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Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.)
Fish & Shellfish Immunology (2002) 12, 283–285 doi:10.1006/fsim.2001.0386 Available online at http://www.idealibrary.com on
SHORT COMMUNICATION Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.) T. J. BOWDEN1*, D. MENOYO-LUQUE1, I. R. BRICKNELL1 H. WERGELAND2 1
FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, Scotland and 2Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway (Received 9 October 2001, accepted 26 October 2001, published electronically 18 January 2002) Atlantic halibut (Hippoglossus hippoglossus L.) is a potentially important new species to cold-water aquaculture. Development of a viable industrial farming technique has been hampered by continued pathogen problems within the rearing cycle and there are several reports that indicated how susceptible juvenile halibut are to bacterial and viral diseases [1]. Interest has been expressed, within the industry, over the possibility of vaccinating suitably sized animals to protect against the more common aquaculture pathogens. Vibrio spp. are of particular concern due to their ubiquitous nature and the relatively frequent occurrence of these pathogens within marine aquaculture. We have previously investigated the susceptibility of Atlantic halibut to infection by Vibrio anguillarum and the e$cacy of intraperitoneal injected delivery of a commercial vaccine in protecting against the disease [2]. Given the very high rate of protection o#ered by immunisation we wanted to investigate the e#ect of alternate routes of administration on the e$cacy of the vaccine. 2002 Elsevier Science Ltd. Key words: Vibrio, halibut, vaccination, challenge.
Juvenile Atlantic halibut (40 g) were obtained from a commercial hatchery and acclimated in 80 cm diameter tanks at the Bergen High Technology Centre. They were maintained on sea water at an average 9·3 C (S.D. =0·63) for the duration of the experiment and fed twice daily on Ewos micro 3 and 4 diets. The V. anguillarum vaccine was prepared by growing the bacterium (MT1637: Marine Laboratory, Aberdeen) in tryptone soy broth (Oxoid) +2% NaCl at 22 C for 48 h in an orbital incubator. Formaldehyde was added to a concentration of 0·2% and the broth incubated for a further 24 h. Animals were anaesthetised with MS222 at 100 mg l 1, vaccinated with bacterial suspension and marked using a fluorescent latex marker (Northwest Marine Technology Inc., Shaw Island, WA, U.S.A.) which was injected into the fin margin (immersion fish were anaesthetised, marked, recovered and then immersed). The fish were vaccinated either by immersion for 1 min in a 1 in 10 dilution of the suspension in sea water, by injection of 100 l of suspension into the peritoneal *Corresponding author. E-mail: [email protected] 283 1050–4648/02/030283+03 $35.00/0
2002 Elsevier Science Ltd.
284
T. J. BOWDEN ET AL.
100 Cumulative mortality (%)
90 80 70 60 50 40 30 20 10 0
1
4
7
10
13
16
19
22
25
28
Days post-challenge Fig. 1. Graph of the cumulative percent mortality of halibut vaccinated via various routes and then challenged with 100 l of V. anguillarum by i.p. injection. Injected and intubated vaccination fish received 100 l of vaccine. Immersion vaccination fish were immersed in a 1 in 10 dilution of the injection vaccine for 1 min. Control fish received 100 l of phosphate bu#ered saline by i.p. injection (n=35). Control (), immersion (), i.p. (), oral intubation (), anal intubation ( ).
cavity, by oral intubation into the stomach, or anal intubation of 100 l of bacterial suspension using a syringe and narrow gauge silicon tubing. Control fish received 100 l of sterile PBS by intraperitoneal (i.p.) injection. The fish (35 per group) were evenly distributed amongst four tanks. After 12 weeks the fish were challenged with 100 l i.p. injection of V. anguillarum (a Norwegian isolate, HI-610 serotype 02) at 1107 cfu ml 1 that had been grown on tryptone soy agar (Oxoid) +2% NaCl (TSA2) for 48 h and resuspended in phosphate bu#ered saline. The experiment was concluded when no further mortalities had occurred for four consecutive days. Moribund fish were sampled by kidney swab for re-isolation of V. anguillarum on tryptone soy agar+2% NaCl plates. The results (Fig. 1) showed that i.p. injection and immersion both provided e#ective vaccination routes with near 100% survival in both groups. Anal intubation gave about 80% survival with oral intubation giving only 50% survival. The challenge proved e#ective by killing 100% of the control fish. Aquatic organisms live in a pathogen rich environment compared to terrestrial organisms and the mucosal surfaces of the gut, skin and gills can provide protection by both humoral and cell mediated mechanisms. Fish lack the defined areas of lymphoid tissue, such as Peyer’s patches within the gut, lymph nodes and germinal centre-like structures. Although lymphoid cells and macrophages have been found in the lamina propria [3, 4]. Previous reports have shown that oral and anal administration of antigens results in the uptake of macromolecules by intraepithelial macrophages in the hindgut [5, 6]. There is evidence from carp for the existence of a mucus specific Ig molecule that would indicate that mucosal immunity is an important facet of the fishes defence system [7]. In addition, after oral administration of V. anguillarum vaccine to carp, specific Ig-secreting cells could be demonstrated in gills and gut using antibodies raised against the mucus specific Ig mentioned [8]. This suggests that there may be an e#ective mucosal immune system operating within the gut, skin and gill. The results from the present study would suggest that halibut also possess an e#ective mucosal immune system and that immersion, i.p. injection and anal intubation all provide an e#ective vaccination route for a systemic challenge with this pathogen. The inability of the oral administration route to provide such good protection probably relates to degeneration of the antigen before it can reach suitable lymphoid tissues and perhaps encapsulation may result in better stimulation of the gut-associated lymphoid tissue and subsequent protection to challenge.
VACCINATION AGAINST VIBRIO ANGUILLARUM IN ATLANTIC HALIBUT
285
This project has been supported by the TMR (Training and Mobility of Researchers) Programme from the European Union through Contract No. ERBFMGECT950013. References 1 Hjeltnes, B & Roberts, R. J. (1993). Vibriosis. In Bacterial Diseases of Fish (V. Inglis, R. J. Roberts & N. R. Bromage, eds) pp. 109–121. Oxford: Blackwell Scientific. 2 Bricknell, I. R., Bowden, T. J., Verner-Je#reys, D. W., Bruno, D. W., Shields, R. W. & Ellis, A. E. (2000). Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and the e$cacy of protection induced by vaccination. Fish & Shellfish Immunology 10, 319–327. 3 Rombout, J. H. W. M., Taverne-Thiele, J. J. & Villena, M. I. (1993). The gut associated lymphoid tissue (GALT) of carp (Cyprinus carpio L.): immunocytochemical analysis. Developmental and Comparative Immunology 15, 55–66. 4 van Muiswinkel, W. B., Lamers, C. H. J. & Rombout, J. H. W. M. (1991). Structural and functional aspects of the spleen in bony fish. Research in Immunology 142, 362–366. 5 Georgopoulou, U. & Vernier, J. M. (1986). Local immunological response in the posterior intestinal segment of the rainbow trout after oral administration of macromolecules. Developmental and Comparative Immunology 10, 529–537. 6 Rombout, J. H. W. M., Bot, H. E. & Taverne-Thiele, J. J. (1989). Immunological importance of the second gut segment of carp. II. Characterisation of mucosal leucocytes. Journal of Fish Biology 35, 167–178. 7 Rombout, J. H. W. M., Taverne, N., Van de Kamp, M. & Taverne-Thiele, J. J. (1993). Di#erences in mucus and serum immunoglobulin of carp (Cyprinus carpio L.). Developmental and Comparative Immunology 17, 309–317. 8 Joosten, P. H. M., Tiemersma, E., Threels, A., Caumartin-Dhieux, C. & Rombout, J. H. W. M. (1997). Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Fish & Shellfish Immunology 7, 471–485.
SHORT COMMUNICATION Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.) T. J. BOWDEN1*, D. MENOYO-LUQUE1, I. R. BRICKNELL1 H. WERGELAND2 1
FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, Scotland and 2Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway (Received 9 October 2001, accepted 26 October 2001, published electronically 18 January 2002) Atlantic halibut (Hippoglossus hippoglossus L.) is a potentially important new species to cold-water aquaculture. Development of a viable industrial farming technique has been hampered by continued pathogen problems within the rearing cycle and there are several reports that indicated how susceptible juvenile halibut are to bacterial and viral diseases [1]. Interest has been expressed, within the industry, over the possibility of vaccinating suitably sized animals to protect against the more common aquaculture pathogens. Vibrio spp. are of particular concern due to their ubiquitous nature and the relatively frequent occurrence of these pathogens within marine aquaculture. We have previously investigated the susceptibility of Atlantic halibut to infection by Vibrio anguillarum and the e$cacy of intraperitoneal injected delivery of a commercial vaccine in protecting against the disease [2]. Given the very high rate of protection o#ered by immunisation we wanted to investigate the e#ect of alternate routes of administration on the e$cacy of the vaccine. 2002 Elsevier Science Ltd. Key words: Vibrio, halibut, vaccination, challenge.
Juvenile Atlantic halibut (40 g) were obtained from a commercial hatchery and acclimated in 80 cm diameter tanks at the Bergen High Technology Centre. They were maintained on sea water at an average 9·3 C (S.D. =0·63) for the duration of the experiment and fed twice daily on Ewos micro 3 and 4 diets. The V. anguillarum vaccine was prepared by growing the bacterium (MT1637: Marine Laboratory, Aberdeen) in tryptone soy broth (Oxoid) +2% NaCl at 22 C for 48 h in an orbital incubator. Formaldehyde was added to a concentration of 0·2% and the broth incubated for a further 24 h. Animals were anaesthetised with MS222 at 100 mg l 1, vaccinated with bacterial suspension and marked using a fluorescent latex marker (Northwest Marine Technology Inc., Shaw Island, WA, U.S.A.) which was injected into the fin margin (immersion fish were anaesthetised, marked, recovered and then immersed). The fish were vaccinated either by immersion for 1 min in a 1 in 10 dilution of the suspension in sea water, by injection of 100 l of suspension into the peritoneal *Corresponding author. E-mail: [email protected] 283 1050–4648/02/030283+03 $35.00/0
2002 Elsevier Science Ltd.
284
T. J. BOWDEN ET AL.
100 Cumulative mortality (%)
90 80 70 60 50 40 30 20 10 0
1
4
7
10
13
16
19
22
25
28
Days post-challenge Fig. 1. Graph of the cumulative percent mortality of halibut vaccinated via various routes and then challenged with 100 l of V. anguillarum by i.p. injection. Injected and intubated vaccination fish received 100 l of vaccine. Immersion vaccination fish were immersed in a 1 in 10 dilution of the injection vaccine for 1 min. Control fish received 100 l of phosphate bu#ered saline by i.p. injection (n=35). Control (), immersion (), i.p. (), oral intubation (), anal intubation ( ).
cavity, by oral intubation into the stomach, or anal intubation of 100 l of bacterial suspension using a syringe and narrow gauge silicon tubing. Control fish received 100 l of sterile PBS by intraperitoneal (i.p.) injection. The fish (35 per group) were evenly distributed amongst four tanks. After 12 weeks the fish were challenged with 100 l i.p. injection of V. anguillarum (a Norwegian isolate, HI-610 serotype 02) at 1107 cfu ml 1 that had been grown on tryptone soy agar (Oxoid) +2% NaCl (TSA2) for 48 h and resuspended in phosphate bu#ered saline. The experiment was concluded when no further mortalities had occurred for four consecutive days. Moribund fish were sampled by kidney swab for re-isolation of V. anguillarum on tryptone soy agar+2% NaCl plates. The results (Fig. 1) showed that i.p. injection and immersion both provided e#ective vaccination routes with near 100% survival in both groups. Anal intubation gave about 80% survival with oral intubation giving only 50% survival. The challenge proved e#ective by killing 100% of the control fish. Aquatic organisms live in a pathogen rich environment compared to terrestrial organisms and the mucosal surfaces of the gut, skin and gills can provide protection by both humoral and cell mediated mechanisms. Fish lack the defined areas of lymphoid tissue, such as Peyer’s patches within the gut, lymph nodes and germinal centre-like structures. Although lymphoid cells and macrophages have been found in the lamina propria [3, 4]. Previous reports have shown that oral and anal administration of antigens results in the uptake of macromolecules by intraepithelial macrophages in the hindgut [5, 6]. There is evidence from carp for the existence of a mucus specific Ig molecule that would indicate that mucosal immunity is an important facet of the fishes defence system [7]. In addition, after oral administration of V. anguillarum vaccine to carp, specific Ig-secreting cells could be demonstrated in gills and gut using antibodies raised against the mucus specific Ig mentioned [8]. This suggests that there may be an e#ective mucosal immune system operating within the gut, skin and gill. The results from the present study would suggest that halibut also possess an e#ective mucosal immune system and that immersion, i.p. injection and anal intubation all provide an e#ective vaccination route for a systemic challenge with this pathogen. The inability of the oral administration route to provide such good protection probably relates to degeneration of the antigen before it can reach suitable lymphoid tissues and perhaps encapsulation may result in better stimulation of the gut-associated lymphoid tissue and subsequent protection to challenge.
VACCINATION AGAINST VIBRIO ANGUILLARUM IN ATLANTIC HALIBUT
285
This project has been supported by the TMR (Training and Mobility of Researchers) Programme from the European Union through Contract No. ERBFMGECT950013. References 1 Hjeltnes, B & Roberts, R. J. (1993). Vibriosis. In Bacterial Diseases of Fish (V. Inglis, R. J. Roberts & N. R. Bromage, eds) pp. 109–121. Oxford: Blackwell Scientific. 2 Bricknell, I. R., Bowden, T. J., Verner-Je#reys, D. W., Bruno, D. W., Shields, R. W. & Ellis, A. E. (2000). Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and the e$cacy of protection induced by vaccination. Fish & Shellfish Immunology 10, 319–327. 3 Rombout, J. H. W. M., Taverne-Thiele, J. J. & Villena, M. I. (1993). The gut associated lymphoid tissue (GALT) of carp (Cyprinus carpio L.): immunocytochemical analysis. Developmental and Comparative Immunology 15, 55–66. 4 van Muiswinkel, W. B., Lamers, C. H. J. & Rombout, J. H. W. M. (1991). Structural and functional aspects of the spleen in bony fish. Research in Immunology 142, 362–366. 5 Georgopoulou, U. & Vernier, J. M. (1986). Local immunological response in the posterior intestinal segment of the rainbow trout after oral administration of macromolecules. Developmental and Comparative Immunology 10, 529–537. 6 Rombout, J. H. W. M., Bot, H. E. & Taverne-Thiele, J. J. (1989). Immunological importance of the second gut segment of carp. II. Characterisation of mucosal leucocytes. Journal of Fish Biology 35, 167–178. 7 Rombout, J. H. W. M., Taverne, N., Van de Kamp, M. & Taverne-Thiele, J. J. (1993). Di#erences in mucus and serum immunoglobulin of carp (Cyprinus carpio L.). Developmental and Comparative Immunology 17, 309–317. 8 Joosten, P. H. M., Tiemersma, E., Threels, A., Caumartin-Dhieux, C. & Rombout, J. H. W. M. (1997). Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Fish & Shellfish Immunology 7, 471–485.