- Email: [email protected]
Studies of antimicrobial peptides in rainbow trout
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
closely related to tetrapods than to bony fishes, and therefore could be considered as the living sister-group of tetrapods. * Corresponding author. E-mail address: [email protected] (F. Buonocore)
O-361. Eel immune response to Vibrio vulnificus infection. Host-pathogen relationship A. Callol 1, 2, *, D. Pajuelo 1, S. MacKenzie 2, 3, C. Amaro 1. 1 Department of Microbiology and Ecology, Faculty of Biology, University of Valencia, Burjassot, Valencia, Spain; 2 Institute of Biotechnology and Biomedicine, University Autonomous of Barcelona, Bellaterra, Barcelona, Spain; 3 Institute of Aquaculture, School of Naural Sciences, University of Stirling, Stirling, Scotland, UK
Abstract The European eel (Anguilla anguilla), has experienced a dangerous decline in recruitment, yield and stock over the last 30 years and this decline is likely to continue into the future. Several major threats are responsible for this situation, including overfishing of glass eels for consumption, new infections by introduced pathogens, dams and blocking of migration routes. The lack of knowledge concerning the biology of this species represents a handicap to the conservation and recovery of the population. Nevertheless, the eel farming industry is increasing, therefore it could be considered one of the major risks for wild eel stock perpetuation, which is threatened principally by the lack of knowledge of the complete reproductive cycle of the European eel and by emerging bacterial, viral and parasite diseases. Vibrio vulnificus is the aetiological agent of warm-water vibriosis, a disease that constitutes the main threat to eels under culture conditions, provoking important outbreaks and can be an opportunistic pathogen for humans. V. vulnificus uses a novel characterized virulence and survival system named MARTX (multifunctional repeat in toxin) that it is supposed to trigger a cytokine storm when it is produced in blood during the infection. It is also involved in the colonization of the gills, to invade internal organs and causes death by septicemia. We sequenced an eel immune-enriched transcriptome with Roche 454, and the data has been used to create new molecular tools for further research. We have also designed a custom eel-specific microarray (4x44K, Agilent). Our research has been focused on eel-vibrio interaction, how the host responds to the challenge and the importance of MARTX system of the bacteria for the colonization. Different challenges were performed using wild type strain (CECT4999) and Rtx double mutant strain (CT285) to evaluate mucosal immunity in the gills, the principal portal of entry. We also studied the early immune response of circulating leukocytes and the role of the erythrocytes, as one of the target cells of the lytic activity of this bacterium. Results obtained by RT-qPCR and ISH on gills demonstrate the immunocompetent function of this tissue, based on the expression and localization of immune-relates genes such as, TLRs, cytokines, chemokines and signalling molecules. Microarray studies describe key genes expressed after vibrio infection analyzed across 12h post-challenge on gills and blood (leukocytes and erythrocytes separately), specific host response against vibrio Rtx toxin, as well as, a possible function of erythrocytes in the immune response. * Corresponding author. E-mail address: [email protected] (A. Callol)
O-241. Interactions between hemolymph components of Mytilus galloprovincialis and different Vibrio species L. Canesi*, T. Balbi, R. Fabbri, A. Smerilli, E. Pezzati, C. Grande, M. Stauder, L. Vezzulli, C. Pruzzo. DISTAV, Dipartimento di Scienze della Terra, dell'Ambiente e della Vita, Università di Genova, Italy
Abstract Marine bivalves are filter-feeding invertebrates that can accumulate large numbers of bacteria, in particular Vibrio species particularly abundant in coastal waters. Persistence of different vibrios in bivalve tissues largely depends on their sensitivity to the bactericidal activity of the hemolymph, resulting from complex interactions between bacteria, circulating hemocytes, and soluble hemolymph components. Host-pathogen interactions have been increasingly investigated in different bivalve species, with the aim of understanding the pathogenesis of diseases in species susceptible to infection by certain Vibrio spp. and strains. V. splendidus LPG32 strains have been associated with the ‘summer mortalities’ syndrome of juvenile oysters. On the other hand, the mussel Mytilus is considered to be particularly resistant to Vibrio infection. In this work, in order to explore the susceptibility of mussels to different Vibrio species, the interactions between hemolymph components of Mytilus galloprovincialis and V. aestuarianus and V. splendidus were investigated. In vitro, the bactericidal activity of whole hemolymph towards different vibrios, the capacity of bacteria to adhere to hemocyte monolayers both in the presence and in the absence of different sugars, as well as their effects on hemocyte lysosomal membrane stability (LMS) were evaluated. In in vivo experiments, mussels were injected with live V. aestuarianus and V. splendidus and bactericidal activity of whole hemolymph, bacterial concentration in mussel tissues, hemocyte LMS and serum lysozyme activity were evaluated at 6, 24 and 96 h post-injection. Moreover, LMS was evaluated as a biomarker of general stress in the digestive gland. Overall, the results indicate distinct surface interactions between mussel hemolymph components and the vibrio strains tested: these interactions lead to killing of V. aestuarianus, whereas V. splendidus was actually able to grow within the hemolymph. Moreover, V. splendidus induced severe decreases in LMS in both circulating hemocytes and digestive gland. The results demonstrate that, although V. splendidus LPG32 is considered not pathogenic to Mytilus, this vibrio strain is able to grow within the mussel host and to cause disruption of the lysosomal function at the cellular and tissue level, thus leading to significant stressful conditions and alteration of mussel health. This work was supported by the EU project BIVALIFE (FP7-KBBE-2010-4). * Corresponding author. E-mail address: [email protected] (L. Canesi)
O-185. Studies of antimicrobial peptides in rainbow trout E. Casadei 1, *, J. Zou 1, S. Wadsworth 2, J.L. González Vecino 2, C.J. Secombes 1. 1
SFIRC, Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK; 2 EWOS Innovation A.S., Dirdal N-4335, Norway Abstract In fish, the innate immune system is the first line of defence against disease and is represented by physical barriers such as the scales, skin, epithelium, mucus and other body fluids. These structures not only provide protection against external agents, they also contain humoral components (complement factors, lysozyme, antimicrobial peptides, immunoglobulin) able to augment the defence of the organism. In particular, antimicrobial peptides (AMPs) can directly interact with the cell wall components of invading microbes, disrupting the membrane integrity of these pathogens. In addition in mammals these AMPs are known to activate the acquired immune response required for a more efficient and long-lasting protection. AMPs may also have value as markers of immunostimulant treatment or as adjuvants to generate more potent vaccines. The purpose of this research was two-fold. Firstly, to study the expression of the known repertoire of rainbow trout AMPs after administration of a novel functional feed containing peptidoglycan, to assess which AMPs are induced and when/where this occurs. Secondly, initial attempts were undertaken to produce the four known trout betadefensins (omDB-1, omDB-2, omDB-3, and omDB-4), using transfection of RTG-2 and establishment of cell lines expressing these AMPs. Our findings shed light on the multiple roles of AMPs in fish not only as a first
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
line of defence (microbicidal activity) but also o their potential involvement in other immunological pathways. * Corresponding author. E-mail address: [email protected] (E. Casadei)
O-417. Proteomic characterization of the common octopus hemolymph: effect of the infection by the protozoan parasite Aggregata octopiana S. Castellanos-Martínez 1, A.P. Diz 2, P. Álvarez-Chaver 3, C. Gestal 1, *. 1 Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello, 6, 36208 Vigo, Spain; 2 Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Vigo, Spain; 3 Unidad de Proteómica, CACTI, Universidad de Vigo, 36310 Vigo, Spain
Abstract The common octopus, Octopus vulgaris (Mollusca: Cephalopoda), is an important species in worldwide fisheries. In recent years, the interest for developing the commercial culture of this cephalopod species has gained increasing attention due to their economic importance and the declining of landings. However, diseases caused by different pathogens affecting this species can result in large economic loses. One of the most important pathogens affecting octopuses is the gastrointestinal parasite Aggregata octopiana. Despite the economical relevance and undeniable potential as an aquaculture species, a limited knowledge exists about the defence mechanisms of this mollusc. Nowadays, molecular tools are useful to assess the health of the organisms and to find specimens resistant to infections, and proteomic offers a new way to complement genomic studies. In this study we use a proteomic approach to analyze the octopus response against the infection by the coccidia Aggregata octopiana. A comparative proteomic analysis of the hemolymph of octopus highly and lowly infected by the parasite showed differences in relative abundance in samples from hemocytes and plasma. The identities of 36 proteins from hemocytes and 5 proteins from plasma were determined by ESI-FTICR and MALDI-TOF-TOF. In addition, 7 proteins were selected by principal component analysis as the major contributors to differences between levels of infection and potential biomarkers. Among these, peroxiredoxin, filamin and fascin are highlighted. The present results lead to obtain the general protein profile of the common octopus hemolymph and suggest that the infection by the parasite influences the activation of the O. vulgaris immune system. Additionally, the study supports the utility of the proteomic to analyze changes in the octopus proteome, contributing to understand the bases of octopus tolerance-resistance to A. octopiana. * Corresponding author. E-mail address: [email protected] (C. Gestal)
O-124. DNA vaccination in fish promotes an early chemokine-related recruitment of B cells to the muscle R. Castro 1, S. Martínez-Alonso 1, U. Fischer 2, N. Álvarez de Haro 3, V. SotoLampe 2, N. Lorenzen 4, E. Lorenzen 4, T. Wang 5, C.J. Secombes 5, C. Tafalla 1,*. 1
Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain; 2 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Insel Riems, Germany; 3 Área de Biología Celular, Universidad de León, León, Spain; 4 National Veterinary Laboratory, Technical University of Denmark, Aarhus, Denmark; 5 Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, UK Abstract In fish, intramuscular injection of plasmid DNA encoding viral proteins has proved as the most effective vaccination strategy against many viral
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pathogens. The efficacy of DNA vaccination in teleost fish is based on a high level of viral antigen expression in muscle cells inducing a strong and longlasting protection. However, the mechanisms through which this protection is conferred in fish are still not understood. Moreover, similarities to mammalian models can not be established since DNA vaccination in mammals induces much lower responses. In this work, we have focused on the characterization of immune cells that infiltrate the muscle at the site of DNA delivery in vaccinated fish and the chemokines that may be involved in their infiltration. It was observed that B lymphocytes, both IgM+ and IgT+, represent a major infiltrating cell type in fish vaccinated with a viral hemorrhagic septicemia virus (VHSV) DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocytes were attracted. While IgM+ cells were the major B cell population at early time points post vaccination, IgT+ cells represented the predominant cell type later on. Among twelve chemokine genes studied in the injected muscle tissues, only CXCL10, CK5B and CK6 were more strongly transcribed in DNA vaccinated fish compared to control fish injected with the corresponding vector backbone. In vitro tests performed with recombinant trout CK5B and CK6 revealed that these chemokines have chemotactic capacities which might explain the recruitment of immune cells to the site of DNA injection. Our results suggest that B cells are involved in the initial phase of the immune response to intramuscular DNA vaccination against VHSV. This appears to be a major difference to what we know from mammalian models where T cells play a major role. * Corresponding author. E-mail address: [email protected] (C. Tafalla)
O-161. Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection R. Castro 1, L. Jouneau 1, H.P. Pham 2, 3, O. Bouchez 4, V. Giudicelli 5, M.P. Lefranc 5, E. Quillet 6, A. Benmansour 1, F. Cazals 7, A. Six 2, S. Fillatreau 8, O. Sunyer 9, P. Boudinot 1, *. 1
Virologie et Immunologie Moléculaires, INRA, Jouy-en-Josas, France; UPMC Univ Paris 06, UMR 7211, “Integrative Immunology” Team, Paris, France; 3 CNRS, UMR 7211, “Immunology, Immunopathology, Immunotherapy”, Paris, France; 4 UMR INRA 0444 Laboratoire de Génétique Cellulaire, GeT-PlaGe core facility, Castanet Tolosan, France; 5 IMGT, The International ImMunoGeneTics Information System, Laboratoire d'ImmunoGénétique Moléculaire LIGM, IGH, UPR CNRS 1142 and Université Montpellier 2, Montpellier, France; 6 Génétique Animale et Biologie Intégrative, INRA, 78352 Jouy-enJosas, France; 7 INRIA Sophia-Antipolis, Méditerranée, Algorithms-Biology-Structure, Sophia-Antipolis, France; 8 Deutsches RheumaForschungszentrum, A Leibniz Institute, Berlin, Germany; 9 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA 2
Abstract Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
closely related to tetrapods than to bony fishes, and therefore could be considered as the living sister-group of tetrapods. * Corresponding author. E-mail address: [email protected] (F. Buonocore)
O-361. Eel immune response to Vibrio vulnificus infection. Host-pathogen relationship A. Callol 1, 2, *, D. Pajuelo 1, S. MacKenzie 2, 3, C. Amaro 1. 1 Department of Microbiology and Ecology, Faculty of Biology, University of Valencia, Burjassot, Valencia, Spain; 2 Institute of Biotechnology and Biomedicine, University Autonomous of Barcelona, Bellaterra, Barcelona, Spain; 3 Institute of Aquaculture, School of Naural Sciences, University of Stirling, Stirling, Scotland, UK
Abstract The European eel (Anguilla anguilla), has experienced a dangerous decline in recruitment, yield and stock over the last 30 years and this decline is likely to continue into the future. Several major threats are responsible for this situation, including overfishing of glass eels for consumption, new infections by introduced pathogens, dams and blocking of migration routes. The lack of knowledge concerning the biology of this species represents a handicap to the conservation and recovery of the population. Nevertheless, the eel farming industry is increasing, therefore it could be considered one of the major risks for wild eel stock perpetuation, which is threatened principally by the lack of knowledge of the complete reproductive cycle of the European eel and by emerging bacterial, viral and parasite diseases. Vibrio vulnificus is the aetiological agent of warm-water vibriosis, a disease that constitutes the main threat to eels under culture conditions, provoking important outbreaks and can be an opportunistic pathogen for humans. V. vulnificus uses a novel characterized virulence and survival system named MARTX (multifunctional repeat in toxin) that it is supposed to trigger a cytokine storm when it is produced in blood during the infection. It is also involved in the colonization of the gills, to invade internal organs and causes death by septicemia. We sequenced an eel immune-enriched transcriptome with Roche 454, and the data has been used to create new molecular tools for further research. We have also designed a custom eel-specific microarray (4x44K, Agilent). Our research has been focused on eel-vibrio interaction, how the host responds to the challenge and the importance of MARTX system of the bacteria for the colonization. Different challenges were performed using wild type strain (CECT4999) and Rtx double mutant strain (CT285) to evaluate mucosal immunity in the gills, the principal portal of entry. We also studied the early immune response of circulating leukocytes and the role of the erythrocytes, as one of the target cells of the lytic activity of this bacterium. Results obtained by RT-qPCR and ISH on gills demonstrate the immunocompetent function of this tissue, based on the expression and localization of immune-relates genes such as, TLRs, cytokines, chemokines and signalling molecules. Microarray studies describe key genes expressed after vibrio infection analyzed across 12h post-challenge on gills and blood (leukocytes and erythrocytes separately), specific host response against vibrio Rtx toxin, as well as, a possible function of erythrocytes in the immune response. * Corresponding author. E-mail address: [email protected] (A. Callol)
O-241. Interactions between hemolymph components of Mytilus galloprovincialis and different Vibrio species L. Canesi*, T. Balbi, R. Fabbri, A. Smerilli, E. Pezzati, C. Grande, M. Stauder, L. Vezzulli, C. Pruzzo. DISTAV, Dipartimento di Scienze della Terra, dell'Ambiente e della Vita, Università di Genova, Italy
Abstract Marine bivalves are filter-feeding invertebrates that can accumulate large numbers of bacteria, in particular Vibrio species particularly abundant in coastal waters. Persistence of different vibrios in bivalve tissues largely depends on their sensitivity to the bactericidal activity of the hemolymph, resulting from complex interactions between bacteria, circulating hemocytes, and soluble hemolymph components. Host-pathogen interactions have been increasingly investigated in different bivalve species, with the aim of understanding the pathogenesis of diseases in species susceptible to infection by certain Vibrio spp. and strains. V. splendidus LPG32 strains have been associated with the ‘summer mortalities’ syndrome of juvenile oysters. On the other hand, the mussel Mytilus is considered to be particularly resistant to Vibrio infection. In this work, in order to explore the susceptibility of mussels to different Vibrio species, the interactions between hemolymph components of Mytilus galloprovincialis and V. aestuarianus and V. splendidus were investigated. In vitro, the bactericidal activity of whole hemolymph towards different vibrios, the capacity of bacteria to adhere to hemocyte monolayers both in the presence and in the absence of different sugars, as well as their effects on hemocyte lysosomal membrane stability (LMS) were evaluated. In in vivo experiments, mussels were injected with live V. aestuarianus and V. splendidus and bactericidal activity of whole hemolymph, bacterial concentration in mussel tissues, hemocyte LMS and serum lysozyme activity were evaluated at 6, 24 and 96 h post-injection. Moreover, LMS was evaluated as a biomarker of general stress in the digestive gland. Overall, the results indicate distinct surface interactions between mussel hemolymph components and the vibrio strains tested: these interactions lead to killing of V. aestuarianus, whereas V. splendidus was actually able to grow within the hemolymph. Moreover, V. splendidus induced severe decreases in LMS in both circulating hemocytes and digestive gland. The results demonstrate that, although V. splendidus LPG32 is considered not pathogenic to Mytilus, this vibrio strain is able to grow within the mussel host and to cause disruption of the lysosomal function at the cellular and tissue level, thus leading to significant stressful conditions and alteration of mussel health. This work was supported by the EU project BIVALIFE (FP7-KBBE-2010-4). * Corresponding author. E-mail address: [email protected] (L. Canesi)
O-185. Studies of antimicrobial peptides in rainbow trout E. Casadei 1, *, J. Zou 1, S. Wadsworth 2, J.L. González Vecino 2, C.J. Secombes 1. 1
SFIRC, Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK; 2 EWOS Innovation A.S., Dirdal N-4335, Norway Abstract In fish, the innate immune system is the first line of defence against disease and is represented by physical barriers such as the scales, skin, epithelium, mucus and other body fluids. These structures not only provide protection against external agents, they also contain humoral components (complement factors, lysozyme, antimicrobial peptides, immunoglobulin) able to augment the defence of the organism. In particular, antimicrobial peptides (AMPs) can directly interact with the cell wall components of invading microbes, disrupting the membrane integrity of these pathogens. In addition in mammals these AMPs are known to activate the acquired immune response required for a more efficient and long-lasting protection. AMPs may also have value as markers of immunostimulant treatment or as adjuvants to generate more potent vaccines. The purpose of this research was two-fold. Firstly, to study the expression of the known repertoire of rainbow trout AMPs after administration of a novel functional feed containing peptidoglycan, to assess which AMPs are induced and when/where this occurs. Secondly, initial attempts were undertaken to produce the four known trout betadefensins (omDB-1, omDB-2, omDB-3, and omDB-4), using transfection of RTG-2 and establishment of cell lines expressing these AMPs. Our findings shed light on the multiple roles of AMPs in fish not only as a first
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
line of defence (microbicidal activity) but also o their potential involvement in other immunological pathways. * Corresponding author. E-mail address: [email protected] (E. Casadei)
O-417. Proteomic characterization of the common octopus hemolymph: effect of the infection by the protozoan parasite Aggregata octopiana S. Castellanos-Martínez 1, A.P. Diz 2, P. Álvarez-Chaver 3, C. Gestal 1, *. 1 Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello, 6, 36208 Vigo, Spain; 2 Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Vigo, Spain; 3 Unidad de Proteómica, CACTI, Universidad de Vigo, 36310 Vigo, Spain
Abstract The common octopus, Octopus vulgaris (Mollusca: Cephalopoda), is an important species in worldwide fisheries. In recent years, the interest for developing the commercial culture of this cephalopod species has gained increasing attention due to their economic importance and the declining of landings. However, diseases caused by different pathogens affecting this species can result in large economic loses. One of the most important pathogens affecting octopuses is the gastrointestinal parasite Aggregata octopiana. Despite the economical relevance and undeniable potential as an aquaculture species, a limited knowledge exists about the defence mechanisms of this mollusc. Nowadays, molecular tools are useful to assess the health of the organisms and to find specimens resistant to infections, and proteomic offers a new way to complement genomic studies. In this study we use a proteomic approach to analyze the octopus response against the infection by the coccidia Aggregata octopiana. A comparative proteomic analysis of the hemolymph of octopus highly and lowly infected by the parasite showed differences in relative abundance in samples from hemocytes and plasma. The identities of 36 proteins from hemocytes and 5 proteins from plasma were determined by ESI-FTICR and MALDI-TOF-TOF. In addition, 7 proteins were selected by principal component analysis as the major contributors to differences between levels of infection and potential biomarkers. Among these, peroxiredoxin, filamin and fascin are highlighted. The present results lead to obtain the general protein profile of the common octopus hemolymph and suggest that the infection by the parasite influences the activation of the O. vulgaris immune system. Additionally, the study supports the utility of the proteomic to analyze changes in the octopus proteome, contributing to understand the bases of octopus tolerance-resistance to A. octopiana. * Corresponding author. E-mail address: [email protected] (C. Gestal)
O-124. DNA vaccination in fish promotes an early chemokine-related recruitment of B cells to the muscle R. Castro 1, S. Martínez-Alonso 1, U. Fischer 2, N. Álvarez de Haro 3, V. SotoLampe 2, N. Lorenzen 4, E. Lorenzen 4, T. Wang 5, C.J. Secombes 5, C. Tafalla 1,*. 1
Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain; 2 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Insel Riems, Germany; 3 Área de Biología Celular, Universidad de León, León, Spain; 4 National Veterinary Laboratory, Technical University of Denmark, Aarhus, Denmark; 5 Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, UK Abstract In fish, intramuscular injection of plasmid DNA encoding viral proteins has proved as the most effective vaccination strategy against many viral
1643
pathogens. The efficacy of DNA vaccination in teleost fish is based on a high level of viral antigen expression in muscle cells inducing a strong and longlasting protection. However, the mechanisms through which this protection is conferred in fish are still not understood. Moreover, similarities to mammalian models can not be established since DNA vaccination in mammals induces much lower responses. In this work, we have focused on the characterization of immune cells that infiltrate the muscle at the site of DNA delivery in vaccinated fish and the chemokines that may be involved in their infiltration. It was observed that B lymphocytes, both IgM+ and IgT+, represent a major infiltrating cell type in fish vaccinated with a viral hemorrhagic septicemia virus (VHSV) DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocytes were attracted. While IgM+ cells were the major B cell population at early time points post vaccination, IgT+ cells represented the predominant cell type later on. Among twelve chemokine genes studied in the injected muscle tissues, only CXCL10, CK5B and CK6 were more strongly transcribed in DNA vaccinated fish compared to control fish injected with the corresponding vector backbone. In vitro tests performed with recombinant trout CK5B and CK6 revealed that these chemokines have chemotactic capacities which might explain the recruitment of immune cells to the site of DNA injection. Our results suggest that B cells are involved in the initial phase of the immune response to intramuscular DNA vaccination against VHSV. This appears to be a major difference to what we know from mammalian models where T cells play a major role. * Corresponding author. E-mail address: [email protected] (C. Tafalla)
O-161. Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection R. Castro 1, L. Jouneau 1, H.P. Pham 2, 3, O. Bouchez 4, V. Giudicelli 5, M.P. Lefranc 5, E. Quillet 6, A. Benmansour 1, F. Cazals 7, A. Six 2, S. Fillatreau 8, O. Sunyer 9, P. Boudinot 1, *. 1
Virologie et Immunologie Moléculaires, INRA, Jouy-en-Josas, France; UPMC Univ Paris 06, UMR 7211, “Integrative Immunology” Team, Paris, France; 3 CNRS, UMR 7211, “Immunology, Immunopathology, Immunotherapy”, Paris, France; 4 UMR INRA 0444 Laboratoire de Génétique Cellulaire, GeT-PlaGe core facility, Castanet Tolosan, France; 5 IMGT, The International ImMunoGeneTics Information System, Laboratoire d'ImmunoGénétique Moléculaire LIGM, IGH, UPR CNRS 1142 and Université Montpellier 2, Montpellier, France; 6 Génétique Animale et Biologie Intégrative, INRA, 78352 Jouy-enJosas, France; 7 INRIA Sophia-Antipolis, Méditerranée, Algorithms-Biology-Structure, Sophia-Antipolis, France; 8 Deutsches RheumaForschungszentrum, A Leibniz Institute, Berlin, Germany; 9 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA 2
Abstract Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct