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A technical report on oral immunization of marine tropical fish against Vibrio anguillarum
Aquaculture, 11 (1977) 281-283 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
281
Short Communication A TECHNICAL REPORT ON ORAL IMMUNIZATION OF MARINE TROPICAL FISH AGAINST VIBR IO ANG UILLA R UM
S.R. PRESCOTT New Technology (Received
Limited,
Penshurst,
Tonbridge,
Kent (Great Britain)
31 December 1976; revised 12 April 1977)
ABSTRACT Presscott, S.R., 1977. A technical report on oral immunization of marine tropical fiih against Vibrio anguillarum. Aquaculture, 11: 281-283. Experiments were conducted to find the efficacy of vaccine of Vibrio anguillarum ministered orally upon several varieties of marine tropical fiih.
ad-
INTRODUCTION
Following upon the work of several researchers in the field of oral immuization of fish against various pathogenic organisms (e.g. Duff, 1942; Fletcher and White, 1973; Harrell et al., 1975), it was decided to investigate the potential prophylactic effect of orally administered vaccine of Vibrio anguillarum upon a selected variety of marine tropical fish to study the immune response to such treatment. The species selected were (1) Eupomacentus leucostictus, Muller, (2) Abedefdul behanii, Bleeker, (3) DascyZZus aruanus D.A., L., and (4)Lutjunus hasmiru, Forskal. METHODS
Twenty specimens of each species were obtained by courtesy of Tropical Marine Centre, Borehamwood, and, in the first experiment, one of each species were placed into four aquaria with a capacity of 25 gallons each. Temperature was 23°C and salinity reading 1.023 specific gravity. Full aeration and sub-gravel filtration were applied in the usual manners. Vibrio culture (NCMB 407) was obtained from Torry Research Station, Aberdeen. A second experiment was carried out using four aquaria of 45 gallons each. Temperature and salinity were as before. This time, four specimens of each species were in aquaria 1 and 2, with an equal number of each species in aquaria 3 and 4 as controls.
282
Preparation of vaccine Vaccine was made up according to the preparations indicated by Fletcher and White (1973). Feeding vaccine was fed to the assorted fishes in two tanks (numbers 1 and 2) over a period of 10 successive days by the addition of 1 g of dried food which had been allowed to soak in 0.4 cc of prepared vaccine for a period of 1 min. The fishes in tanks 3 and 4 were fed the same diet without vaccine as controls. The concentration of cells must be witheld at this time for proprietary reasons. Challenge of fish Twenty days after commencement of the programme, all fish were challenged by subcutaneous injection of 0.4 cc each of live bacteria with a similar concentration of cells to that in the sonicated vaccine. Reaction to the live injections became apparent in tanks 3 and 4 after 36 h; the fishes
TABLE I Results of the two trials
-. Tank 1 M
Results
of first expertmen
Tank 2
S _.___
NS
M
Tank 4
Tank 3 NS
S
M
S
NS
M
S
NS -
t
4 X Eupomacentus
leucostictus 4 X Abedefduf behanii 4 X Dascyllus arunnus D.A. 4 x Lutjanus kaamim
2
1
1
2
0
4
0
0
3
1
0
0
1
3
1
2
2
2
0
3
1
0
1
0
3
0
0
1
1
2
2
0
2
0
0
4
0
1
2
2
0
3
1
0
Totals
3
2
11
3
3
9
5
2
11
3
2
2
4
10
3
4
11
5
0
9
6
1
0
2
14
2
3
9
I
0
8
I
1
1
3
12
0
4
8
6
2
7
5
4
0
2
14
0
1
I
7
2
10
6
0
3
11
50
5
12
35
25
4
34
24
6
Reeulta of second experiment 16 X Eupomacentus
kucostictus 16 X Abedefduf behanii 16 X Dascyllus orunnus D.A. 16 X Lutjanus kasmim Totals
M = symptoms followed by mortality; S = symptoms only; NS = no symptoms.
283
showed haemorrhagic lesions and blotchiness in many cases and mortality usually ensued within 48 h of symptoms appearing. No reaction in tanks 1 and 2 was apparent for 6 days and then the symptoms and mortalities were very considerably lower than those in the control tanks. DISCUSSION
The results, whilst varying, show a considerable degree of protection for each of the four species tested. The degree of protection would appear to be somewhat greater in species Abedefduf and Lutjunus than in the other two but, neverthless, the test, which for cost reasons had to be limited to the numbers mentioned, strongly confirms the previous evidence in this field. The results when statistically analysed by computer show a probability factor of P < 0.001. Research is continuing with other species and also with other methods of administration of the vaccine after the work of Amend and Fender (1976). This further work will be the subject of another report at a later date.
REFERENCES Amend, D.F. and Fender, D.C., 1976. Uptake of bovine serum albumin by rainbow trout from hyperosmotic solutions: A model for vaccinating fish. Science, 192: 793-794. Duff, D.C.B., 1942. The oral immunisation of trout against Bacterium sulmonicida. J. Immunol., 44 (1): 87-93. Fletcher, T. C. and White, A., 1973. Antibody production in the plaice (Pleuronectes platessa L.) after oral and perenteral immunization with Vibrio anguillarum antigens. Aquaculture, 1: 417-428. Harrell, L.W., Etlinger, H.M. and Hodgins, H.O., 1975. Humoral factors important in resistance of salmonid fiih to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo gairdneri) against vibriosis. Aquaculture, 6: 211-218. Ross, A.J. and Klontz, G.W., 1965. Oral immunisation of rainbow trout (Salmo gairdneri) against an etiologic agent of “Redmouth disease”. J. Fish. Res. Board Can., 22 (3): 713-719.
281
Short Communication A TECHNICAL REPORT ON ORAL IMMUNIZATION OF MARINE TROPICAL FISH AGAINST VIBR IO ANG UILLA R UM
S.R. PRESCOTT New Technology (Received
Limited,
Penshurst,
Tonbridge,
Kent (Great Britain)
31 December 1976; revised 12 April 1977)
ABSTRACT Presscott, S.R., 1977. A technical report on oral immunization of marine tropical fiih against Vibrio anguillarum. Aquaculture, 11: 281-283. Experiments were conducted to find the efficacy of vaccine of Vibrio anguillarum ministered orally upon several varieties of marine tropical fiih.
ad-
INTRODUCTION
Following upon the work of several researchers in the field of oral immuization of fish against various pathogenic organisms (e.g. Duff, 1942; Fletcher and White, 1973; Harrell et al., 1975), it was decided to investigate the potential prophylactic effect of orally administered vaccine of Vibrio anguillarum upon a selected variety of marine tropical fish to study the immune response to such treatment. The species selected were (1) Eupomacentus leucostictus, Muller, (2) Abedefdul behanii, Bleeker, (3) DascyZZus aruanus D.A., L., and (4)Lutjunus hasmiru, Forskal. METHODS
Twenty specimens of each species were obtained by courtesy of Tropical Marine Centre, Borehamwood, and, in the first experiment, one of each species were placed into four aquaria with a capacity of 25 gallons each. Temperature was 23°C and salinity reading 1.023 specific gravity. Full aeration and sub-gravel filtration were applied in the usual manners. Vibrio culture (NCMB 407) was obtained from Torry Research Station, Aberdeen. A second experiment was carried out using four aquaria of 45 gallons each. Temperature and salinity were as before. This time, four specimens of each species were in aquaria 1 and 2, with an equal number of each species in aquaria 3 and 4 as controls.
282
Preparation of vaccine Vaccine was made up according to the preparations indicated by Fletcher and White (1973). Feeding vaccine was fed to the assorted fishes in two tanks (numbers 1 and 2) over a period of 10 successive days by the addition of 1 g of dried food which had been allowed to soak in 0.4 cc of prepared vaccine for a period of 1 min. The fishes in tanks 3 and 4 were fed the same diet without vaccine as controls. The concentration of cells must be witheld at this time for proprietary reasons. Challenge of fish Twenty days after commencement of the programme, all fish were challenged by subcutaneous injection of 0.4 cc each of live bacteria with a similar concentration of cells to that in the sonicated vaccine. Reaction to the live injections became apparent in tanks 3 and 4 after 36 h; the fishes
TABLE I Results of the two trials
-. Tank 1 M
Results
of first expertmen
Tank 2
S _.___
NS
M
Tank 4
Tank 3 NS
S
M
S
NS
M
S
NS -
t
4 X Eupomacentus
leucostictus 4 X Abedefduf behanii 4 X Dascyllus arunnus D.A. 4 x Lutjanus kaamim
2
1
1
2
0
4
0
0
3
1
0
0
1
3
1
2
2
2
0
3
1
0
1
0
3
0
0
1
1
2
2
0
2
0
0
4
0
1
2
2
0
3
1
0
Totals
3
2
11
3
3
9
5
2
11
3
2
2
4
10
3
4
11
5
0
9
6
1
0
2
14
2
3
9
I
0
8
I
1
1
3
12
0
4
8
6
2
7
5
4
0
2
14
0
1
I
7
2
10
6
0
3
11
50
5
12
35
25
4
34
24
6
Reeulta of second experiment 16 X Eupomacentus
kucostictus 16 X Abedefduf behanii 16 X Dascyllus orunnus D.A. 16 X Lutjanus kasmim Totals
M = symptoms followed by mortality; S = symptoms only; NS = no symptoms.
283
showed haemorrhagic lesions and blotchiness in many cases and mortality usually ensued within 48 h of symptoms appearing. No reaction in tanks 1 and 2 was apparent for 6 days and then the symptoms and mortalities were very considerably lower than those in the control tanks. DISCUSSION
The results, whilst varying, show a considerable degree of protection for each of the four species tested. The degree of protection would appear to be somewhat greater in species Abedefduf and Lutjunus than in the other two but, neverthless, the test, which for cost reasons had to be limited to the numbers mentioned, strongly confirms the previous evidence in this field. The results when statistically analysed by computer show a probability factor of P < 0.001. Research is continuing with other species and also with other methods of administration of the vaccine after the work of Amend and Fender (1976). This further work will be the subject of another report at a later date.
REFERENCES Amend, D.F. and Fender, D.C., 1976. Uptake of bovine serum albumin by rainbow trout from hyperosmotic solutions: A model for vaccinating fish. Science, 192: 793-794. Duff, D.C.B., 1942. The oral immunisation of trout against Bacterium sulmonicida. J. Immunol., 44 (1): 87-93. Fletcher, T. C. and White, A., 1973. Antibody production in the plaice (Pleuronectes platessa L.) after oral and perenteral immunization with Vibrio anguillarum antigens. Aquaculture, 1: 417-428. Harrell, L.W., Etlinger, H.M. and Hodgins, H.O., 1975. Humoral factors important in resistance of salmonid fiih to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo gairdneri) against vibriosis. Aquaculture, 6: 211-218. Ross, A.J. and Klontz, G.W., 1965. Oral immunisation of rainbow trout (Salmo gairdneri) against an etiologic agent of “Redmouth disease”. J. Fish. Res. Board Can., 22 (3): 713-719.