Rosario Castro

Effect of oral administration of glucans on the resistance of gilthead seabream to pasteurellosis

Abstract The present study evaluated the effect of oral administration of three types of glucan on the resistance of gilthead seabream to Photobacterium damselae subsp. piscicida and on the activity of its phagocytes. Groups of fish were fed with a basal diet (controls) or with the basal diet supplemented with glucan for two different periods of time before being bath challenged with the bacterium. Groups of fish fed with 1 and 10 g kg −1 of glucan for a short period (2 weeks with glucan and 1 week with the basal diet) showed a higher degree of protection against pasteurellosis than the control group. This was especially pronounced in groups of fish fed with the higher concentrations of glucan. The respiratory burst and phagocytic activity of spleen phagocytes varied with time but no significant differences were found between groups in the third week when the challenge was carried out. The group of fish fed with 1 g kg −1 of glucan for longer periods of time (2 weeks with glucan, 1 week with the basal diet, 2 weeks with glucan, and 1 week with the basal diet) also showed enhanced protection against P. damselae . However, the group fed with 5 g kg −1 had mortality rates comparable to the control group, and protection in the group fed with 10 g kg −1 of glucan was significantly lower than in the control group. These results suggest that glucans can be used in the diet to prevent or reduce mortalities in gilthead seabream due to pasteurellosis, and they show the importance of the concentration and the period of administration of glucan to obtain optimal protection against this disease.

Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection

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 repertoires for IgM, IgD and IgT, respectively, with a few amplified μ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.

The Astonishing Diversity of Ig Classes and B Cell Repertoires in Teleost Fish

With lymphoid tissue anatomy different than mammals, and diverse adaptations to all aquatic environments, fish constitute a fascinating group of vertebrate to study the biology of B cell repertoires in a comparative perspective. Fish B lymphocytes express immunoglobulin (Ig) on their surface and secrete antigen-specific antibodies in response to immune challenges. Three antibody classes have been identified in fish, namely IgM, IgD, and IgT, while IgG, IgA, and IgE are absent. IgM and IgD have been found in all fish species analyzed, and thus seem to be primordial antibody classes. IgM and IgD are normally co-expressed from the same mRNA through alternative splicing, as in mammals. Tetrameric IgM is the main antibody class found in serum. Some species of fish also have IgT, which seems to exist only in fish and is specialized in mucosal immunity. IgM/IgD and IgT are expressed by two different sub-populations of B cells. The tools available to investigate B cell responses at the cellular level in fish are limited, but the progress of fish genomics has started to unravel a rich diversity of IgH and immunoglobulin light chain locus organization, which might be related to the succession of genome remodelings that occurred during fish evolution. Moreover, the development of deep sequencing techniques has allowed the investigation of the global features of the expressed fish B cell repertoires in zebrafish and rainbow trout, in steady state or after infection. This review provides a description of the organization of fish Ig loci, with a particular emphasis on their heterogeneity between species, and presents recent data on the structure of the expressed Ig repertoire in healthy and infected fish.

Water-soluble seaweed extracts modulate the respiratory burst activity of turbot phagocytes

Abstract In this study, we investigated the effects of water-soluble extracts (WSE) from seaweed species Ulva rigida C. Agardh, Enteromorpha sp., Codium tomentosum (Huds) Stackh., Fucus vesiculosus L., Pelvetia canaliculata (L.) Decne et Thur, Dictyota dichotoma (Huds) Lamour, Chondrus crispus Stackh and Porphyra umbilicalis (L.) J. Agardh, on the respiratory burst of turbot phagocytes, in search of biologically active substances with immunostimulating capacities. The stimulatory capacities of the extracts varied greatly, depending on their origin, the concentrations used and the time of incubation. The best responses were induced by the extracts obtained from U. rigida , Enteromorpha sp. and C. crispus . Pre-incubation of the phagocyte cells with U. rigida and C. crispus extracts and subsequent incubation with phorbol myristate acetate (PMA) showed that these cells responded to further stimulation by this substance and, at some concentrations, they showed higher respiratory burst activity than control cells stimulated by PMA alone, suggesting that the extracts had a priming effect. We also tested the effects of protein-free water-soluble extracts (PF-WSE) and of the polysaccharide fractions (PSF) obtained from the PF-WSE of U. rigida and C. crispus . These extracts induced an increase in the respiratory burst activity of turbot phagocytes, suggesting that most of the stimulatory capacity of the water-soluble extracts was associated with polysaccharides.

T cell diversity and TcR repertoires in teleost fish.

In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses.

The pyloric caeca area is a major site for IgM+ and IgT+ B cell recruitment in response to oral vaccination in rainbow trout

Although previous studies have characterized some aspects of the immune response of the teleost gut in response to diverse pathogens or stimuli, most studies have focused on the posterior segments exclusively. However, there are still many details of how teleost intestinal immunity is regulated that remain unsolved, including the location of IgM+ and IgT+ B cells along the digestive tract and their role during the course of a local stimulus. Thus, in the current work, we have studied the B cell response in five different segments of the rainbow trout (Oncorhynchus mykiss) digestive tract in both naïve fish and fish orally vaccinated with an alginate-encapsulated DNA vaccine against infectious pancreatic necrosis virus (IPNV). IgM+ and IgT+ cells were identified all along the tract with the exception of the stomach in naïve fish. While IgM+ cells were mostly located in the lamina propria (LP), IgT+ cells were primarily localized as intraepithelial lymphocytes (IELs). Scattered IgM+ IELs were only detected in the pyloric caeca. In response to oral vaccination, the pyloric caeca region was the area of the digestive tract in which a major recruitment of B cells was demonstrated through both real time PCR and immunohistochemistry, observing a significant increase in the number of both IgM+ and IgT+ IELs. Our findings demonstrate that both IgM+ and IgT+ respond to oral stimulation and challenge the paradigm that teleost IELs are exclusively T cells. Unexpectedly, we have also detected B cells in the fat tissue associated to the digestive tract that respond to vaccination, suggesting that these cells surrounded by adipocytes also play a role in mucosal defense.

CCR7 Is Mainly Expressed in Teleost Gills, Where It Defines an IgD+IgM− B Lymphocyte Subset

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyers patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7+ cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7+ cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7+ cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD+ cells in the gills expressed CCR7. Intriguingly, the IgD+CCR7+ population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7+ cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD+memIgM− B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD+IgM− subsets in mammals are discussed.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.

Antigenic and cross-protection studies on two turbot scuticociliate isolates

The protection induced in turbot by inactivated vaccines containing either of two isolates (I(1) and C(1)) of the scuticociliate parasite Philasterides dicentrarchi, which causes important mortalities in turbot cultures, was evaluated in the present study. The results obtained after challenging the fish with the two isolates show that vaccination protected fish only against the homologous isolate, but did not confer cross-protection. The two isolates constitute two serotypes, as shown in the immobilization tests with mouse and turbot anti-I(1) and anti-C(1) antisera, in which only the homologous antisera immobilized the ciliates. ELISA assays, using total antigen free of proteases (TAWP), cytosolic antigens (CYA), ciliar antigens (CA) or membrane protein fraction (MPF), were also carried out. Differences in the levels of antibodies produced in mouse against the homologous and heterologous antigens were observed; these differences were significantly different when the antigen preparations used in the ELISA were TAWP, CYA or CA. Nevertheless, ELISA assays using turbot sera against TAWP did not show significant differences in the levels of antibodies against the homologous and heterologous antigens. Antigenic cross-reactivity was also detected in the Western blot assays, as well as significant differences in the patterns of antigenic recognition in the two isolates - in both reduced and non-reduced TAWP antigens, but which was noteworthy when mouse antisera were used. The results obtained in the present study demonstrate for the first time the existence of serotypes of the ciliate parasite of turbot Philasterides dicentrarchi that display clear antigenic differences, which must be taken into consideration in the future development of a vaccine against scuticociliatosis.

Early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (VHSV) infection.

Among the essential metabolic functions of the liver, in mammals, a role as mediator of systemic and local innate immunity has also been reported. Although the presence of an important leukocyte population in mammalian liver is well documented, the characterization of leukocyte populations in the teleost liver has been only scarcely addressed. In the current work, we have confirmed the presence of IgM+, IgD+, IgT+, CD8α+, CD3+ cells, and cells expressing major histocompatibility complex (MHC-II) in rainbow trout (Oncorhynchus mykiss) liver by flow cytometry and/or immunohistochemistry analysis. Additionally, the effect of viral hemorrhagic septicemia virus (VHSV) on the liver immune response was assessed. First, we studied the effect of viral intraperitoneal injection on the transcription of a wide selection of immune genes at days 1, 2 and 5 post-infection. These included a group of leukocyte markers genes, pattern recognition receptors (PRRs), chemokines, chemokine receptor genes, and other genes involved in the early immune response and in acute phase reaction. Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV. Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver. No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations. In addition, a strong up-regulation in the transcription levels of several PRRs and chemokines was observed from the second day of infection, indicating an important role of these factors in the response of the liver to viral infections.