before the experiment was only 3.6% but a large percentage of sick fish were noted. Water temperature was 23”C, flow rate of 400 l/h and the biomass I.3 g/l. A batch of 1 360 fish was moved into
319
a 400-l tank at 18”C, with a flow rate of 40 l/h and a biomass of 2.3 g/l, and received no treatment (tank A’). The remaining fish in tank A received a Furanace treatment. Both tanks received the same Furanace treatment 9 days later.. Experiment 3 was carried out to test a Furoxone treatment on handled and unhandled fish. It was started in 2-m3 tanks with 96-day-old (fish from the same spawn; tank B contained 3 274 fish, water at lS”C, had a flow rate of 400 l/h and a biomass 1.7 g/l. Tank C contained 1 265 fish in water at 24°C had a flow rate of 400 l/h and a biomass 0.6 g/l. In the last 5 days before the experiment, tank B had a disease mortality of 15.2% and tank C 15.9%. A batch of 1 064 fish was transferred from tank B to a similar one (tank B’) and then both were treated with Furoxone. Tank C received no treatment. Initial biomass in tank B was 1.2 g/l; that in tank B’ and in tank C, 0.6 g/l. For technical reasons, in experiments 2 and 3 it was not possible to work at the same temperature. The evolution of the biomass in each tank was calculated from the growth curve of weight and the daily number of fish in the tank. RESULTS
The results of experiment 1 are plotted in Fig. 1. The mortality after 8 days was 28% in the control tank, 30.4% in the batch whose pellets were supplemented with vitamins and oligoelements, 20% in the malachite green-formalin treated tank, 9.2% in the Furoxone treated tank and 6.4% in the Furanace treated one. % MORTALITY
T
Fig. 1. Mortality curves of experiment 1: control tank ooo ; vitamins and oligoelements supplemented tank - * a;malachite green-formalin in treated tank -W-V--; Furoxone treated tank -; Furanace treated tank - - - -. l
320
For experiment 2, the mortality curves are plotted in Fig. 2 a and the changes in the biomass in each tank are shown in Fig. 2 b. Eight days after the beginning of the experiment, the mortality was 5% in the treated original tank A and 35% in the transferred untreated batch (A’). For experiment 3, the mortality curves are plotted in Fig. 3 a and the changes in the biomass in Fig. 3 b. Six days after the beginning of the experiment, the mortality was 8% in the untreated tank C; in the Furoxone treated tank B it was 11.6%. a little higher than in the transferred and Furoxone treated batch B’, where it was5.4%. q/t
% MORTALITY T
,
3.0
42_
I I l,‘e
35 -
)c--
28 2.5
34_
2,4
/!
30_ /
I
2,2
26 22_
ti;
Ia_ _ 14 _ IO 1
iI $1 =I I I I
I’ I I
11111,11111,,,,,1,,11,,,,
93
95
97
99
IO1 103 105 107 109 Ill
85
57
59
91
93
95
(days)
97
99
IO1 103 I05 107 109 (days 1
Fig, 2. (a) Mortality curves of experiment 2; treated tank -; untreated tank ----. (b) Changes in the biomass in experiment 2: original tank ***a; Furanace treated tank A p; untreated tank A’ - - - .
%MORTALITY
pr/l
,,I-
T
Fl 96
I
97
98
I
I
I
I)
99 100 IO1 IO2
.I
lllll’lr,,,,, AGE (days)
91
93
95
97
99
IO1
)AGE (days1
Fig. 3. (a) Mortality curves of experiment 3: untreated tank C .*.*; unhandled Furoxone treated tank B-I-*-* , handled Furoxone treated tank B’ - - - . (b) Changes in the biomass in experiment 3 : original tank B -. original tank C .*.*; unhandled Furoxone treated tank B -+-*; handled Puroxone treaded tank B’ ---.
321
DISCUSSION
In the first experiment, both nitrofuran derivatives showed similarly good efficiencies in the control of disease. The antiseptic solution (malachite green-formalin) showed about half of their efficiency. The complement to the pellets of 4% more vitamins and oligoelements had no effect. These results can be compared to the cost of the different treatments: for a cubic metre of treated water, it costs roughly 0.54 F for the malachite greenformalin, 0.90 F for the Furoxone and 18.00 F for the Furanace. In the second experiment, the Furanace confirmed its good efficiency in the control of disease in the original tank, while a transfer to a new tank did not prove to be useful. In the third experiment, the results disagree with those of the first experiment; they seem to indicate that the Furoxone is less effective than the Furanace at the same concentration, Furthermore, the effect of the biomass might be more important in the development of the disease than the effect of handling the fish; of both Furoxone treated tanks, tank B, with the higher biomass and unhandled fish, had higher mortality than handled fish in tank B’ with the lower biomass. In the untreated tank C, with a low biomass, the mortality remained low. Furoxone should be tested a little more before being used as a routine method in the treatment of bacterial disease. These experiments lead us to the conclusion that the bacterial disease in reared sea bass can be efficiently controlled by a 20-min bath treatment of Furanace at 12 mg/l. A higher concentration of Furoxone might be needed to always obtain the same efficiency. One could put forward the hypothesis that malachite green-formalin treatments might control the disease before, or at very early stages of, its external symptoms. The causes of stress in our laboratory are not established, but crowding and imbalance of the pellet formulation could be important factors in the development of disease. Research on these factors must be continued, as well as studies on prophylaxis. ACKNOWLEDGEMENTS
The author acknowledges the fellowship received from the French Government through the Cultural Services of the French Embassy in Lisbon. Also, F.L. Baudin, G. Tixerant and J. Thery for their advice on pathology, L. Laubier, M. Girin, I. Paperna, F.L. Baudin and M. Francheteau for their critical reading of the manuscript, and G. Nedelec for his technical help.
REFERENCES Amend, D.F., 1970. Myxobacterial infections of salmonids: prevention and treatment. In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 258-264.
322
Amend, D.F. and Ross, A.J., 1970. Experimental control of columnaris disease with a new nitrofuran drug, P-7138. Prog. Fish Cult., 32(l): 19-25. Akazawa, H., 1968. Bacterial disease of marine fish. In: Symposium on Bacterial Fish Diseases. Bull. Jpn. Sot. Sci. Fish., 34(3): 271-272. Barahona-Fernandes, M.H. and Girin, M., 1976. Preliminary tests on the optimal pelletadaptation age for sea bass larvae (Pisces, Dicentrarchus labrax L. 1758). Aquaculture, 8: 283-290. Bullock, G.L. and McLaughlin, J.J.A., 1970. Advances in knowledge concerning bacteria pathogenic to fishes (1954-1968). In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 231-242. Girin, M., Barahona-Fernandes, M.H. and Le Roux, A., 1975. Larval Rearing of Sea Bass (Dicentrarchus labrax (L.)) with a High Survival. Presented at Int. Count. Explor. Sea, Comm. Meet. 1975/G: 14,8 pp. Holt, R.A., Conrad, J.F. and Fryer, J.L., 1975. Furanace for control of Cytophaga psychrophilu, the causitive agent of cold water disease in coho salmon. Prog. Fish Cult., 37(3): 137-139. Leteux, F. and Meyer, F.P., 1972. Mixtures of malachite green and formalin for controlling Zchthyophthirius and other protozoan parasites of fish. Prog. Fish Cult., 34(l): 21-26. Metailler, R. and Girin, M., 1976. Croissance de jeunes soles (Solea solea) nees en laboratoire et conditionnees B l’aliment compos& Int. Count. Explor. Sea, 2nd Meet. Work. Group Maricult., Hamburg, May 1976,12 pp. Oppenheimer, C.H., 1962. On marine fish diseases. In: G. Borgston (Editor), Fish as Food, vol. 2. Academic Press, New York, N.Y., 541 pp. Pacha, R.E. and Ordal, E.Y., 1970. Myxobacterial diseases of salmonids. In: S.F. Snieszko (Editor), Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ. No. 5, pp. 243-255. Pearse, L., Pullin, R.S.V., Conroy, D.A. and McGregor, D., 1974. Observations on the use of furanace for the control of Vibrio disease in marine flatfish. Aquaculture, 3: 295302. Sindermann, C.J., 1970. Principal Diseases of Marine Fish and Shellfish. Academic Press, New York, N.Y., London, 369 pp. Shimizu, M. and Takase, Y., 1967. A potent chemotherapeutic agent against fish diseases: 6-Hydroxymethyl-2-[ 2-(5-nitro-2-furyl) vinyl] pyridine (P-7138). Bull. Jpn. Sot. Sci. Fish., 33(6): 544-554. Takase, Y., Shimizu, M. and Kubota, S.S., 1968. The absorption and distribution of a chemotherapeutic agent, P-7138, in fishes. Bull. Jpn. Sot. Sci. Fish., 34(2): 1118-1123 Van Duijn Jr, C., 1972. Diseases of Fishes. Iliffe Books, London, 3rd edn, 372 pp.