Immunology and Cell Biology (1992) 70: 391-395
Serological responses and immunity produced in salmonids by vaccination with Australian strains of Vibrio anguillarum B. L. MUNDAY,' J. CARSON,^ R. WHITTINGTON^ and J. ALEXANDER'* ^National Key Centre for Teaching and Research in Aquaculture, Launceston, Tasmania, '^Department of Primary Industry and Fisheries, Kings Meadows, Tasmania, ^Elizabeth Macarthur Agricultural Institute, Camden, New South Wales and ^Safcol, Margate, Tasmania, Australia Summary The Australian populations of rainbow trout {Onchorhynchm mykiss) and Atlantic salmon (Salmo salar) were found to have similar immunological responses to local strains of Vibrio anguillarum as those reported for the more genetically diverse populations of these fish and strains of V. anguillarum found in the Northern Hemisphere. In addition, our studies more precisely defined the respective responses of rainbow trout and Atlantic salmon to immersion vaccination by the bath and dip methods. Key words: ELISA, immunity, salmonids, vaccination, yibrio anguillarlum.
Introduction Vibriosis due to Vibrio anguillarum is a major cause of mortality in farmed marine fish worldwide.' The importance of the disease is related to a wide range of factors including the species farmed, the genetic susceptibility within species, environmental conditions and the availability of effective therapeutic agents and vaccines.^ In contrast to the situation in most other countries, particularly those in the Northern Hemisphere, little information is available on vibriosis vaccines in this country. In fact, the Australian situation in relation to vibriosis vaccination of salmonids is probably unique as the genetic variation in these populations is restricted due to limited introductions,"^ and, to date, only one serotype of V. anguillarum has been isolated from outbreaks of clinical vibriosis."* Consequently, this paper not only reports on observations that contribute to the existing body of knowledge on the immunology of vibriosis, but also provides information relating to the response of fish of presumed
uniform genotype kept in an environment significantly different from those used for farming of salmonids in the major aquaculture countries of the Northern Hemisphere. In particular, temperatures encountered in Australian waters used for salmonid aquaculture are generally higher than those in Norway and Scotland and this could have significant effect on the expression of the disease. The observations were undertaken in two distinct parts. One part focused on the humoral response to an Australian isolate of inactivated V. anguillarum when administered parenteraliy. The other investigated the protective value of a vaccine produced from the same serovar when administered by immersion of the fish in a suspension of this vaccine.
Materials and methods Humoral responses One year old rainbow trout (Onchorhynchus mykiss) weighing 200 g were used as experimental animals. These were maintained in
Correspondence: B. L. Munday, PO Box 1214, Launceston, Tasmania 7250, Australia. Accepted for publication 13 August 1992.
392
B. L. Munday et al.
freshwater in temperature-controlled tanks (two replicates) at 17°C. The V. anguillarum inoculum was prepared from a Tasmanian strain of the organism that was serological ly identical with serovar C (also designated 01).^ This was inactivated with 0.5% formalin and adjusted to a density of 2 mg/mL wet weight of cells before mixing with an equal volume of Freund's complete adjuvant. The vaccination and bleeding schedule for the fish is shown in Table 1. The fish were vaccinated intraperitoneally with 0.1 mL of the antigen preparation, and individual fish were bled only once. Antibodies against V. anguillarum were detected with both an indirect, enzyme-linked immunosorbent assay (ELISA) and an agglutination test.^ In both instances, whole cell antigens were used in the tests.
Treatments used were null, bath vaccination and dip vaccinatioii. In the first year about 200 fish ot^ each species were allocated to each treatment. In the second year 100 fish of each species were allocated to each treatment. Bath vaccination involved holding the fish in a 1 : 500 dilution of vaccine for 1 h. This necessitated interrupting the water flow and oxygenating the water, but did not involve translocation of the fish. Dip vaccination involved dipping the fish in a 1 : 10 dilution of vaccine for 30 s. This involved netting and transferring the fish. The trial was replicated over time with challenges being given at 4 weeks and 5 weeks after vaccination in the first year and 4 weeks after vaccination in the second year. Live V. anguillarum was administered by intraperitoneal injection at the dose rates shown in Tables 2 and 3. All mortalities were collected and kidney material cultured on sheep's blood agar to verify that mortalities were due to vibriosis.
Protective vaccination Underyearling rainbow trout and Atlantic salmon {Salmo salar) maintained in flowthrough tanks at ambient temperature (mean 16''C) were used. In the first year, the mean weights for rainbow trout and Atlantic salmon were 46.1 and 25.6 g, respectively. In the second year the mean weights were 11.7 and 10.5 g, respectively, at the time of vaccination. Anguillivac-C® containing 1 x 10 cells /inL of formalin-inactivated V. anguiilarum serovar C was used, as described above. A Tasmanian isolate of V. anguillarum was passaged once through juvenile rainbow trout before being used in challenge trials. Table 1. Vaccination and bleeding schedule for each replicate. Week Null (« = 25)
0 4 6 8.5
5 bled 5 bled 5 hied 4 hied
Group V. anguillarum Obled 5 bled 5 bled, 20 revaccinated 4 bled from fish vaccinated once and 5 bled from fish vaccinated twice
Results Humoral responses It was decided that the standard optical density (O.D.) for the ELISA would be the mean of the values obtained for the prevaccination samples when diluted 1 ; 200. All other results were compared to this standard and the results for the various groups are shown in Fig. 1. There was a lO-fofd increase in antibody readings 4 weeks after vaccination, which further increased to week 6 and then declined slightly at week 8.5. At all these times there was a significant difference between the vaccinated and the null groups, but no significant difference between the once and twice vaccinated groups (Student's f-test). As had been experienced previously (J. Carson, unpubl. data), the agglutination test showed a lack of accuracy, although there was an obvious differentiation between the vaccinates and the controls 6 weeks after vaccination (Fig. 2). Because of the superiority of the ELISA, the agglutination test was abandoned after week 6. Protective vaccination First year's results The mortality rates due to vibriosis are documented in Table 2. It is
393
Vaccination against vibriosis Table 2. Challenge levels of V. anguillarum and resulting mortalities in year 1. Atlantic salmon
Rainhow trout Challenge level (cells/fish)
Null
Bath
Dip
4 weeks p.v. 1.5x10" 5.0x10" 1.8x10" 2.5x10^
3/25 8/25 6/25 3/25
1/25 1/25 2/25 0/25
Fish died of misadventure
5 weeks p.v. 5.3 X 10* 1.0x10^ 7.6x10^ 2.3 X 10^
26/27 23/27 16/27 7/27
23/27 11/26 3/27 1/27
11/12 3/24 1/24 0/24
Challenge level (cells/fish)
Null
Bath
Dip
4 weeks p.v. 1.5x10^ 5.0x10" 1.8 X 10" 2.5 X 10^
15/25 19/25 19/25 22/25
9/25 16/25 7/25 U/23
4/25 1/23 2/24 2/24
5 weeks p.v. 2.3x10^ 1.1x10^ 2.8 X 10" 6.8x10^
17/27 22/27 16/27 10/27
Mill 18/24 A/21 6/26
4/25 4/26 2/26 1/27
Null
Bath
Dip
21/25 18/25 18/25 7/25
4/24 5/25 4/25 3/25
1/25 0/25 1/25 0/25
p.v., post-vaccination.
Table 3. Challenge levels of V. anguillarum and resulting mortalities in year 2. Atlantic salmon
Rainbow trout Challenge level (cells/fish) 4 weeks p.v. 7.3 X 10^ 1.2x10'^ 7.4x10^ 1.2x10"
Null
Bath
Dip
23/25 22/25 22/25 21/25
19/25 1/25 2/25 1/25
12/25 2/25 2/25 0/25
Challenge level (cells/fish) 4 weeks p.v. 5.6x10^ 1.2x10^ 9.6x10'* 1.2x10"
p.v., post-vaccination.
20n
C. 8 0 0 £•
15-
.y
10-
control Single dose vaccine two doses vaccine
600
.E 400 5 -
200 -
8.5
Time after vaccination (weeks)
Fig. 1. Vibrio ELISA results for various groups over time.
Time after vaccination (weeks)
Fig. 2. Vibrio agglutination test results at 4 and 6 weeks after vaccination.
394
B. L. Munday et al.
immediately apparentthat abnormal mortalithe immune response. Our trials, in which ties occurred in the null and bath vaccinated both species were subjected to the same procedAtlantic salmon. This was assumed to be due ures at the same time, do not appear to have to bacterial [V. anguillarum) invasion of tissue been performed previously and quite clearly show that the responses of rainbow trout and damaged due to dye-marking on the anterior Atlantic salmon are very comparable, with and middle trunk region. This vi^as later conboth vaccination procedures eliciting strong firmed experimentally (J. Alexander, unpubl. immunity. This response was seen despite the data). very high blood cortisol levels associated with However, the rainbow trout exhibited no dip vaccination (J. Alexander, unpubl. data), such problem, presumably due to their greater and it is probable that this stress response was size, and easily met the criteria of Cardella and of such limited duration that it did not have a Eimers for potency testing of Vibrio (i.e. a significant effect on the fish's lymphocyte minimum survival of 60% of the vaccinated populations. fish when the challenge killed 70% or more The results of this research have been transof the null group).** Even though 24-27 lated into practical recommendations for the rather than 30 fish were used in our groups, vaccination of salmonids in Tasmania. The the results are sufficiently emphatic to comresults have been heartening with clinical pensate for the smaller group sizes in the vibriosis only being recorded in vaccinated instance of the dip vaccination at least. fish when they have been subjected to unreasonable stress (J. Carson, unpubl. data). Second year's results The mortality rates due to vibriosis are documented in Table 3 and quite clearly illustrate the protective value of Acknowledgements the vaccine. It is noteworthy that this pattern applied to both rainbow trout and Atlantic The authors wish to acknowledge the consalmon. siderable assistance provided by the staff and students in the National Key Centre for Teaching and Research in Aquaculture and Discussion the staff of Salmon Enterprises of Tasmania. It is apparent from the results that the Australian (Tasmanian) strains of V. anguillarum serovar C are highly immunogenic in both the serologica! and protective senses. The ELISA results were very similar to those reported by Swedish workers using a bivalent (serotypes 01 and 02) vibrio vaccine produced by the Swedish National Veterinary Institute.^ This suggests that the Australian isolates and rainbow trout stocks are similar to those in the Northern Hemisphere, and an extrapolation of results from other countries should be valid despite the degree of inbreeding in the Australian stocks. One ofthe aims ofthe vaccination trial was to determine if rainbow trout and Atlantic salmon would behave similarly to both bath and dip vaccination, because Atlantic salmon have only recently been domesticated and would be expected to be severely stressed by dip vaccination with possible compromise of
References 1. Colwell, R. R. and Grimes, D. J. 1984. Vihrio diseases of marine fish populations. Helgoldnder Meeresunters 37: 265-287. 2. Austin, B. and Austin, D. A. 1987. Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. Ellis
Horwood Ltd., Chichester. 3. Hortle, M. E. 1988. The Quarantine of Atlantic Salmon (Saimo salar L.) in Tasmania 1984-86. Technical Report 29, Department of Sea Fisheries, Hobart. 4. Carson, J. 1990. Microbial diseases of fm fish. In Fin Fish Diseases. D. j . Brydcn (ed.). Postgraduate Committee in Veterinary Science of the University of Sydney, pp. 56-80. 5. Han-ell, L. W., Etiinger, H. M. and Hodglns, H. O. 1975. Humoral factors important in resistance of salmonid fish to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo ^airdneri) against vibriosis. Aquaculture 6:
211-2i9.
Vaccination against vibriosis 6. Cardella, M. A. and Eimers, M. E. 1990. Safety and potency testing of. Federal licensed fish bacterins.y. Aquat. Animal Health 2: 49-55. 1. Thuvandet. A., Hongslo, T., Jansson E. and
395
Sundquist, B. 1987. Duration of protective immunity and antibody titres measured by ELISA after vaccination of rainbow trout, Salmo gairdneri, against vibriosis./ Fish Dis. 10: 479-486.
Serological responses and immunity produced in salmonids by vaccination with Australian strains of Vibrio anguillarum B. L. MUNDAY,' J. CARSON,^ R. WHITTINGTON^ and J. ALEXANDER'* ^National Key Centre for Teaching and Research in Aquaculture, Launceston, Tasmania, '^Department of Primary Industry and Fisheries, Kings Meadows, Tasmania, ^Elizabeth Macarthur Agricultural Institute, Camden, New South Wales and ^Safcol, Margate, Tasmania, Australia Summary The Australian populations of rainbow trout {Onchorhynchm mykiss) and Atlantic salmon (Salmo salar) were found to have similar immunological responses to local strains of Vibrio anguillarum as those reported for the more genetically diverse populations of these fish and strains of V. anguillarum found in the Northern Hemisphere. In addition, our studies more precisely defined the respective responses of rainbow trout and Atlantic salmon to immersion vaccination by the bath and dip methods. Key words: ELISA, immunity, salmonids, vaccination, yibrio anguillarlum.
Introduction Vibriosis due to Vibrio anguillarum is a major cause of mortality in farmed marine fish worldwide.' The importance of the disease is related to a wide range of factors including the species farmed, the genetic susceptibility within species, environmental conditions and the availability of effective therapeutic agents and vaccines.^ In contrast to the situation in most other countries, particularly those in the Northern Hemisphere, little information is available on vibriosis vaccines in this country. In fact, the Australian situation in relation to vibriosis vaccination of salmonids is probably unique as the genetic variation in these populations is restricted due to limited introductions,"^ and, to date, only one serotype of V. anguillarum has been isolated from outbreaks of clinical vibriosis."* Consequently, this paper not only reports on observations that contribute to the existing body of knowledge on the immunology of vibriosis, but also provides information relating to the response of fish of presumed
uniform genotype kept in an environment significantly different from those used for farming of salmonids in the major aquaculture countries of the Northern Hemisphere. In particular, temperatures encountered in Australian waters used for salmonid aquaculture are generally higher than those in Norway and Scotland and this could have significant effect on the expression of the disease. The observations were undertaken in two distinct parts. One part focused on the humoral response to an Australian isolate of inactivated V. anguillarum when administered parenteraliy. The other investigated the protective value of a vaccine produced from the same serovar when administered by immersion of the fish in a suspension of this vaccine.
Materials and methods Humoral responses One year old rainbow trout (Onchorhynchus mykiss) weighing 200 g were used as experimental animals. These were maintained in
Correspondence: B. L. Munday, PO Box 1214, Launceston, Tasmania 7250, Australia. Accepted for publication 13 August 1992.
392
B. L. Munday et al.
freshwater in temperature-controlled tanks (two replicates) at 17°C. The V. anguillarum inoculum was prepared from a Tasmanian strain of the organism that was serological ly identical with serovar C (also designated 01).^ This was inactivated with 0.5% formalin and adjusted to a density of 2 mg/mL wet weight of cells before mixing with an equal volume of Freund's complete adjuvant. The vaccination and bleeding schedule for the fish is shown in Table 1. The fish were vaccinated intraperitoneally with 0.1 mL of the antigen preparation, and individual fish were bled only once. Antibodies against V. anguillarum were detected with both an indirect, enzyme-linked immunosorbent assay (ELISA) and an agglutination test.^ In both instances, whole cell antigens were used in the tests.
Treatments used were null, bath vaccination and dip vaccinatioii. In the first year about 200 fish ot^ each species were allocated to each treatment. In the second year 100 fish of each species were allocated to each treatment. Bath vaccination involved holding the fish in a 1 : 500 dilution of vaccine for 1 h. This necessitated interrupting the water flow and oxygenating the water, but did not involve translocation of the fish. Dip vaccination involved dipping the fish in a 1 : 10 dilution of vaccine for 30 s. This involved netting and transferring the fish. The trial was replicated over time with challenges being given at 4 weeks and 5 weeks after vaccination in the first year and 4 weeks after vaccination in the second year. Live V. anguillarum was administered by intraperitoneal injection at the dose rates shown in Tables 2 and 3. All mortalities were collected and kidney material cultured on sheep's blood agar to verify that mortalities were due to vibriosis.
Protective vaccination Underyearling rainbow trout and Atlantic salmon {Salmo salar) maintained in flowthrough tanks at ambient temperature (mean 16''C) were used. In the first year, the mean weights for rainbow trout and Atlantic salmon were 46.1 and 25.6 g, respectively. In the second year the mean weights were 11.7 and 10.5 g, respectively, at the time of vaccination. Anguillivac-C® containing 1 x 10 cells /inL of formalin-inactivated V. anguiilarum serovar C was used, as described above. A Tasmanian isolate of V. anguillarum was passaged once through juvenile rainbow trout before being used in challenge trials. Table 1. Vaccination and bleeding schedule for each replicate. Week Null (« = 25)
0 4 6 8.5
5 bled 5 bled 5 hied 4 hied
Group V. anguillarum Obled 5 bled 5 bled, 20 revaccinated 4 bled from fish vaccinated once and 5 bled from fish vaccinated twice
Results Humoral responses It was decided that the standard optical density (O.D.) for the ELISA would be the mean of the values obtained for the prevaccination samples when diluted 1 ; 200. All other results were compared to this standard and the results for the various groups are shown in Fig. 1. There was a lO-fofd increase in antibody readings 4 weeks after vaccination, which further increased to week 6 and then declined slightly at week 8.5. At all these times there was a significant difference between the vaccinated and the null groups, but no significant difference between the once and twice vaccinated groups (Student's f-test). As had been experienced previously (J. Carson, unpubl. data), the agglutination test showed a lack of accuracy, although there was an obvious differentiation between the vaccinates and the controls 6 weeks after vaccination (Fig. 2). Because of the superiority of the ELISA, the agglutination test was abandoned after week 6. Protective vaccination First year's results The mortality rates due to vibriosis are documented in Table 2. It is
393
Vaccination against vibriosis Table 2. Challenge levels of V. anguillarum and resulting mortalities in year 1. Atlantic salmon
Rainhow trout Challenge level (cells/fish)
Null
Bath
Dip
4 weeks p.v. 1.5x10" 5.0x10" 1.8x10" 2.5x10^
3/25 8/25 6/25 3/25
1/25 1/25 2/25 0/25
Fish died of misadventure
5 weeks p.v. 5.3 X 10* 1.0x10^ 7.6x10^ 2.3 X 10^
26/27 23/27 16/27 7/27
23/27 11/26 3/27 1/27
11/12 3/24 1/24 0/24
Challenge level (cells/fish)
Null
Bath
Dip
4 weeks p.v. 1.5x10^ 5.0x10" 1.8 X 10" 2.5 X 10^
15/25 19/25 19/25 22/25
9/25 16/25 7/25 U/23
4/25 1/23 2/24 2/24
5 weeks p.v. 2.3x10^ 1.1x10^ 2.8 X 10" 6.8x10^
17/27 22/27 16/27 10/27
Mill 18/24 A/21 6/26
4/25 4/26 2/26 1/27
Null
Bath
Dip
21/25 18/25 18/25 7/25
4/24 5/25 4/25 3/25
1/25 0/25 1/25 0/25
p.v., post-vaccination.
Table 3. Challenge levels of V. anguillarum and resulting mortalities in year 2. Atlantic salmon
Rainbow trout Challenge level (cells/fish) 4 weeks p.v. 7.3 X 10^ 1.2x10'^ 7.4x10^ 1.2x10"
Null
Bath
Dip
23/25 22/25 22/25 21/25
19/25 1/25 2/25 1/25
12/25 2/25 2/25 0/25
Challenge level (cells/fish) 4 weeks p.v. 5.6x10^ 1.2x10^ 9.6x10'* 1.2x10"
p.v., post-vaccination.
20n
C. 8 0 0 £•
15-
.y
10-
control Single dose vaccine two doses vaccine
600
.E 400 5 -
200 -
8.5
Time after vaccination (weeks)
Fig. 1. Vibrio ELISA results for various groups over time.
Time after vaccination (weeks)
Fig. 2. Vibrio agglutination test results at 4 and 6 weeks after vaccination.
394
B. L. Munday et al.
immediately apparentthat abnormal mortalithe immune response. Our trials, in which ties occurred in the null and bath vaccinated both species were subjected to the same procedAtlantic salmon. This was assumed to be due ures at the same time, do not appear to have to bacterial [V. anguillarum) invasion of tissue been performed previously and quite clearly show that the responses of rainbow trout and damaged due to dye-marking on the anterior Atlantic salmon are very comparable, with and middle trunk region. This vi^as later conboth vaccination procedures eliciting strong firmed experimentally (J. Alexander, unpubl. immunity. This response was seen despite the data). very high blood cortisol levels associated with However, the rainbow trout exhibited no dip vaccination (J. Alexander, unpubl. data), such problem, presumably due to their greater and it is probable that this stress response was size, and easily met the criteria of Cardella and of such limited duration that it did not have a Eimers for potency testing of Vibrio (i.e. a significant effect on the fish's lymphocyte minimum survival of 60% of the vaccinated populations. fish when the challenge killed 70% or more The results of this research have been transof the null group).** Even though 24-27 lated into practical recommendations for the rather than 30 fish were used in our groups, vaccination of salmonids in Tasmania. The the results are sufficiently emphatic to comresults have been heartening with clinical pensate for the smaller group sizes in the vibriosis only being recorded in vaccinated instance of the dip vaccination at least. fish when they have been subjected to unreasonable stress (J. Carson, unpubl. data). Second year's results The mortality rates due to vibriosis are documented in Table 3 and quite clearly illustrate the protective value of Acknowledgements the vaccine. It is noteworthy that this pattern applied to both rainbow trout and Atlantic The authors wish to acknowledge the consalmon. siderable assistance provided by the staff and students in the National Key Centre for Teaching and Research in Aquaculture and Discussion the staff of Salmon Enterprises of Tasmania. It is apparent from the results that the Australian (Tasmanian) strains of V. anguillarum serovar C are highly immunogenic in both the serologica! and protective senses. The ELISA results were very similar to those reported by Swedish workers using a bivalent (serotypes 01 and 02) vibrio vaccine produced by the Swedish National Veterinary Institute.^ This suggests that the Australian isolates and rainbow trout stocks are similar to those in the Northern Hemisphere, and an extrapolation of results from other countries should be valid despite the degree of inbreeding in the Australian stocks. One ofthe aims ofthe vaccination trial was to determine if rainbow trout and Atlantic salmon would behave similarly to both bath and dip vaccination, because Atlantic salmon have only recently been domesticated and would be expected to be severely stressed by dip vaccination with possible compromise of
References 1. Colwell, R. R. and Grimes, D. J. 1984. Vihrio diseases of marine fish populations. Helgoldnder Meeresunters 37: 265-287. 2. Austin, B. and Austin, D. A. 1987. Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. Ellis
Horwood Ltd., Chichester. 3. Hortle, M. E. 1988. The Quarantine of Atlantic Salmon (Saimo salar L.) in Tasmania 1984-86. Technical Report 29, Department of Sea Fisheries, Hobart. 4. Carson, J. 1990. Microbial diseases of fm fish. In Fin Fish Diseases. D. j . Brydcn (ed.). Postgraduate Committee in Veterinary Science of the University of Sydney, pp. 56-80. 5. Han-ell, L. W., Etiinger, H. M. and Hodglns, H. O. 1975. Humoral factors important in resistance of salmonid fish to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo ^airdneri) against vibriosis. Aquaculture 6:
211-2i9.
Vaccination against vibriosis 6. Cardella, M. A. and Eimers, M. E. 1990. Safety and potency testing of. Federal licensed fish bacterins.y. Aquat. Animal Health 2: 49-55. 1. Thuvandet. A., Hongslo, T., Jansson E. and
395
Sundquist, B. 1987. Duration of protective immunity and antibody titres measured by ELISA after vaccination of rainbow trout, Salmo gairdneri, against vibriosis./ Fish Dis. 10: 479-486.
