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Many pathogens exploit the olfactory route to infect the lower respiratory tract or the central nervous system. Understanding the mechanisms by which the nasal mucosa halts pathogen progression is vital for the control of many human diseases. We have recently developed a model for the study of neuroimmune interactions in the olfactory organ of rainbow trout based on our initial discovery of a nasopharynx-associated lymphoid tissue in bony fish. Our studies indicate that viruses activate neurons in the olfactory epithelium and olfactory bulb of rainbow trout and that neuronal-viral interactions are required for the onset of ultra-rapid innate immune responses. These responses, that occur within minutes, have been overlooked thus far, and are characterized by the influx of non-conventional CD8 T cells that express TCRa/b. Further studies indicate that these response occur in response to a wide-range of microbial stimuli, albeit at lesser magnitude than in the case of live viruses. Current research efforts in our laboratory involve the validation of this model using human-relevant pathogens and vaccines such as the Flu vaccine. These results underscore the importance of understanding neuronal immune responses to pathogens and vaccines in order to develop better immunotherapies that exploit the nasal route.

Symbiotic bacteria and mucosal immunoglobulins have co-evolved for millions of years in vertebrate animals. Symbiotic bacteria products are known to modulate different aspects of the host immune system. We recently reported that Flectobacillus major is a predominant species that lives in the gill and skin mucosal surfaces of rainbow trout (Oncorhynchus mykiss). F. major is known to produce sphingolipids of a unique molecular structure. Here we propose a role for F. major and its sphingolipids in the regulation of B cell populations in rainbow trout, as well as an essential role for sphingolipids in trout mucosal homeostasis. We found that F. major-specific IgT titers are confined to the gill and skin mucus, whereas F. major-specific IgM titers are only detected in serum. Live F. major cells are able to stimulate sustained IgT expression and secretion in gills. F. major sphingolipids modulate the growth of trout total skin and gill symbiotic bacteria. In vivo systemic administration of F. major sphingolipids changes the proportion of IgT+ to IgM+ B cells in trout HK. These results demonstrate the key role of the symbiont F. major and its sphingolipids in mucosal homeostasis via the modulation of mucosal and systemic Igs and B cells.

Resumen: La menor participación de las mujeres en la industria minera predomina a nivel internacional. En Chile, a pesar de las mayores remuneraciones, del nivel tecnológico y del déficit proyectado de capital humano en el sector, las mujeres representan el 4,2% en las áreas operacionales. El objetivo de este trabajo es analizar el rol de las jefaturas mineras para revertir esta condición. Con un estudio de caso, en la Compañía minera Esperanza perteneciente al grupo Antofagasta Minerals, ubicada en el norte de Chile, se entrevistaron en profundidad a nueve jefes de áreas operacionales. Los resultados reportan lo imperativo de ejercer un liderazgo transformacional con perspectiva de género y con ello flexibilizar la gestión del recurso humano, facilitar la adaptación y proyección de las mujeres, promover las jefaturas femeninas y adjudicar a los jefes la responsabilidad de una gestión más simétrica. Estos hallazgos son centrales para una gestión estratégica en una industria que experimenta una alta demanda y un intenso dinamismo a nivel internacional. Clasificación JEL: J16, J81, B54. Abstract: A low participation of women in the mining industry is a prevailing issue around the world. In Chile, despite higher wages, technological level and the projected deficit of human capital in the sector, women represent 4.2% in operational areas. Therefore, the aim of this paper is to analyze the role of mining industry managers to reverse this condition. By means of a case study in Compañía Minera Esperanza, part of the group Antofagasta minerals, located in northern Chile, nine (9) heads of operational areas were in-depth interviewed. Results reported the need of executing a transformational leadership with a gender perspective and thus a more flexible human resource management, as well as facilitating the adaptation and projection of women, promoting managerial positions for women and assigning heads of areas with responsibilities to a more symmetrical management. These findings are central to strategic management in an industry experiencing high demand and intense dynamism at international level. Clasificación JEL: J16, J81, B54. Resumo: A menor participação das mulheres na indústria de mineração predomina a nível internacional. No Chile, apesar das maiores remunerações, do nível tecnológico e do déficit projetado de capital humano no setor, as mulheres representam 4,2% nas áreas operacionais. O objetivo deste trabalho é analisar o papel das chefias de mineração para reverter essa condição. Com um estudo de caso, na Compañía minera Esperanza pertencente ao grupo Antofagasta minerals, localizada no norte do Chile, entrevistaramse em profundidade a 9 chefes de áreas operacionais. Os resultados informam a necessidade de exercer uma liderança transformacional com perspectiva de gênero e com isso flexibilizar a gestão do recurso humano, facilitar a adaptação e projeção das mulheres, promover as chefias femininas e adjudicar aos chefes a responsabilidade de uma gestão mais simétrica. Essas descobertas são centrais para uma gestão estratégica numa indústria que experimenta uma alta demanda e um intenso dinamismo a nível internacional. Clasificación JEL: J16, J81, B54. Résumé: La faible participation des femmes dans l'industrie minière domine la scène internationale. Au Chili, malgré des salaires plus élevés, le niveau technologique et le déficit projeté du capital humain dans le secteur, les femmes représentent 4,2 % dans les sièges opérationnels. Le but de cet article est d'analyser le rôle des directions minières pour retourner cette condition. Au cours d'une étude de cas dans la Compañía Minera Esperanza, appartenant au groupe Antofagasta minerals, situé dans le nord du Chili, on a interviewé en profondeur neuf chefs de sièges opérationnels. Les résultats indiquent qu'il est impératif d'exercer le leadership transformationnel avec une perspective de sexe et, ce faisant, d'assouplir la gestion des ressources humaines, de faciliter l'adaptation et la projection des femmes, de promouvoir les directions féminines et d'attribuer aux directeurs la responsabilité d'une gestion plus symétrique. Ces résultats sont au cceur de la gestion stratégique dans un secteur connaissant une forte demande et de dynamisme intense au niveau international. Clasificación JEL: J16, J81, B54.

Mucosal surfaces require balancing different physiological roles and immune functions. To effectively achieve multifunctionality, mucosal epithelia have evolved unique microenvironments that create unique regional immune responses without impairing other normal physiological functions. Whereas examples of regional immunity are known in other mucosal epithelia, to date, no immune microenvironments have been described in the nasal mucosa, a site where the complex functions of olfaction and immunity need to be orchestrated. In this study we identified the presence of CD8α+ cells in the rainbow trout (Oncorhynchus mykiss) nasal epithelium. Nasal CD8α+ cells display a distinct phenotype suggestive of CD8+ T cells with high integrin β2 expression. Importantly, nasal CD8α+ cells are located in clusters at the mucosal tip of each olfactory lamella but scattered in the neuroepithelial region. The grouping of CD8α+ cells may be explained by the greater expression of CCL19, ICAM-1, and VCAM-1 in the mucosal tip compared with the neuroepithelium. Whereas viral Ag uptake occurred via both tip and lateral routes, tip-resident MHC class II+ cells are located significantly closer to the lumen of the nasal cavity than are their neuroepithelial counterparts, therefore having quicker access to invading pathogens. Our studies reveal compartmentalized mucosal immune responses within the nasal mucosa of a vertebrate species, a strategy that likely optimizes local immune responses while protecting olfactory sensory functions.

This study investigates the ontogeny of New Zealand groper (Polyprion oxygeneios) immune system, a new species for aquaculture in the Southern Pacific Ocean. In the eggs, both lysozyme and IgM were detected. Egg IgM was found at 1.07-1.56 μg/g wet weight and consisted of monomers compared to the polymerized IgM found in adult serum. In larvae, the head-kidney (HK) was first observed at 6 dph, followed by the spleen at 16 dph, and thymus at 20 dph, and within these organs IgM(+) cells were first detected in the HK (12 dph), then the spleen (32 dph) and finally in the thymus and the gastrointestinal tract (45 dph). Low levels of Igμ heavy chain transcripts were detected at 2 and 3 dph and they increased at 9 dph. Igμ expression further increased from day 45 onwards. In juveniles (115 dph), the HK and blood showed similar percentages of IgM(+) cells as the adult groper. These results highlight the important maturation steps that occur during the development of the immune system in the marine teleost P. oxygeneios.

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus , a pathogen of New Zealand groper ( Polyprion oxygeneios ). The aim of this study was to characterise both the host (groper)-parasite ( M. avidus ) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO + cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus , which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals. Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg −1 ) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo . Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite.

As physical barriers that separate teleost fish from the external environment, mucosae are also active immunological sites that protect them against exposure to microbes and stressors. In mammals, the sites where antigens are sampled from mucosal surfaces and where stimulation of naive T and B lymphocytes occurs are known as inductive sites and are constituted by mucosa-associated lymphoid tissue (MALT). According to anatomical location, the MALT in teleost fish is subdivided into gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), and gill-associated lymphoid tissue (GIALT). All MALT contain a variety of leukocytes, including, but not limited to, T cells, B cells, plasma cells, macrophages and granulocytes. Secretory immunoglobulins are produced mainly by plasmablasts and plasma cells, and play key roles in the maintenance of mucosal homeostasis. Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD, in which IgM was thought to be the only one responding to pathogens both in systemic and mucosal compartments. However, a third teleost immunoglobulin class, IgT/IgZ, was discovered in 2005, and it has recently been shown to behave as the prevalent immunoglobulin in gut mucosal immune responses. The purpose of this review is to summarise the current knowledge of mucosal immunoglobulins and B cells of fish MALT. Moreover, we attempt to integrate the existing knowledge on both basic and applied research findings on fish mucosal immune responses, with the goal to provide new directions that may facilitate the development of novel vaccination strategies that stimulate not only systemic, but also mucosal immunity.

Vertebrate olfactory sensory neurons (OSNs) are located in direct contact with the external environment and therefore they directly exposed to microorganisms such as viruses. Teleost fish have four types of OSNs, including crypt neurons, a cell type with enigmatic function. Crypt neurons express tyrosine receptor kinase A (TrkA)-like molecule and ORA4. In this study, we hypothesized that TrkA expressing crypt neurons are fast sensors of viruses in the olfactory mucosa of fish serving as an ultra-rapid mechanism to elicit local antiviral immune responses. Our results show that, within minutes, nasal delivery of rhadboviruses induces caspase dependent apoptosis in crypt neurons along with neuronal activation in rainbow trout olfactory organ (OO) and olfactory bulb (OB). Induction of chemokine CCL19 and prostaglandin synthase expression was detected 15 minutes after nasal delivery of virus in OO; whereas, in parallel, significant downregulation of CCL19 expression was measured in the OB. Pharmacological blocking of TrkA abolished these responses. CD8+ T cells infiltrated the OO within minutes of nasal viral delivery and this response was abrogated when TrkA was blocked. We identified that the microvasculature surrounding the OB and not the peripheral blood is the source of the infiltrating CD8 T cells suggesting a rapid shunt of these immune cells from the OB microvasculature into the OO. In support, ablation of crypt neurons in zebrafish results in increased susceptibility to rhabdoviral challenge. Combined, our results indicate a novel function for a subset of teleost OSNs, crypt neurons, and the importance of sensory neurons as a first layer of danger detection in vertebrates.

The symbiosis between metazoans and prokaryotes is one of the most ancient and successful relationships found in nature. Mucosal surfaces are the main interface between animal hosts and their environment, and these surfaces are colonized by unique microbial communities that shape every physiological system of the host. Teleost fish have both an innate and adaptive immune system and are the most ancient vertebrates with a dedicated mucosal immunoglobulin (Ig), known as IgT/IgZ. IgT is secreted via the polymeric Ig receptor across teleost mucosal epithelia, including the gills, skin, nose and gut. Recent studies have shed light on the interactions between mucosal Igs and microbiota in teleosts both during health and disease. Teleost fish symbiotic bacteria are coated by both secretory Igs (sIgT and sIgM) as well as pIgR. Using two different models, a stress model and an IgT depletion model, we provide evidence that any alterations in the levels of mucosal Igs result in different states of dysbiosis in rainbow trout (Oncorhynchus mykiss). Dysbiosis in trout is characterized by losses of certain taxa and subsequent invasion of new species in several mucosal sites. Unlike in mammalian models of IgA deficiency, where IgM compensatory responses occur, no compensatory IgM or IgD responses were observed in IgT-depleted rainbow trout. These results demonstrate that teleost fish, similar to mammals, utilize specialized mucosal Igs to maintain harmonic relationships with symbiotic bacteria.

Intensive fish production worldwide has increased the risk of infectious diseases. However, before any infection can be established, pathogens must penetrate the primary barrier. In fish, the three major routes of infection are the skin, gills and gastrointestinal (GI) tract. The GI tract is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit a regional variation in structure and function. In the last two decades, our understanding of the endocytosis and translocation of bacteria across this mucosa, and the sorts of cell damage caused by pathogenic bacteria, has increased. Electron microscopy has made a valuable contribution to this knowledge. In the fish-farming industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp. salmonicida) and vibriosis [agent, Vibrio (Listonella) anguillarum]. This article provides an overview of the GI tract of fish from an electron microscopical perspective focusing on cellular damage (specific attack on tight junctions and desmosomes) caused by pathogenic bacteria, and interactions between the ‘good’ intestinal bacteria [e.g. lactic acid bacteria (LAB)] and pathogens. Using different in vitro methods, several studies have demonstrated that co-incubation of Atlantic salmon (Salmo salar L.) foregut (proximal intestine) with LAB and pathogens can have beneficial effects, the cell damage caused by the pathogens being prevented, to some extent, by the LAB. However, there is uncertainty over whether or not similar effects are observed in other species such as Atlantic cod (Gadus morhua L.). When discussing cellular damage in the GI tract of fish caused by pathogenic bacteria, several important questions arise including: (1) Do different pathogenic bacteria use different mechanisms to infect the gut? (2) Does the gradual development of the GI tract from larva to adult affect infection? (3) Are there different infection patterns between different fish species? The present article addresses these and other questions.

Microorganisms isolated from fish can be used as prophylactic tools for aquaculture in the form of probiotic preparations. The purpose of this study was to evaluate the effects of dietary administration of the live yeast Debaryomyces hansenii CBS 8339 on the gilthead seabream (Sparus aurata L.) innate immune responses. Seabream were fed control or D. hansenii-supplemented diets (10(6) colony forming units, CFU g(-1)) for 4 weeks. Humoral (seric alternative complement and peroxidase activities), and cellular (peroxidase, phagocytic, respiratory burst and cytotoxic activities) innate immune parameters and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) were measured from serum, head-kidney leucocytes and liver, respectively, after 2 and 4 weeks of feeding. Expression levels of immune-associated genes, Hep, IgM, TCR-beta, NCCRP-1, MHC-II alpha, CSF-1R, C3, TNF-alpha and IL-1 beta, were also evaluated by real-time PCR in head-kidney, liver and intestine. Humoral immune parameters were not significantly affected by the dietary supplementation of yeast at any time of the experiment. On the other hand, D. hansenii administration significantly enhanced leucocyte peroxidase and respiratory burst activity at week 4. Phagocytic and cytotoxic activities had significantly increased by week 2 of feeding yeast but unchanged by week 4. A significant increase in liver SOD activity was observed at week 2 of feeding with the supplemented diet; however CAT activity was not affected by the dietary yeast supplement at any time of the experiment. Finally, the yeast supplemented diet down-regulated the expression of most seabream genes, except C3, in liver and intestine and up-regulated all of them in the head-kidney. These results strongly support the idea that live yeast Debaryomyces hansenii strain CBS 8339 can stimulate the innate immune parameters in seabream, especially at cellular level.

Unmethylated CpG motifs present in bacterial DNA are recognized by leucocyte receptors triggering an immune response. We have evaluated herein the immunomodulatory actions of a CpG motif in an important commercial fish, the gilthead seabream. Thus, 1, 3 and 7 days after intraperitoneal injection of the CpG motif the seabream immune parameters and gene expression profile were evaluated. Firstly, humoral innate immune responses were unaffected by CpG ODN 1668. On the other hand, ODN injection significantly enhanced the number of peritoneal leucocytes (PELs) 1 day after injection and increased the main innate immune parameters of PELs and HKLs (head-kidney leucocytes). Thus, injection of ODN 1668 significantly increased respiratory burst, peroxidase, cytotoxic and phagocytic activities, with variations in increment and time. The cytotoxic activity of HKLs was the most increased (up to 4.2-fold). Moreover, the expression profile of immune-relevant genes in head-kidney was affected, with substantial up-regulation of TLR9, IL-1β, Mx, TGFβ and Gal8 gene expression. These results demonstrate that unmethylated CpG motifs prime the fish immune response with promising applications for aquaculture.

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrueckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72 h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.

En el presente artículo se exponen algunos de los antecedentes centrales del proyecto de investigación "Los Discursos de la Masculinidad": El Empoderamiento Femenino desde el Análisis Critico del Discurso.1 Utilizando un enfoque cualitativo, se desarrollaron entrevistas abiertas con el objetivo de comprender el significado que le asignan los hombres al liderazgo femenino. Los discursos fueron analizados en base a la Teoría Fundamentada de Anselm Strauss y Juliet Corbin (2003).2 Los resultados muestran que los varones coinciden en términos generales respecto a los cambios que ha habido en los roles de género, en que las mujeres tienen y deben tener más oportunidades, en que hay una mayor incorporación de éstas en el mercado laboral y en la necesidad de tener relaciones más equitativas al interior de la familia. Así mismo, emergen tensiones que configuran el malestar de la masculinidad, traspasando la esfera íntima y cuestionando las relaciones de intergénero actuales. También hay discursos más progresistas en los varones, que se aventuran hacia un reconocimiento de las capacidades y roles que han ido asumiendo las mujeres en el espacio público, aunque se trata de un reconocimiento vigilante, ambivalente, mediatizado por una percepción de confianza/desconfianza, eficiencia/incompetencia, hacia lo que ellas son capaces de lograr.The present contribution deals with the meaning of the feminine leadership from a masculine perspective. By using a qualitative approach, open interviews were developed to understand the meaning that men assign to the feminine leadership. The speeches were analyzed on the basis of the Strauss/Corbin-theory (2003). The results show that men agree with the changes experienced by the gender roles. In a specific way, men agree that women need to have more societal-economic opportunities, which are necessary for more equitable relationship inside the family. However, within this positive framework emerging tensions configure a dissatisfaction of the masculinity that, in turn, impact the intimate sphere questioning the present day inter-gender relationship. Finally, there is a men's group with more progressive speeches which venture towards a positive evaluation of the women's capacities and their assumed public space-roles. However this positive evaluation is vigilant, ambivalent, and influenced by a perception of confidence/uncertainty, efficiency/inefficiency of the effective societal contribution that women are able to reach.

The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h post-injection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens.

Adenosine (A) and its derivatives have important biological functions, including the inhibition of immune responses and apoptosis induction. The aim of this work was to investigate whether A and N 6 -cyclohexyladenosine (CHA) regulate apoptosis in leukocyte cultures from the teleost, gilthead seabream ( Sparus aurata L.) by flow cytometry using a double fluorescence staining method. Head kidney leukocytes (HKLs) were cultured for 0, 24 or 48 h. The kinetics of FDA + /PI − (viable), FDA − /PI − (apoptotic) and FDA − /PI + (necrotic) leukocyte subpopulations were followed during the culture process. Apoptosis was induced by addition of resveratrol or staurosporine to the culture media and the study was validated by transmission electron microscopy. A and CHA did not induce but decreased apoptosis of seabream HKLs, in particular HK lymphocytes (L), after 24 h in culture. Results raise the question whether apoptosis is differently modulated by purine nucleosides in fish L and phagocytes and also in fish compared to mammalian cells.

The olfactory organs of vertebrates are not only extraordinary chemosensory organs but also a powerful defense system against infection. Nasopharynx-associated lymphoid tissue (NALT) has been traditionally considered as the first line of defense against inhaled antigens in birds and mammals. Novel work in early vertebrates such as teleost fish has expanded our view of nasal immune systems, now recognized to fight both water-borne and air-borne pathogens reaching the olfactory epithelium. Like other mucosa-associated lymphoid tissues (MALT), NALT of birds and mammals is composed of organized lymphoid tissue (O-NALT) (i.e., tonsils) as well as a diffuse network of immune cells, known as diffuse NALT (D-NALT). In teleosts, only D-NALT is present and shares most of the canonical features of other teleost MALT. This review focuses on the evolution of NALT in vertebrates with an emphasis on the most recent findings in teleosts and lungfish. Whereas teleost are currently the most ancient group where NALT has been found, lungfish appear to be the earliest group to have evolved primitive O-NALT structures.

The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout ( Oncorhynchus mykiss ). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis , two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.

Determining the earliest age at which farmed fish can be successfully vaccinated is a very important question for fish farmers. Nasal vaccines are novel mucosal vaccines that prevent aquatic infectious diseases of finfish. The present study investigates the ontogeny of the olfactory organ of rainbow trout by histology and aims to establish the earliest age for vaccination against infectious hematopoietic necrosis (IHN) and enteric red mouth (ERM) disease using the nasal route. Rainbow trout (Oncorhynchus mykiss) were vaccinated intranasally (I.N) at three different ages: 1050° days (DD) (group A); 450 DD (group B); and 360 DD (group C), or 70, 30 and 24 days post-hatch (dph), respectively. The mean weights of groups A, B and C were 4.69 g, 2.9 g and 2.37 g, respectively. Fish received either a live attenuated IHN virus vaccine, ERM formalin killed bacterin or saline (mock vaccinated). Fish were challenged to the corresponding live pathogen 28 days post-vaccination. IHN vaccine delivery at 360 DD resulted in 40% mortality likely due to residual virulence of the vaccine. No mortality was observed in the ERM nasal delivery groups. Following challenge, very high protection rates against IHN virus were recorded in all three age groups with survivals of 95%, 100% and 97.5% in groups A, B and C, respectively. Survival against ERM was 82.5%, 87.5% and 77.5% in groups A, B and C, respectively. Survival rates did not differ among ages for either vaccine. Our results indicate the feasibility and effectiveness of nasal vaccination as early as 360 DD and vaccination-related mortalities when a live attenuated viral vaccine was used in the youngest fish.

Transportation of live fish is a common practice among aquaculture facilities. Many studies have previously reported how transport elicits physiological stress responses and increases disease susceptibility in farmed fish. The aim of this work is to investigate the changes that the skin of rainbow trout (Oncorhynchus mykiss) experiences due to stress. Since NaCl is commonly added to transport water as a stress mitigator, the effects of salt addition on the skin mucosa and skin-associated bacteria were also examined. Three experimental groups (Control, post-transport no salt (PTNS) and post-transport with salt (PTS)) were analyzed in a 5-hour transport acute stress model. Results indicate that the skin mucosa and the skin-associated bacteria are affected by transport stress. Total numbers of culturable skin-associated bacteria increased by ~10-fold and ~50-fold in the PTS and PTNS groups, respectively. Compared to controls, MUC2 expression was increased by 5-fold and 2-fold in the PTNS and PTS groups, respectively. Claudin-7, 8d and 12 expression levels were higher in both PTNS and PTS groups whereas antimicrobial peptide gene expression was lower than controls. Expression of the anti-inflammatory cytokine TGF-β but not IL-1β, IL-6 and TNF-α was up-regulated 2-3 fold in both the PTS and PTNS groups. The addition of salt diminished some of the physiological responses measured including the numbers of skin-associated bacteria. The responses recorded here appeared to be efficient at controlling bacterial translocation since stress did not lead to significant presence of bacteria in the liver or spleen of rainbow trout. When examining the ability of skin mucus to inhibit or promote growth of the bacterial pathogen Vibrio anguillarum, the skin mucus of PTS trout was more efficient at inhibiting V. anguillarum growth (20% inhibition) compared to control or PTNS mucus (11-12% inhibition). Our data clearly indicate the skin and skin microbiota of rainbow trout undergo important physiological responses during stress. The reduction in the magnitude of the skin responses recorded when salt was added to the transport water explains a new mechanism by which salt is an effective stress mitigator in some fish species. Aquaculture specialists will benefit from the present study by taking into consideration the importance of skin health during live transport.

Much of the history of modern immunology has focused on a relatively small number of species, most of which are placental mammals such as mice. This is not surprising given that they represent the closest models for the study of human immunology and diseases. Since the publication of the previous edition of this text, however, substantial progress has been made in the field of mucosal immunology of nonmammalian vertebrates. This has economic significance for the aquaculture industry's need for vaccine development, as well as increasing the understanding of the origin and function of mucosal immune systems. Most striking has been some of the lineage-specific adaptations that have occurred to solve unique problems different vertebrate lineages face with respect to how they interact with their environment.

In the present chapter, we provide an in-depth review of knowledge related to the intestinal mucosal immune compartment of fish. The anatomical and physiological makeup is described along with a comprehensive overview of the innate and adaptive cellular and molecular effectors that participate in the gastrointestinal immune response. Emerging tools and laboratory models for the study of mucosal immunity are highlighted.

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 x 10(4) or 6 x 10(6) viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer's solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

The in vitro effects of four heat-inactivated bacterial species on the cellular innate immune responses of the gilthead seabream (Sparus aurata L.) were investigated. Head-kidney leucocytes were isolated and incubated for 30 min with two bacteria isolated from seabream skin (Pdp11 and 51M6; members of the Vibrionaceae and in the genus Shewanella) and two bacteria used as probiotics in humans and cattle (Lactobacillus delbrüeckii subsp. lactis and Bacillus subtilis) at 5 x 10(5), 5 x 10(6) or 5 x 10(7)CFU/ml. After incubation, different cellular innate immune parameters (leucocyte peroxidase content, phagocytosis, respiratory burst activity and cytotoxicity) were evaluated. The leucocyte peroxidase content was significantly higher after incubation with 51M6 at 5 x 10(7)CFU/ml. Head-kidney phagocytes were able to engulf the four bacterial species in all four cases, L. delbrüeckii subsp. lactis and B. subtilis being the most actively phagocytized. The incubation of seabream leucocytes with 51M6, L. delbrüeckii subsp. lactis and B. subtilis resulted in a great increase in respiratory burst activity. Cytotoxic activity was generally stimulated in a dose-dependent manner, the enhancement obtained with 5 x 10(7)CFU/ml being statistically significant. The usefulness of in vitro assays for screening and selecting candidate probiotic bacteria, as well as for optimising their effective dose, is discussed in relation with their immune-modulatory properties.

The possible immuneregulatory effects of two purine nucleotides, adenosine and cyclohexyladenosine, on gilthead seabream ( Sparus aurata L.) leucocytes were investigated. Leucocytes isolated from the head-kidney were incubated with five different concentrations (ranging from 0 to 100 μM) of adenosine or cyclohexyladenosine for 30, 180 and 360 min and the effect on leucocyte viability and some of the main innate cellular immune responses was evaluated. Adenosine did not significantly affect the viability or the innate immune parameters of seabream leucocytes at any of the assayed concentrations or incubation times. High concentrations of cyclohexyladenosine, on the other hand, caused a significant drop in the respiratory burst activity of head-kidney leucocytes at all the times assayed. In addition, phagocytosis of yeast cells was significantly rapidly depressed (30 min) after incubation with 100 μM of this analogue. The present results demonstrate that cyclohexyladenosine at high concentrations is capable of down-regulating certain innate immune responses in seabream leucocytes, suggesting that telesot fish immune cells, like their mammal counterparts, possess receptors for purine nucleotides.

The humoral innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei has been studied. At 10, 22, 38, 52 and 108 days of cohabitation fish were sampled to examine gut histology and to determine serum innate immune parameters and the mRNA expression of pro-inflammatory cytokines (IL-1β and TNFα) in head-kidney. The parasite was successfully transmitted to 45% of the recipient fish and prevalence reached a maximum (62.5%) at the last sampling time (108 days). Recipient fish started to die after 74 days of cohabitation. In general, alternative complement activity was higher whereas the peroxidase level was lower in recipient fish than in controls. Moreover, IL-1β mRNA expression increased while the TNFα gene expression decreased in recipient fish. These data demonstrate the involvement of complement activity in the defence mechanisms of the gilthead seabream against the myxosporean E. leei. Within the recipient fish group, few differences were observed in the studied immune parameters between E. leei-parasitized and non-parasitized recipient fish. Parasitological and immunological implications of E. leei infections in Mediterranean fish farms are discussed., This work has been funded by an EU Project (QLRT2001-00722 MyxFishControl). A. C. and I. S. are beneficiaries of Fundación CajaMurcia and Fundación Séneca fellowships, respectively. Primers for cytokines were designed by Dr V. Mulero of the Fish Innate Immune System Group of the University of Murcia.

The aims of this study were (i) to identify alternative Mx stimulatory compounds in Atlantic salmon (Salmo salar L.) and to characterise the kinetics and intensity of the stimulated responses and (ii) to investigate the effect of temperature on such responses by semi-quantitative RT-PCR. Mx transcripts were measured in Atlantic salmon parr kept at 14 degrees C and injected with either LPS, the synthetic double-stranded polyribonucleotide poly I:C, Vibrio anguillarum serotypes I and II-ordalii bacterin, beta-glucan, whole yeast cells or yeast RNA. Sampling periods lasted until transcripts were undetectable or up to three weeks after immunisation. The effect of temperature on poly I:C-induced Mx response was studied by injecting parr kept at 6 degrees C. Newly hatched salmon fry were immersed once, twice or three times in the Vibrio bacterin diluted five or 10 times and sampled for three weeks. None of the yeast compounds induced Mx expression in Atlantic salmon parr. LPS induced a very low Mx response 2 and 3 days after injection. The Vibrio bacterin administered by injection in parr (but not by immersion in fry) resulted in strong Mx induction on days 2 and 3, disappearing by day 6. Poly I: C-induced Mx responses that were more intense and longer lasting than those induced by the bacterin, peaking on day 3 and lasting over 6 days, disappearing by day 9 at 14 degrees C. Lower temperature caused a longer lasting Mx response to poly I:C (at least 21 days), which peaked on days 7-14, with a similar intensity and no delayed onset as compared with the response at 14 degrees C. However, some toxicity of the poly I:C was indicated in treatments at 6 degrees C.

Mucosal surfaces are multifunctional barriers that need to orchestrate a variety of physiological functions. One of the mechanisms by which mucosal surfaces carry immunological functions without compromising other physiological roles is the establishment of regional immunity. Regional immunity can be generated by selective expression of chemokines, chemokine receptors and adhesion molecules within mucosal microenvironments. In the olfactory organ of animals, immune responses may result in damage to the olfactory sensory neurons and, as a consequence, compromise olfaction. The olfactory organ of teleost fish is histologically divided into two distinct areas, a sensory or neuroepithelial region in the lateral regions and valleys if the lamellae and a non-sensory region located at the tip of each lamella characterized by presence of goblet cells. Thus, we hypothesized the presence of two unique immune microenvironments corresponding with these two regions in rainbow trout. In line with our hypothesis, we found clusters of CD8+ T cells at the mucosal regions of lamellae whereas few numbers were found in the neuroepithelium. Nasal CD8+ T cells displayed a mucosal phenotype similar to those found in the gut. Laser capture microdissected tip versus lateral samples of control trout olfactory tissue were collected and RT-qPCR revealed a higher expression of the chemokine and chemokine receptor CCL19 and CCR7, the adhesion molecules L-selectin, ICAM, VCAM, and integrin β2 and several T cell markers in the mucosal tip compared to the neuroepithelium. Moreover, MHC-II immunostaining indicated that the mucosal tip preferentially participates in antigen presentation within the trout nasal mucosa. Finally, in response to nasal vaccination, cell proliferation occurs in the tip mucosal region and not the neuroepithelium. Our results support the idea of compartmentalized immune responses within the nasal mucosa of teleosts that likely evolved as a sparing mechanism to protect olfactory sensory function.

Cultured fish represent a critical food resource and efficient means for providing protein for human consumption. Nevertheless, disease limits the sustainability and production of this industry. A major factor restricting the advancement of basic and applied research for fish health is the lack of immunological reagents. These reagents are required for evaluating shifts in immunity during infection and vaccination against pathogens that limit the full potential of aquaculture. CIRNAS (http://biology.unm.edu/CIRNAS) is a collaborative network with a goal of serving the aquaculture community by advancing the availability of immunological resources and knowledge base for fish health. Four fish species: Atlantic salmon, rainbow trout, channel catfish and Nile tilapia will be targeted. Antibody panels and immune assays will be developed to assess the contribution of different leukocyte subsets and effector molecules that cannot be currently measured during disease and vaccination due to lack of corresponding reagents. Specific focus areas include the development of reagents for mucosal immunity and vaccinology. Importantly, a major objective of CIRNAS is to establish an international, collaborative network for improving health, safety and production of aquacultured fish by partnering with other fish health researchers. By doing so, the project will advance the development of collaborative research projects worldwide for diseases impacting production and fish health. Current and proposed research will be presented.

Interactions among host, microbiota and viral pathogens are complex and poorly understood. The goal of the present study is to assess the changes in the skin microbial community of Atlantic salmon (Salmo salar L.) in response to experimental infection with salmonid alphavirus (SAV). The salmon skin microbial community was determined using 16S rDNA pyrosequencing in five different experimental groups: control, 7 days after infection with low-dose SAV, 14 days after infection with low-dose SAV, 7 days after infection with high-dose SAV, and 14 days after infection with high-dose SAV. Both infection treatment and time after infection were strong predictors of the skin microbial community composition. Skin samples from SAV3 infected fish showed an unbalanced microbiota characterized by a decreased abundance of Proteobacteria such as Oleispira sp. and increased abundances of opportunistic taxa including Flavobacteriaceae, Streptococcaceae and Tenacibaculum sp. These results demonstrate that viral infections can result in skin dysbiosis likely rendering the host more susceptible to secondary bacterial infections.

The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT(+) B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.

Commensal microorganisms live in association with the mucosal surfaces of all vertebrates. The skin of teleost fish is known to harbor commensals. In this study we report for the first time the presence of an intracellular Gram positive bacteria, Staphylococcus warneri that resides in the skin epidermis of rainbow trout (Oncorhynchus mykiss). S. warneri was isolated from healthy hatchery trout skin epithelial cells. In situ hybridization confirmed the intracellular nature of the bacterium. Skin explants exposed in vitro to S. warneri or the extracellular pathogen Vibrio anguillarum show that S. warneri is able to induce an anti-inflammatory cytokine status via TGF-β 1b compared to the pro-inflammatory responses (IL-1β , IL-6 and TNF-α) elicited by V. anguillarum. In vivo experiments showed that S. warneri is not pathogenic to rainbow trout when injected intraperitoneally at high concentrations. However, S. warneri is able to stimulate V. anguillarum growth and biofilm formation on rainbow trout scales. Our results demonstrate that rainbow trout skin commensals such as S. warneri have the potential to become indirect pathobionts by enhancing growth and biofilm formation of pathogens such as V. anguillarum. These results show that fish farming practices (i.e. handling and other manipulations) can alter the skin microbiota and compromise the skin health of rainbow trout.

Skin homeostasis is critical to preserve animal integrity. Although the skin of most vertebrates is known to contain a skin-associated lymphoid tissue (SALT), very little is known about skin B-cell responses as well as their evolutionary origins. Teleost fish represent the most ancient bony vertebrates containing a SALT. Due to its lack of keratinization, teleost skin possesses living epithelial cells in direct contact with the water medium. Interestingly, teleost SALT structurally resembles that of the gut-associated lymphoid tissue, and it possesses a diverse microbiota. Thus, we hypothesized that, because teleost SALT and gut-associated lymphoid tissue have probably been subjected to similar evolutionary selective forces, their B-cell responses would be analogous. Confirming this hypothesis, we show that IgT, a teleost immunoglobulin specialized in gut immunity, plays the prevailing role in skin mucosal immunity. We found that IgT + B cells represent the major B-cell subset in the skin epidermis and that IgT is mainly present in polymeric form in the skin mucus. Critically, we found that the majority of the skin microbiota are coated with IgT. Moreover, IgT responses against a skin parasite were mainly limited to the skin whereas IgM responses were almost exclusively detected in the serum. Strikingly, we found that the teleost skin mucosa showed key features of mammalian mucosal surfaces exhibiting a mucosa-associated lymphoid tissue. Thus, from an evolutionary viewpoint, our findings suggest that, regardless of their phylogenetic origin and tissue localization, the chief immunoglobulins of all mucosa-associated lymphoid tissue operate under the guidance of primordially conserved principles.

Lungfishes (Dipnoi) represent the closest ancestor of tetrapods. Dipnoi have dual breathing modes extracting oxygen from water and air. The primitive lungs of lungfishes are exposed to external antigens including viruses. To date, the immune response of lungfishes against viruses has not been investigated. During viral immune responses, cell exposure to type I interferon induces the replacement of the constitutive proteasome with LMP2, LMP7 and MECL-1 beta subunits forming the immunoproteasome and enhancing antigen presentation to MHC class I molecules. In order to study the immune defense system of the lungfish lung, we have characterized for the first time the three immunoproteasome subunits in the sarcopterygian fish, the Nigerian spotted lungfish (Protopterus dolloi). LMP2, LMP7 and MECL-1 were identified in P. dolloi and their sequences encoded predicted proteins of 216, 275 and 278 amino acids, respectively. The mRNA of these three genes was expressed in multiple tissues, including the lung, with the highest abundance observed in kidney and post-pyloric spleen. In vitro stimulation of lungfish lung and kidney primary cell cultures with PolyI:C for 4 and 12 h resulted in increased LMP2, LMP7 and MECL-1 expression in both tissues. These results suggest a central role of these genes in the activation of an antiviral immune response in lungfish. Importantly, they indicate that the primitive lung of the common ancestor of all tetrapods is capable of inducing the expression of these genes in response to viral stimulation.

The field of mucosal immunology research has grown fast over the past few years, and our understanding on how mucosal surfaces respond to complex antigenic cocktails is expanding tremendously. With the advent of new molecular sequencing techniques, it is easier to understand how the immune system of vertebrates is, to a great extent, orchestrated by the complex microbial communities that live in symbiosis with their hosts. The commensal microbiota is now seen as the “extended self” by many scientists. Similarly, fish immunologist are devoting important research efforts to the field of mucosal immunity and commensals. Recent breakthroughs on our understanding of mucosal immune responses in teleost fish open up the potential of teleosts as animal research models for the study of human mucosal diseases. Additionally, this new knowledge places immunologists in a better position to specifically target the fish mucosal immune system while rationally designing mucosal vaccines and other immunotherapies. In this review, an updated view on how teleost skin, gills and gut immune cells and molecules, function in response to pathogens and commensals is provided. Finally, some of the future avenues that the field of fish mucosal immunity may follow in the next years are highlighted.

J chain is a small polypeptide responsible for immunoglobulin (Ig) polymerization and transport of Igs across mucosal surfaces in higher vertebrates. We identified a J chain in dipnoid fish, the African lungfish (Protopterus dolloi) by high throughput sequencing of the transcriptome. P. dolloi J chain is 161 aa long and contains six of the eight Cys residues present in mammalian J chain. Phylogenetic studies place the lungfish J chain closer to tetrapod J chain than to the coelacanth or nurse shark sequences. J chain expression occurs in all P. dolloi immune tissues examined and it increases in the gut and kidney in response to an experimental bacterial infection. Double fluorescent in-situ hybridization shows that 88.5% of IgM⁺ cells in the gut co-express J chain, a significantly higher percentage than in the pre-pyloric spleen. Importantly, J chain expression is not restricted to the B-cell compartment since gut epithelial cells also express J chain. These results improve our current view of J chain from a phylogenetic perspective.

The vast majority of fish pathogens enter their host through mucosal surfaces. The skin, gut and gill represent the largest mucosal surfaces in fish and they all contain a mucosal-associated lymphoid tissue (MALT). We have previously demonstrated that pathogen-specific mucosal antibody responses in the gut, skin and gill are overwhelmingly mediated by the IgT antibody class. In addition, we have shown that IgT is the main immunoglobulin isotype coating commensal bacteria and thus, it plays a key function in immune exclusion. While these IgT activities point to a pivotal role of this immunoglobulin in teleost mucosal immunity, whether IgT is required for pathogen clearance and microbiota homeostasis remains to be demonstrated. To address this critical question we have developed a unique IgT B-cell depletion trout model. Upon one depletion treatment, IgT B cells from all analyzed mucosal and systemic lymphoid organs were depleted by over 95% for a 3-4 week period. The application of an additional depletion treatment executed at day 21 was able to extend the depletion status by 3-4 weeks. Interestingly, the % of IgM B cells did not change throughout the 7 week depletion period. Upon IgT B-cell depletion, fish where sublethally challenged with Ichthyophthirius multifiliis or Flavobacterium columnare. After two weeks post-challenge, a significant percentage of mortality occurred in the IgT B-cell depleted groups (25-50%). Critically, pathogen load was dramatically higher in the IgT B-cell depleted groups when compared to control fish. Moreover, IgT coating of microbiota had for the most part disappeared 3 weeks post-depletion treatment. More importantly, the microbiome at mucosal surfaces was significantly changed. Interestingly we could never observe IgM or IgD compensatory responses against the pathogens or commensals. In conclusion we demonstrate that IgT is essential for pathogen clearance at mucosal surfaces, and it plays a critical role in the maintenance of microbiota homeostasis. This represents the first non-mammalian model in which a specialized mucosal immunoglobulin and the B cells producing it are depleted. Importantly, this novel IgT B-cell depletion model will be critical to understand further the role of IgT in host-pathogen interactions at fish mucosal surfaces. At the more practical level, this new capacity to elucidate whether a pathogen requires mucosal IgT responses for its clearance will direct efforts to the development of vaccines that not only stimulate systemic IgM but also mucosal IgT responses against that particular pathogen.

As key effector molecules of jawed vertebrate’s adaptive immune system, immunoglobulins are produced by B lymphocytes, either as a secretory form (antibody) or as a membrane form (B cell receptor). Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD. In addition, IgM in these species was thought to be the only immunoglobulin isotype responding to pathogens both in systemic or mucosal compartments. However, the unexpected discovery of IgT, a new teleost immunoglobulin unearthed in 2005, has provided for new opportunities to analyze further roles of teleost immunoglobulins in these two physiologically distinct compartments. The smoke about the potential function of IgT has cleared recently with the finding that this immunoglobulin appears to be specialized in gut mucosal immunity. Significantly, the new capability of measuring not only IgM but also IgT responses will greatly facilitate the evaluation and understanding of fish immune responses as well as the protective effects of fish vaccines. The purpose of this review is to summarize the molecular characterization of new IgT orthologs and subtypes in teleosts, as well as to describe the new findings concerning the protein structure of IgT, the B cells producing it, and its role in mucosal immunity.

Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.

The effects of the oral administration of heat-inactivated Lactobacillus delbrueckii ssp. lactis and Bacillus subtilis, individually or combined, on gilthead seabream immune responses were investigated both systemically and locally in the gut. In a first experiment, seabream (65 g) were fed for 3 weeks different diets supplemented with 1 x 10(7)CFU g(-1)Lactobacillus, 1 x 10(7)CFU g(-1)Bacillus, or 0.5 x 10(7)CFU g(-1)Lactobacillus plus 0.5 x 10(7)CFU g(-1)Bacillus. Controls were fed non-supplemented diet. Six fish per group were sampled at the end of the trial and some humoral and cellular systemic innate immune parameters were evaluated. Feeding the mixture of the two killed bacteria species significantly increased natural complement, serum peroxidase and phagocytic activities compared with controls. In a second experiment, juvenile seabream (13 g) were fed for 3 weeks the same experimental diets and total serum IgM and numbers of gut IgM(+) cells and acidophilic granulocytes were evaluated. All these parameters were significantly higher in the multispecies probiotic group compared to monospecies and control fed groups. The advantages provided by administration of killed probiotic bacteria as well as multispecies versus monospecies formulations are discussed in light of the results obtained and for their possible application in aquacultural practices.

The present contribution deals with the meaning of the feminine leadership from a masculine perspective. By using a qualitative approach, open interviews were developed to understand the meaning that men assign to the feminine leadership. The speeches were analyzed on the basis of the Strauss/Corbin-theory (2003). The results show that men agree with the changes experienced by the gender roles. In a specific way, men agree that women need to have more societal-economic opportunities, which are necessary for more equitable relationship inside the family. However, within this positive framework emerging tensions configure a dissatisfaction of the masculinity that, in turn, impact the intimate sphere questioning the present day inter-gender relationship. Finally, there is a men’s group with more progressive speeches which venture towards a positive evaluation of the women’s capacities and their assumed public space-roles. However this positive evaluation is vigilant, ambivalent, and influenced by a perception of confidence/ uncertainty, efficiency/inefficiency of the effective societal contribution that women are able to reach. En el presente artículo se exponen algunos de los antecedentes centrales del proyecto de investigación “Los Discursos de la Masculinidad”: El Empoderamiento Femenino desde el Análisis Critico del Discurso.1 Utilizando un enfoque cualitativo, se desarrollaron entrevistas abiertas con el objetivo de comprender el significado que le asignan los hombres al liderazgo femenino. Los discursos fueron analizados en base a la Teoría Fundamentada de Anselm Strauss y Juliet Corbin (2003).2 Los resultados muestran que los varones coinciden en términos generales respecto a los cambios que ha habido en los roles de género, en que las mujeres tienen y deben tener más oportunidades, en que hay una mayor incorporación de éstas en el mercado laboral y en la necesidad de tener relaciones más equitativas al interior de la familia. Así mismo, emergen tensiones que configuran el malestar de la masculinidad, traspasando la esfera íntima y cuestionando las relaciones de intergénero actuales. También hay discursos más progresistas en los varones, que se aventuran hacia un reconocimiento de las capacidades y roles que han ido asumiendo las mujeres en el espacio público, aunque se trata de un reconocimiento vigilante, ambivalente, mediatizado por una percepción de confianza/desconfianza, eficiencia/ incompetencia, hacia lo que ellas son capaces de lograr

Probiotic bacteria increase the host health status and protect mucosal tissue against pathogen-caused damage in mammalian models. Using an in vitro (intestinal sac) method this study aimed to address (a) the in vitro ability of Lactobacillus delbrueckii subsp. lactis to remain in the gastrointestinal tract of Atlantic salmon (Salmo salar L.) and (b) its ability to prevent cellular damage caused by successive incubation with Aeromonas salmonicida subsp. salmonicida the causative agent of furunculosis. Short in vitro incubation of salmon foregut with (TRITC)-labelled L. delbrueckii subsp. lactis showed that the probiont was able to colonize the enterocyte surface as studied by confocal microscopy. Furthermore, foregut incubated with the probiotic bacteria only, resulted in a healthy intestinal barrier whereas exposure to A. salmonicida disrupted its integrity. However, pre-treatment of salmon intestine with L. delbrueckii subsp. lactis prevented Aeromonas damaging effects. These results are promising in the context of the use of non-autochthonous probiotic bacteria as prophylactic agents against fish bacterial infections in the gastrointestinal tract.

The cellular innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by maintaining uninfected fish (recipients) together with infected animals. A group of fish not exposed to the infection served as controls. After 10, 22, 38, 52 and 108 days, control and recipient fish were sampled and leucocyte subpopulations and cellular immune responses (leucocyte peroxidases, phagocytosis, respiratory burst and cytotoxicity) of the head-kidney leucocytes were determined. The percentage of acidophilic granulocytes was significantly lower in non-parasitized and parasitized recipient fish than in control fish after 22 days but no significant differences were seen between non-parasitized and parasitized recipient animals. The leucocyte peroxidase content, phagocytosis and respiratory burst activity were seen to have decreased significantly at different sampling times in both non-parasitized and parasitized recipient fish with respect to the controls, whereas cytotoxic activity was up to 2.3 times higher than in control fish. Within the recipient group, little difference was observed in the studied parameters between non-parasitized and parasitized fish. These data demonstrate that cytotoxic activity may have an important role in the defence of gilthead seabream against the myxosporean E. leei. Immunological implications of E. leei infections are discussed., This work has been funded by an EU Project (QLRT-2001-00722 MyxFishControl). A. Cuesta and I. Salinas are beneficiaries of Fundación CajaMurcia and Fundación Séneca fellowships, respectively.

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.