- Email: [email protected]
Relationships between cell migration, adhesion, apoptosis and gene expression in free and attached peritoneal cells in turbot after administration of vaccines containing P. dicentrarchi antigen and different adjuvants
64
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
The use of a second panel of antibodies enabled us to distinguish between various populations of both B and T lymphocytes, allowing the study of their specific roles during vaccination and infection. Using this panel we were able to characterise kinetics of recruitment of specific B and T lymphocyte populations to local sites after vaccination, as well as their distribution in systemic and other mucosal immune organs. Besides the characterization of local and systemic immune responses after oral DNA vaccination, using a wide array of techniques including RT-qPCR, histology, flow cytometry and in vitro macrophage-T cell cultures, we are currently studying the induction of immune memory after oral vaccination. This combination of techniques enables us to establish an unique overview of the kinetics of the immune response of carp after oral DNA vaccination against SVCV, at cell, protein and gene expression level and in a broad selection of immune-relevant organs. Together we will present the promising potential of an oral DNA vaccine against SVCV to not only induce an early local immune response, but also a systemic responses followed by long-term memory formation. Keywords: DNA vaccination, SVCV, oral vaccination, T lymphocytes, immune memory x Corresponding author. Tel.: þ31 317483708; Fax: þ31 317483962. E-mail address: [email protected] (C.WE. Embregts). * These authors have contributed equally to this work.
O-017. Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare Miles D. Lange, Benjamin H. Beck, Jason D. Brown, Bradley D. Farmer, L. Matthew Barnett, Carl D. Webster
Abstract Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination is considered an efficient, environmentally safe and economically sustainable alternative for control of ectoparasite infestations. Although emerging sea lice proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials with unsuccessful results. On the other hand, more than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit and essential for cell viability. A vaccination-challenge trial with an immunogenic peptide of Rhipicephalus sanguineus protein P0 reduced survival of ticks with an overall efficacy of 90%, suggesting that it might be a promising antigen candidate for the control of ectoparasite. We have identified an immunogenic region of the ribosomal protein P0 from Caligus rogercresseyi and Lepeophtheirus salmonis that is not very conserved compared to host P0. We developed several vaccine candidates based on this peptide and produced in E. coli. These antigens were able to elicit a high specific antibody response after intraperitoneal (ip) immunization using tilapia as teleost fish model. It has been shown that ip immunization with some antigens cannot significantly reduce larval lice numbers despite enhanced specific antibody titers were raised. In this context, we have been developed for first time several tools to monitor tilapia immune response (IgT, IFN-g, IL-4, CD154) to vaccination in order to perform a more complete evaluation of the impact of the vaccinate candidates on the fish immune system. These findings will be finally validated in an immunization-challenge trial in Salmo salar. The overall results are relevant in the development of an effective vaccine against sea lice. Keywords: Sea lice, vaccine, immune response, cytokines, ribosomal protein * Corresponding author. Tel.: (53-7)2504423;. E-mail address: [email protected] (M.P. Estrada).
U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR, USA Vaccination remains a viable alternative for bacterial disease protection in fish; however additional work is required to understand the mechanisms of adaptive immunity in the channel catfish. To assess the humoral immune response to Flavobacterium columnare; a group of channel catfish were first immunized with F. columnare LV-359-01 cultured in iron-replete media, before being challenged with wild type F. columnare LV-359-01.The immunization protocol did not confer increased protection against F. columnare; however both control and immunized responders generated serum and skin IgM antibodies against F. columnare proteins. Western blot analyses of individuals from both groups showed that IgM antibodies were generated to the same 70 kDa extracellular protein, which was identified to be the bacterial chaperonin protein DNAk. Antibodies generated were cross reactive to DNAk proteins found in other gram negative bacteria. Our data suggests that DNAk is the dominant epitope in the channel catfish B-cell response to F. columnare.
O-018. Sea lice vaccine development based on an immunogenic peptide derived from the ribosomal protein P0 Yamila Carpio 1, Janet Velazquez 1, Yeny Leal 1, Naylin Herrera 1, Claudia García 1, Jannel Acosta 1, Antonio Morales 1, Fumio Takizawa 2, Oriol Sunyer 2, Mark Fast 3, Mario Pablo Estrada 1, * tica y Biotecnología (CIGB), Habana, Cuba Centro de Ingeniería Gene School of Veterinary Medicine, University of Pennsylvania, USA 3 Atlantic Veterinary College, University of Prince Edward Island, Canada 1
2
O-019. Relationships between cell migration, adhesion, apoptosis and gene expression in free and attached peritoneal cells in turbot after administration of vaccines containing P. dicentrarchi antigen and different adjuvants Francisco Fontenla 1, Jos e M. Leiro 2, Manuel Noia 1, Iria Folgueira 1, mez-Tato 3, Bel nica Blanco-Abad 1, Antonio Go en G. Pardo 4, Vero Paulino Martínez 4, Jesús Lamas 1, * 1
Department of Cell Biology and Ecology, Spain Laboratory of Parasitology, Department of Microbiology and Parasitology, Spain 3 Department of Geometry and Topology, Spain 4 Department of Genetics, University of Santiago de Compostela, Spain 2
Abstract Intraperitoneal (i.p.) injection is the most effective method of vaccinating fish in terms of antibody production and the degree of protection induced, and most current vaccines are administered in fish via this route. Vaccines are known to generate an inflammatory reaction in the peritoneal cavity, which is mainly caused by the adjuvant. Vaccination induces bidirectional movement of cells in and out of the peritoneal cavity where free cells and cells attached to the mesothelium and forming cell-vaccine masses can be observed. However, many aspects of the cellular immune response generated in the peritoneal cavity, such as the processes that provoke those cell responses, remain unknown. The present study analyses the cell traffic, adhesion, apoptosis and changes in gene expression in free and attached peritoneal cells after administration of vaccines containing P. dicentrarchi antigen and different types of adjuvants. Turbot were injected with PBS, PBS plus P. dicentrarchi membrane antigen, microspheres of chitosan-PVMMA containing membrane antigen, or with a mixture of antigen and one the following adjuvants: Montanide Isa 763 A,
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
aluminium hydroxide gel or Matrix-Q. A control group also included non injected fish. Fish were sampled at several times after vaccination. The peritoneal cavity was washed with 5 mL of cold PBS to obtain the free peritoneal cells. Cells forming cell-vaccine masses and cells attached to the peritoneal mesothelium were also obtained and disaggregated by treatment with trypsin and EDTA. Cells were analysed by light microscopy, immunohistochemistry and flow cytometry. Gene expression was determined in free peritoneal cells by using a microarray rich in genes related to the immune response and to the cell cytoskeleton. In addition, gene expression was analysed by real-time PCR analysis of attached peritoneal cells and of spleen and kidney cells. The results indicate intense cell traffic to and from the peritoneal cavity, including free cells, cells attached to the mesothelium and others migrating to lymphoid organs. Gene expression analysis revealed intense upregulation of genes associated with cytoskeleton, cell adhesion and cell surface receptors. Apoptosis was observed from day one, with upregulation of genes involved in apoptosis and cell death. However, lymphocytes were scarce, and downregulation of genes related to lymphocyte activity and lymphocyte chemotaxis was observed. Finally, we compared the responses generated by the different adjuvants. Keywords: Turbot, vaccination, immune response, peritoneal cells, gene expression, Philasterides dicentrarchi * Corresponding author. Tel: þ34881816951. E-mail address: [email protected] (J. Lamas).
O-020. Vaccination with outer membrane vesicles against intracellular pathogens for aquaculture the case of francisellosis in a zebrafish model L. Lagos 1, *, J. Tandberg 1, U. Repnik 2, N. Roos 2, H.C. Winther-Larsen 1 1 Center of Integrative Microbial Evolution and Department of Pharmaceutical Biosciences, School of Pharmacy, Faculty of Mathematics and Natural Science, University of Oslo, Norway 2 Department of Biosciences, Faculty of Mathematics and Natural Science, University Oslo, Norway
Abstract Vaccine development against many extracellular bacteria has been a success for the sustainability of the aquaculture industry. In contrast, infection of fish with intracellular pathogens remains largely an unresolved problem. Francisellosis is a bacterial disease in fish that presents nonspecific clinical signs and can cause high mortalities. Francisella noatunensis (Fn), the causative agents of francisellosis, is a non-motile, Gram-negative, facultative intracellular bacterium. Fn consists of two subspecies; F. noatunensis subsp. orientalis (Fno) that causes disease in “warm-water” fish, like tilapia, while F. noatunensis subsp. noatunensis (Fnn) causes disease in fish living in colder waters, like cod. Production of membrane vesicles by cells is a conserved mechanism occurring throughout all domains of life. Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria are spherical, 10-300 nm in diameter, and consist of a phospholipid bilayer with outer membrane proteins, endotoxin and a lumen with periplasmic proteins. Their secretion is associated with a variety of traits including the discharge of virulence factors during infections. Isolated OMVs are promising vaccine candidates against diseases caused by intracellular bacteria and have been used successfully as a vaccine against meningitis in humans. In the present work, we show that intact OMVs can be isolated from broth-cultured Fno. Proteomic analyses reveal that the vesicles include large sets of proteins involved in the host immune response, such as GroEL, OmpA and ClpB, which play an important role in the initial infection stages and the bacteria’s overall virulence and survival within a host. Furthermore, Fno OMV was tested as a vaccine in an Fno-zebrafish infection model, confirming its capacity to significantly reduce the development of francisellosis, inducing the secretion of cytokines such as TNFa, IL-1b and IFNg. Moreover, we report the transcriptional profile of
65
important immune markers such as mhcii (major histocompatibility complex class 2) and mpeg (macrophage expressed gene), demonstrating that Fno mainly infect macrophages as their replication site in zebrafish. Our research unfolds new possibilities to study the pathogenesis and to develop treatments against intracellular pathogens by using zebrafish as an infection model. Keywords: OMVs, vaccine, francisellosis, zebrafish, immune response * Corresponding author. Tel.: þ47 41079225. E-mail address: [email protected] (L. Lagos).
O-021. Adoptive transfer of immunity against red mouth disease in rainbow trout (Oncorhynchus mykiss) Takuya Yamaguchi, Uwe Fischer§ Federal Research Institute for Animal Health, Friedrich-LoefflerInstitut, Südufer 10, 17393, Greifswald-Insel Riems, Germany Enteric Red Mouth Disease (ERM) caused by Yersinia ruckeri is among the bacterial diseases in rainbow trout aquaculture that can be controlled by registered vaccines and part of these vaccines can be delivered by immersion which is the most convenient way of application inducing good antibody responses and ensuring decreased handling costs. However, the mechanisms by which fish are protected against Yersinia ruckeri are unclear. To shed light on this question we have bath vaccinated clonal rainbow trout donors by using a commercially available vaccine, and leukocytes have been isolated from vaccinated donors followed by adoptive cell transfer to naïve fish recipients of the same trout clone. Control recipients were injected with same numbers of leukocytes from nonvaccinated clonal donors. Transfer between genetically identical donors and recipients ensured that immune cells were not rejected by the recipients. After transfer, both groups of recipients were bath infected with a virulent field strain of Yersinia ruckeri. While rainbow trout that have received leukocytes from vaccinated donors showed no mortality, less than 40% of control recipients survived the challenge. Flow cytometry using leukocytes from recipient survivors revealed that the percentage of IgMþ B cells was considerably higher in recipients that were injected with leukocytes from vaccinated donors when compared with control recipients, while no differences in the percentages of CD8þ cells among groups were recorded. Being the first report on adoptive cellular transfer of immunity in a bacterial infection in fish these data suggest that immunity was rather transferred by IgMþ B cells, but not by CD8þ cytotoxic T cells. . It can be further suggested that bath vaccination has caused an effective systemic immune response since protection could be transferred by nonmucosal leukocytes. Keywords: rainbow trout, Yersinia ruckeri, adoptive transfer, B cells, T cells x Corresponding author. Tel.: þ49 38351 71175; Fax: þ49 38351 71226. E-mail address: uwe.fischer@fli.bund.de (U. Fischer).
O-022. Oil adjuvant elevates protection of rainbow trout (Oncorhynchus mykiss) following injection vaccination against Yersinia ruckeri Rzgar M. Jaafar a, $, Jiwan K. Chettri a, Inger Dalsgaard b, Azmi Al-Jubury a, Per W. Kania a, Jakob Skov a, Kurt Buchmann a a Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark b National Veterinary Institute, Technical University of Denmark,
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
The use of a second panel of antibodies enabled us to distinguish between various populations of both B and T lymphocytes, allowing the study of their specific roles during vaccination and infection. Using this panel we were able to characterise kinetics of recruitment of specific B and T lymphocyte populations to local sites after vaccination, as well as their distribution in systemic and other mucosal immune organs. Besides the characterization of local and systemic immune responses after oral DNA vaccination, using a wide array of techniques including RT-qPCR, histology, flow cytometry and in vitro macrophage-T cell cultures, we are currently studying the induction of immune memory after oral vaccination. This combination of techniques enables us to establish an unique overview of the kinetics of the immune response of carp after oral DNA vaccination against SVCV, at cell, protein and gene expression level and in a broad selection of immune-relevant organs. Together we will present the promising potential of an oral DNA vaccine against SVCV to not only induce an early local immune response, but also a systemic responses followed by long-term memory formation. Keywords: DNA vaccination, SVCV, oral vaccination, T lymphocytes, immune memory x Corresponding author. Tel.: þ31 317483708; Fax: þ31 317483962. E-mail address: [email protected] (C.WE. Embregts). * These authors have contributed equally to this work.
O-017. Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare Miles D. Lange, Benjamin H. Beck, Jason D. Brown, Bradley D. Farmer, L. Matthew Barnett, Carl D. Webster
Abstract Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination is considered an efficient, environmentally safe and economically sustainable alternative for control of ectoparasite infestations. Although emerging sea lice proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials with unsuccessful results. On the other hand, more than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit and essential for cell viability. A vaccination-challenge trial with an immunogenic peptide of Rhipicephalus sanguineus protein P0 reduced survival of ticks with an overall efficacy of 90%, suggesting that it might be a promising antigen candidate for the control of ectoparasite. We have identified an immunogenic region of the ribosomal protein P0 from Caligus rogercresseyi and Lepeophtheirus salmonis that is not very conserved compared to host P0. We developed several vaccine candidates based on this peptide and produced in E. coli. These antigens were able to elicit a high specific antibody response after intraperitoneal (ip) immunization using tilapia as teleost fish model. It has been shown that ip immunization with some antigens cannot significantly reduce larval lice numbers despite enhanced specific antibody titers were raised. In this context, we have been developed for first time several tools to monitor tilapia immune response (IgT, IFN-g, IL-4, CD154) to vaccination in order to perform a more complete evaluation of the impact of the vaccinate candidates on the fish immune system. These findings will be finally validated in an immunization-challenge trial in Salmo salar. The overall results are relevant in the development of an effective vaccine against sea lice. Keywords: Sea lice, vaccine, immune response, cytokines, ribosomal protein * Corresponding author. Tel.: (53-7)2504423;. E-mail address: [email protected] (M.P. Estrada).
U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR, USA Vaccination remains a viable alternative for bacterial disease protection in fish; however additional work is required to understand the mechanisms of adaptive immunity in the channel catfish. To assess the humoral immune response to Flavobacterium columnare; a group of channel catfish were first immunized with F. columnare LV-359-01 cultured in iron-replete media, before being challenged with wild type F. columnare LV-359-01.The immunization protocol did not confer increased protection against F. columnare; however both control and immunized responders generated serum and skin IgM antibodies against F. columnare proteins. Western blot analyses of individuals from both groups showed that IgM antibodies were generated to the same 70 kDa extracellular protein, which was identified to be the bacterial chaperonin protein DNAk. Antibodies generated were cross reactive to DNAk proteins found in other gram negative bacteria. Our data suggests that DNAk is the dominant epitope in the channel catfish B-cell response to F. columnare.
O-018. Sea lice vaccine development based on an immunogenic peptide derived from the ribosomal protein P0 Yamila Carpio 1, Janet Velazquez 1, Yeny Leal 1, Naylin Herrera 1, Claudia García 1, Jannel Acosta 1, Antonio Morales 1, Fumio Takizawa 2, Oriol Sunyer 2, Mark Fast 3, Mario Pablo Estrada 1, * tica y Biotecnología (CIGB), Habana, Cuba Centro de Ingeniería Gene School of Veterinary Medicine, University of Pennsylvania, USA 3 Atlantic Veterinary College, University of Prince Edward Island, Canada 1
2
O-019. Relationships between cell migration, adhesion, apoptosis and gene expression in free and attached peritoneal cells in turbot after administration of vaccines containing P. dicentrarchi antigen and different adjuvants Francisco Fontenla 1, Jos e M. Leiro 2, Manuel Noia 1, Iria Folgueira 1, mez-Tato 3, Bel nica Blanco-Abad 1, Antonio Go en G. Pardo 4, Vero Paulino Martínez 4, Jesús Lamas 1, * 1
Department of Cell Biology and Ecology, Spain Laboratory of Parasitology, Department of Microbiology and Parasitology, Spain 3 Department of Geometry and Topology, Spain 4 Department of Genetics, University of Santiago de Compostela, Spain 2
Abstract Intraperitoneal (i.p.) injection is the most effective method of vaccinating fish in terms of antibody production and the degree of protection induced, and most current vaccines are administered in fish via this route. Vaccines are known to generate an inflammatory reaction in the peritoneal cavity, which is mainly caused by the adjuvant. Vaccination induces bidirectional movement of cells in and out of the peritoneal cavity where free cells and cells attached to the mesothelium and forming cell-vaccine masses can be observed. However, many aspects of the cellular immune response generated in the peritoneal cavity, such as the processes that provoke those cell responses, remain unknown. The present study analyses the cell traffic, adhesion, apoptosis and changes in gene expression in free and attached peritoneal cells after administration of vaccines containing P. dicentrarchi antigen and different types of adjuvants. Turbot were injected with PBS, PBS plus P. dicentrarchi membrane antigen, microspheres of chitosan-PVMMA containing membrane antigen, or with a mixture of antigen and one the following adjuvants: Montanide Isa 763 A,
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
aluminium hydroxide gel or Matrix-Q. A control group also included non injected fish. Fish were sampled at several times after vaccination. The peritoneal cavity was washed with 5 mL of cold PBS to obtain the free peritoneal cells. Cells forming cell-vaccine masses and cells attached to the peritoneal mesothelium were also obtained and disaggregated by treatment with trypsin and EDTA. Cells were analysed by light microscopy, immunohistochemistry and flow cytometry. Gene expression was determined in free peritoneal cells by using a microarray rich in genes related to the immune response and to the cell cytoskeleton. In addition, gene expression was analysed by real-time PCR analysis of attached peritoneal cells and of spleen and kidney cells. The results indicate intense cell traffic to and from the peritoneal cavity, including free cells, cells attached to the mesothelium and others migrating to lymphoid organs. Gene expression analysis revealed intense upregulation of genes associated with cytoskeleton, cell adhesion and cell surface receptors. Apoptosis was observed from day one, with upregulation of genes involved in apoptosis and cell death. However, lymphocytes were scarce, and downregulation of genes related to lymphocyte activity and lymphocyte chemotaxis was observed. Finally, we compared the responses generated by the different adjuvants. Keywords: Turbot, vaccination, immune response, peritoneal cells, gene expression, Philasterides dicentrarchi * Corresponding author. Tel: þ34881816951. E-mail address: [email protected] (J. Lamas).
O-020. Vaccination with outer membrane vesicles against intracellular pathogens for aquaculture the case of francisellosis in a zebrafish model L. Lagos 1, *, J. Tandberg 1, U. Repnik 2, N. Roos 2, H.C. Winther-Larsen 1 1 Center of Integrative Microbial Evolution and Department of Pharmaceutical Biosciences, School of Pharmacy, Faculty of Mathematics and Natural Science, University of Oslo, Norway 2 Department of Biosciences, Faculty of Mathematics and Natural Science, University Oslo, Norway
Abstract Vaccine development against many extracellular bacteria has been a success for the sustainability of the aquaculture industry. In contrast, infection of fish with intracellular pathogens remains largely an unresolved problem. Francisellosis is a bacterial disease in fish that presents nonspecific clinical signs and can cause high mortalities. Francisella noatunensis (Fn), the causative agents of francisellosis, is a non-motile, Gram-negative, facultative intracellular bacterium. Fn consists of two subspecies; F. noatunensis subsp. orientalis (Fno) that causes disease in “warm-water” fish, like tilapia, while F. noatunensis subsp. noatunensis (Fnn) causes disease in fish living in colder waters, like cod. Production of membrane vesicles by cells is a conserved mechanism occurring throughout all domains of life. Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria are spherical, 10-300 nm in diameter, and consist of a phospholipid bilayer with outer membrane proteins, endotoxin and a lumen with periplasmic proteins. Their secretion is associated with a variety of traits including the discharge of virulence factors during infections. Isolated OMVs are promising vaccine candidates against diseases caused by intracellular bacteria and have been used successfully as a vaccine against meningitis in humans. In the present work, we show that intact OMVs can be isolated from broth-cultured Fno. Proteomic analyses reveal that the vesicles include large sets of proteins involved in the host immune response, such as GroEL, OmpA and ClpB, which play an important role in the initial infection stages and the bacteria’s overall virulence and survival within a host. Furthermore, Fno OMV was tested as a vaccine in an Fno-zebrafish infection model, confirming its capacity to significantly reduce the development of francisellosis, inducing the secretion of cytokines such as TNFa, IL-1b and IFNg. Moreover, we report the transcriptional profile of
65
important immune markers such as mhcii (major histocompatibility complex class 2) and mpeg (macrophage expressed gene), demonstrating that Fno mainly infect macrophages as their replication site in zebrafish. Our research unfolds new possibilities to study the pathogenesis and to develop treatments against intracellular pathogens by using zebrafish as an infection model. Keywords: OMVs, vaccine, francisellosis, zebrafish, immune response * Corresponding author. Tel.: þ47 41079225. E-mail address: [email protected] (L. Lagos).
O-021. Adoptive transfer of immunity against red mouth disease in rainbow trout (Oncorhynchus mykiss) Takuya Yamaguchi, Uwe Fischer§ Federal Research Institute for Animal Health, Friedrich-LoefflerInstitut, Südufer 10, 17393, Greifswald-Insel Riems, Germany Enteric Red Mouth Disease (ERM) caused by Yersinia ruckeri is among the bacterial diseases in rainbow trout aquaculture that can be controlled by registered vaccines and part of these vaccines can be delivered by immersion which is the most convenient way of application inducing good antibody responses and ensuring decreased handling costs. However, the mechanisms by which fish are protected against Yersinia ruckeri are unclear. To shed light on this question we have bath vaccinated clonal rainbow trout donors by using a commercially available vaccine, and leukocytes have been isolated from vaccinated donors followed by adoptive cell transfer to naïve fish recipients of the same trout clone. Control recipients were injected with same numbers of leukocytes from nonvaccinated clonal donors. Transfer between genetically identical donors and recipients ensured that immune cells were not rejected by the recipients. After transfer, both groups of recipients were bath infected with a virulent field strain of Yersinia ruckeri. While rainbow trout that have received leukocytes from vaccinated donors showed no mortality, less than 40% of control recipients survived the challenge. Flow cytometry using leukocytes from recipient survivors revealed that the percentage of IgMþ B cells was considerably higher in recipients that were injected with leukocytes from vaccinated donors when compared with control recipients, while no differences in the percentages of CD8þ cells among groups were recorded. Being the first report on adoptive cellular transfer of immunity in a bacterial infection in fish these data suggest that immunity was rather transferred by IgMþ B cells, but not by CD8þ cytotoxic T cells. . It can be further suggested that bath vaccination has caused an effective systemic immune response since protection could be transferred by nonmucosal leukocytes. Keywords: rainbow trout, Yersinia ruckeri, adoptive transfer, B cells, T cells x Corresponding author. Tel.: þ49 38351 71175; Fax: þ49 38351 71226. E-mail address: uwe.fischer@fli.bund.de (U. Fischer).
O-022. Oil adjuvant elevates protection of rainbow trout (Oncorhynchus mykiss) following injection vaccination against Yersinia ruckeri Rzgar M. Jaafar a, $, Jiwan K. Chettri a, Inger Dalsgaard b, Azmi Al-Jubury a, Per W. Kania a, Jakob Skov a, Kurt Buchmann a a Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark b National Veterinary Institute, Technical University of Denmark,