A comprehensive coding and microRNA transcriptome of vertebral bone in postlarvae and juveniles of Senegalese sole (Solea senegalensis).

Understanding vertebral bone development is essential to prevent skeletal malformations in farmed fish related to genetic and environmental factors. This is an important issue in Solea senegalensis, with special impact of spinal anomalies in postlarval and juvenile stages. Vertebral bone transcriptomics in farmed fish mainly comes from coding genes, and barely on miRNA expression. Here, we used RNA-seq of spinal samples to obtain the first comprehensive coding and miRNA transcriptomic repertoire for postlarval and juvenile vertebral bone, covering different vertebral phenotypes and egg-incubation temperatures related to skeleton health in S. senegalensis. Coding genes, miRNA and pathways regulating bone development and growth were identified. Differential transcriptomic profiles and suggestive mRNA-miRNA interactions were found between postlarvae and juveniles. Bone-related genes and functions were associated with the extracellular matrix, development and regulatory processes, calcium binding, retinol and lipid metabolism or response to stimulus, including those revealed by the miRNA targets related to signaling, cellular and metabolic processes, growth, cell proliferation and biological adhesion. Pathway enrichment associated with fish skeleton were identified when comparing postlarvae and juveniles: growth and bone development functions in postlarvae, while actin cytoskeleton, focal adhesion and proteasome related to bone remodeling in juveniles. The transcriptome data disclosed candidate coding and miRNA gene markers related to bone cell processes, references for functional studies of the anosteocytic bone of S. senegalensis. This study establishes a broad transcriptomic foundation to study healthy and anomalous spines under early thermal conditions across life-stages in S. senegalensis, and for comparative analysis of skeleton homeostasis and pathology in fish and vertebrates.

Early innate immune responses in European sea bass (Dicentrarchus labrax L.) following Tenacibaculum maritimum infection.

Introduction: The marine aquaculture industry has been witnessing a worldwide emergence of tenacibaculosis, a poorly understood bacterial disease caused by Tenacibaculum maritimum that affects commercially important fish. So far, knowledge on the T. maritimum virulence mechanisms is scarce and the pathogen-host interaction operating in tenacibaculosis remain to be disclosed. This study aimed at contributing to a better understanding of this disease, by evaluating the early innate immune response triggered in European sea bass (Dicentrarchus labrax) by a bath-challenge with T. maritimum. Methods: Groups of sea bass were bath-challenged with T. maritimum (challenged fish) or mock-challenged. Undisturbed fish were used as controls (time 0). Samples of blood, liver and mucosal organs (skin, gills and posterior-intestine) were collected at 0 h (control) and at 6, 24, 48 and 72 h post-challenge (n=12). Mucosal organs were used for analyzing the expression of immune-related genes by RT-qPCR, as well as blood samples for assessing haematological and innate humoral parameters and liver for oxidative stress assessment. Results: An increased expression of il-1ß, il8, mmp9 and hamp1 was detected in all mucosal organs of infected fish when compared with control and mock-challenged fish, suggesting a pro-inflammatory response against T. maritimum transversal to all organs. The faster induction of these pro-inflammatory genes was observed in the gills. Regarding the systemic response, challenged fish presented neutrophilia, monocytosis, signs of anemia, and a decrease of bactericidal and lysozyme activities in plasma. Almost no variations were observed regarding hepatic oxidative stress. Discussion/Conclusions: The present study suggests that T. maritimum induces a local innate immune response upon bath infection not only in the skin of European sea bass, but also in the gills and posterior-intestine, likely triggered by the T. maritimum's capacity to adhere, colonize and damage these organs that can function as entry ways to bacteria, leading ultimately to the seen host's systemic response.

Envejecimiento, bases para una titulación / Aging, bases for a degree / Envelhecimiento, bases para um diploma

OPS en su Serie Recursos Humanos para la Salud No 59 informa las dificultades para formar RRHH en Gerontología Geriatría (G-G) dados por aspectos educativos, escasas ofertas laborales y centros asistenciales específicos. El saber en el tema es vasto, no es así el conocimiento que se tiene frente a la problemática en la formación de RRHH identificada en la serie No 59. El objetivo como formadores de enfermeros posgraduados que dejó en "Stand by" una especialidad en G-G, estructurada en la diversidad de la problemática del adulto mayor AM cuyo currículum oculto no colmó las expectativas de la comunidad educativa. La inquietud como profesores, gestores educativos de enfermería, en un departamento que profundiza los saberes sobre le cuidado AM, está en buscar que el programa perfile a un profesional con valores, competencias para el cuidado, contextualizado al Sistema Nacional Integrado Salud (SNIS), lenguaje analítico-crítico y virtual, transdiciplinar entre otras. Que contribuyan a la calidad de vida del AM, la familia, los aspectos sociales, el hábitat. Además de conocer la opinión de los licenciados clínicos. La revisión descriptiva de investigaciones de estudiantes y docentes del departamento en el período 2015-2019 dan cuenta de enunciados textuales y conceptos útiles para este fin, que conjugado a la experiencia docente informa sobre la tendencia buscada. ¿Qué información se ha recogido en las Tesis Finales de Investigación (TFI) que puedan enriquecer una nueva propuesta de formación posbásica en G-G.? La demografía, la experticia de los profesores, la estrategia de posgrados articulados fundamenta la necesidad de un programa posbásico adaptado, que contemple al "life long learning", un aspirante en G-G que aprende métodos para aprender durante toda su vida. La proyección para el 2050 en el Uruguay es de casi 825.000 AM en una población de 3,7 millones, mientras que los menores de 15 serán más de 570.000 la epidemiología misma demandará cuidado enfermero y cambios en la seguridad social.

PAHO in its Human Resources for Health Series No. 59 reports the difficulties to train HR in educational aspects, few job offer and specific care centers in Gerontology Geriatrics (GG) Knowledge on the subject is vast, not so the knowledge that one has in front of the problem in the formation of RR identified in series No 59. The objective as postgraduate nurse educators who left a specialty in G-G in "Stand by", structured in the diversity of the problems of the elderly AM whose hidden curriculum did not exceed the expectations of the educational community. the concern as teachers, nursing educational managers, in a department that deepens knowledge about AM care, is to seek that the program profiles a professional with values, skills for care, contextualized to the SNIS, analytical-critical and virtual language, transdisciplinary among others. That they contribute to the quality of life of the MA, the family, the social aspects, the habit. In addition to knowing the opinion of clinical graduates. the descriptive review of investigations of students and teachers of the department in the period 2015-2019 reveal textual statements and useful concepts for this purpose, which together with the teaching experience reports on the trend sought. What information has been collected in the TFIs that can enrich a new proposal for post-basic training in G-G? The demographics, the expertise of the professors, the articulated postgraduate strategy support the need for an adapted post-basic program, which includes the "lifelong learning", a G-G candidate who learns methods to learn throughout his life. The projection of Uruguay (almost 825,000 AM) in a population of 3.7 million, while those under 15 will be more than 570,000 the epidemiology itself will demand nursing care and changes in social security.

A OPAS, em sua Série de Recursos Humanos em Saúde nº 59, relata as dificuldades para formar RH em aspectos educacionais, poucas ofertas de empregos e centros de atendimento específicos em Gerontologia Geriatria (GG) O conhecimento sobre o assunto é vasto, não o que se tem. diante do problema na formação do RR identificados na série nº 59. O objetivo como pós-graduandos enfermeiros educadores que saíram de uma especialidade em G-G em "Stand by", estruturados na diversidade dos problemas do idoso AM cujo currículo oculto não superava as expectativas da comunidade educacional. A preocupação como docentes, gestores do ensino de enfermagem, em um setor que aprofunda conhecimentos sobre o cuidado em AM, é buscar que o programa perfile um profissional com valores, habilidades para o cuidado, contextualizado ao SNIS, linguagem analítico-crítica e virtual , transdisciplinar entre outros. Que contribuam para a qualidade de vida do MA, da família, dos aspectos sociais, do hábito. Além de saber a opinião dos graduados em clínica. A revisão descritiva de investigações de alunos e docentes do departamento no período 2015-2019 revelam enunciados textuais e conceitos úteis para esse fim, que juntamente com a experiência docente relata a tendência buscada. Quais informações foram coletadas no TFI que podem enriquecer uma nova proposta de treinamento pós-básico em G-G? A demografia, a expertise dos professores, a estratégia articulada da pós-graduação sustentam a necessidade de um programa pós-básico adaptado, que inclui o "life long learning", um candidato do G-G que aprende métodos para aprender ao longo da vida. A projeção de no Uruguai (quase 825.000 AM) em uma população de 3,7 milhões, enquanto os menores de 15 anos serão mais de 570.000 a própria epidemiologia exigirá cuidados de enfermagem e mudanças na previdência social.

Blood Transcriptomics of Turbot Scophthalmus maximus: A Tool for Health Monitoring and Disease Studies.

Blood transcriptomics is emerging as a relevant tool to monitor the status of the immune system and assist in diagnosis, prognosis, treatment and pathogenesis studies of diseases. In fish pathology, the potential of transcriptome profiling of blood is still poorly explored. Here, RNA sequencing was applied to analyze the blood transcriptional profile of turbot (Scophthalmus maximus), the most important farmed flatfish. The study was conducted in healthy specimens and specimens parasitized by the myxozoan Enteromyxum scophthalmi, which causes one of the most devastating diseases in turbot aquaculture. The blood of healthy turbot showed a transcriptomic profile mainly related to erythrocyte gas transportation function, but also to antigen processing and presentation. In moderately infected turbot, the blood reflected a broad inhibition of the immune response. Particularly, down-regulation of the B cell receptor signaling pathway was shared with heavily parasitized fish, which showed larger transcriptomic changes, including the activation of the inflammatory response. Turbot response to enteromyxosis proved to be delayed, dysregulated and ineffective in stopping the infection. The study evinces that blood transcriptomics can contribute to a better understanding of the teleost immune system and serve as a reliable tool to investigate the physiopathological status of fish.

Morphopathology and gill recovery of Atlantic salmon during the parasitic detachment of Margaritifera margaritifera.

During the conservation aquaculture of the freshwater mussel Margaritifera margaritifera, fish health has become a concern due to the need of mussel larvae (glochidia) to parasitize the salmonid gills and metamorphose into juveniles. However, there is a lack of information about the impact on fish during the juvenile detachment and the subsequent gill healing. To evaluate the morphopathological changes and gill recovery after the parasitism of M. margaritifera, 51 Atlantic salmon fry (Salmo salar), infested with around 22 larvae/fish g, were necropsied during the synchronized detachment of the mussel juveniles, and gills were assessed by stereomicroscopy and by light and scanning electron microscopy. Salmon showed no clinical signs during the trial and gills recovered their normal morphology almost completely in a short time, suggesting a minimal impact on fish health after glochidiosis. In this sense, the non-erosive droplet detachment and the goblet cell hyperplasia favoured an effective gill remodelling mediated by apoptosis, polarization and cell shedding of the gill epithelia, providing insights to the defence, clearing and healing mechanisms of the gill. These morphopathological techniques could also be implemented to preserve fish welfare and to optimize the artificial breeding programmes of endangered freshwater mussels.

Skeletal Anomalies in Senegalese Sole (Solea senegalensis, Kaup) Fed with Different Commercial Enriched Artemia: A Study in Postlarvae and Juveniles.

The high incidence of skeletal anomalies in Senegalese sole (Solea senegalensis) still constitutes a bottleneck constraining its production. There are diverse commercially available products for the enrichment of live preys, but few reports of their influence on skeletogenesis in Senegalese sole. This study evaluated the presence of vertebral anomalies in postlarvae and juvenile Senegalese sole fed with Artemia spp. metanauplii enriched with four commercial products (EA, EB, EC, and ED) in a fish farm. The most frequent alterations consisted of deformations of the neural/haemal arches and spines and fusions and deformations of hypurals, epural, or parhypural. The correspondence analysis ordered fish from each age in separated semiaxis, indicating the presence of different anomaly patterns for the two sampled stages. The results showed only very light changes in the frequency of vertebral abnormalities among tested enrichment products, i.e., individuals from EC and EA lots displayed less vertebral body anomalies and/or vertebral column deviations at 31 and 105 days after hatching, respectively. The existence of a large shared malformation pattern in all the experimental groups leads to impute to the rearing conditions as the main driving factor of the onset of such group of anomalies, probably masking some dietary effect.

The Teleost Thymus in Health and Disease: New Insights from Transcriptomic and Histopathological Analyses of Turbot, Scophthalmus maximus.

The thymus is a primary lymphoid organ that plays a pivotal role in the adaptive immune system. The immunobiology of the thymus in fish is considered to be similar to that of mammals, but it is actually poorly characterized in several cultured teleost species. In particular, while investigations in human and veterinary medicine have highlighted that the thymus can be affected by different pathological conditions, little is known about its response during disease in fish. To better understand the role of the thymus under physiological and pathological conditions, we conducted a study in turbot (Scophthalmus maximus), a commercially valuable flatfish species, combining transcriptomic and histopathological analyses. The myxozoan parasite Enteromyxum scophthalmi, which represents a major challenge to turbot production, was used as a model of infection. The thymus tissues of healthy fish showed overrepresented functions related to its immunological role in T-cell development and maturation. Large differences were observed between the transcriptomes of control and severely infected fish. Evidence of inflammatory response, apoptosis modulation, and declined thymic function associated with loss of cellularity was revealed by both genomic and morphopathological analyses. This study presents the first description of the turbot thymus transcriptome and provides novel insights into the role of this organ in teleosts' immune responses.

Early stages of Margaritifera margaritifera glochidiosis in Atlantic salmon: Morphopathological characterization.

Freshwater mussels of the order Unionida encyst into the fish mucosa to metamorphose and complete their life cycle, causing a parasitic disease known as glochidiosis. This parasitic stage represents a bottleneck for the survival of naiads, particularly for critically endangered species as Margaritifera margaritifera; however, little is known about the events occurring during this critical stage. Therefore, this study aimed to histologically characterize the development of M. margaritifera glochidiosis in Atlantic salmon to get insight into the pathogenesis of this interaction. Fish exposed to glochidia were sampled during the first 44 days post-exposure, and organs were observed by stereomicroscopy and light microscopy. Glochidia attached to the gills by pinching the lamellar epithelium, whereupon an acute proliferative branchitis engulfed most of the larvae. However, during the first 14 days, a severe detachment of unviable glochidia occurred, associated with the presence of pleomorphic inflammatory infiltrate and epithelial degeneration. In the cases where larvae remained attached, a chronification of the lesions with none to scarce inflammation was observed. These results provide key information to better understand the complex host-parasite interaction during the early stages of glochidiosis and provide valuable information to optimize artificial rearing of naiads in conservation of threatened freshwater mussel populations.

Corrigendum: Integrating Genomic and Morphological Approaches in Fish Pathology Research: The Case of Turbot (Scophthalmus Maximus) Enteromyxosis.

[This corrects the article DOI: 10.3389/fgene.2019.00026.].

Integrating Genomic and Morphological Approaches in Fish Pathology Research: The Case of Turbot (Scophthalmus maximus) Enteromyxosis.

Enteromyxosis, caused by Enteromyxum scophthalmi, is one of the most devastating diseases stemming from myxozoan parasites in turbot (Scophthalmus maximus L.), being a limiting factor for its production. The disease develops as a cachectic syndrome, associated to catarrhal enteritis and leukocytic depletion, with morbidity and mortality rates usually reaching 100%. To date, no effective treatment exists and there are different unknown issues concerning its pathogenesis. The gross and microscopic lesions associated to enteromyxosis have been thoroughly described, and several morphopathological studies have been carried out to elucidate the mechanisms of this host-parasite interaction. More recently, efforts have been focused on a multidisciplinary approach, combining histopathology and transcriptome analysis, which has provided significant advances in the understanding of the pathogenesis of this parasitosis. RNA-Seq technology was applied at early and advanced stages of the disease on fishes histologically evaluated and classified based on their lesional degree. In the same way, the transcriptomic data were analyzed in relation to the morphopathological picture and the course of the disease. In this paper, a comprehensive review of turbot enteromyxosis is presented, starting from the disease description up to the most novel information extracted by an integrated approach on the infection mechanisms and host response. Further, we discuss ongoing strategies toward a full understanding of host-pathogen interaction and the identification of suitable biomarkers for early diagnosis and disease management strategies.

Skeletal Anomalies in Senegalese Sole ( Solea senegalensis), an Anosteocytic Boned Flatfish Species.

Skeletal anomalies affect animal welfare and cause important economic problems in aquaculture. Despite the high frequency of skeletal problems in reared Solea senegalensis, there is lack of information regarding the histological features of normal and deformed vertebrae in this flatfish. The aim of this study was to describe the histopathological and radiographical appearance of vertebral body anomalies. Sixty-seven juvenile fish were radiographically examined 104 or 105 days after hatching. Through radiographic images, vertebral segments were selected and processed for histopathological examination from 7 normal and 7 affected fish. Alterations in bone shape and vertebral fusion were the most significant anomalies in the vertebral bodies. These alterations occurred most frequently between the last 3 abdominal vertebrae and the first 10 caudal centra. Radiographically, deformed vertebrae showed flattening of the endplates and narrowing of the intervertebral spaces. The radiographic findings concurred with the histological lesions where affected vertebrae exhibited irregular endplates and changes in trabecular bone. Radiolucent cartilaginous tissue was evident in the endplates of the deformed vertebra and, in some cases, the cartilaginous material extended from the growth zone into the intervertebral space. These changes were likely the primary alterations that led to vertebral fusion. Fused vertebrae were often reshaped and showed a reorganization of the trabeculae. The formation of metaplastic cartilage is frequent in a variety of anomalies affecting teleost species.

First description of a natural infection with spleen and kidney necrosis virus in zebrafish.

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross-species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.

Susceptibility of Zebrafish to Vesicular Stomatitis Virus Infection.

The zebrafish, Danio rerio, has become recognized as a valuable model for infectious diseases. Here we evaluated the susceptibility of zebrafish to be infected with the mammalian vesicular stomatitis virus (VSV). Both zebrafish cells and embryos were highly susceptible to VSV infection. Mortalities exceeded 80% in infected embryos and were preceded by the invasion of the central nervous system by VSV. Live imaging of the infection with GFP-VSV as well as virus titration from infected fish confirmed the viral replication. Immunohistochemical analysis of embryonic fish provided evidence of viral antigens as well as of the apoptosis marker caspase-3 in the brain, eye, liver, pronephros, and skeletal muscle. So far, this is the first report describing the susceptibility of zebrafish to the mammalian virus VSV.

Immunohistochemical study of inducible nitric oxide synthase and tumour necrosis factor alpha response in turbot (Scophthalmus maximus) experimentally infected with Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida represents one of the major threats in aquaculture, especially in salmonid fish and turbot farming. In order to fight bacterial infections, fish have an immune system composed by innate and specific cellular and humoral elements analogous to those present in mammals. However, innate immunity plays a primordial role against bacterial infections in teleost fish. Among these non-specific mechanisms, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) pathway and the tumour necrosis factor-alpha (TNFα) produced by mononuclear phagocytes, are two of the main immune effectors to eliminate bacterial pathogens. In this study, the distribution and kinetic of iNOS and TNFα-producing cells of kidney and spleen of turbot experimentally inoculated with A. salmonicida was assessed by immunohistochemistry. In control and challenged fish, individual iNOS(+) and TNFα(+) cells, showing a similar pattern of distribution, were detected. In challenged fish, the number of immunoreactive cells was significantly increased in the evaluated organs, as well as the melanomacrophage centres showed variable positivity for both antigens. These results indicate that A. salmonicida induced an immune response in challenged turbot, which involved the increase of the activity of iNOS and TNFα in the leukocytic population from kidney and spleen.

RNA-seq analysis of early enteromyxosis in turbot (Scophthalmus maximus): new insights into parasite invasion and immune evasion strategies.

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host-parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosis.

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization.

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.

Immunohistochemical detection and gene expression of TNFα in turbot (Scophthalmus maximus) enteromyxosis.

Enteromyxum scophthalmi (Myxozoa) constitutes one of the most devastating pathogens for turbot (Scophthalmus maximus, L.) aquaculture. This parasite causes a severe intestinal parasitosis that leads to a cachectic syndrome with high morbidity and mortality rates for which no therapeutic options are available. Presence of inflammatory infiltrates, increased apoptotic rates and epithelial detaching have been described at intestinal level, as well as leukocyte depletion in lymphohaematopoietic organs. Previous investigations on enteromyxosis in turbot showed the high susceptibility of this species to the parasite and reported the existence of a dysregulated immune response against the parasite. The pleiotropic cytokine tumour necrosis factor alpha (TNFα) plays a major role in immune response and is involved in a wide range of biological activities. In teleost, the gene expression of this cytokine has been found regulated under several pathological conditions. Teleost TNFα shows some analogous functions with its mammalian counterparts, but the extent of its activities is still poorly understood. Cytokines are generally considered as a double-edge sword and TNFα has been implicated in the pathogenesis of different inflammatory diseases as well as in wasting syndromes described in mammals. The aim of this work was to analyse the expression of TNFα during enteromyxosis with molecular (Q-PCR) and morphological (immunohistochemistry) tools. Kidney, spleen and pyloric caeca from turbot with moderate and severe infections were analysed and compared to healthy naïve fish. TNFα expression was increased in both spleen and kidney in the earlier stages of the disease, whereas in severely infected fish, the expression decreased, especially in kidney. At the intestinal level, an increase in the number of TNFα-positive cells was noticed, which was proportional to the infiltration of inflammatory cells. The results demonstrate the involvement of TNFα in the immune response to E. scophthalmi in turbot, which could be related to the development of the clinic signs and lesions.

Immunolocalization of tumor necrosis factor alpha in turbot (Scophthalmus maximus, L.) tissues.

Tumor necrosis factor alpha (TNFα) is a cytokine involved in a broad spectrum of cellular and organismal responses. Its main function, as a potent pro-inflammatory mediator, has been demonstrated in numerous teleost species and there are many reports on the modulation of TNFα gene expression under pathological conditions. Nevertheless, there is still scarce knowledge about the tissue distribution and type of cells that express this cytokine in fish species, which would help to further investigate its biological activities. These studies are hampered by the lack of molecular markers for teleost that hinder the development of morphological techniques, like immunohistochemistry. The aim of this work was to develop an immunohistochemical technique for the detection of TNFα in paraffin-embedded organs from healthy turbot (Scophthalmus maximus), an economically-important marine fish species. A commercial anti-human TNFα antibody, whose specificity was confirmed by western blot analysis, was used. Immunoreactive cells were observed in higher numbers in the lymphohematopoietic organs, kidney, spleen and thymus, although TNFα-positive cells were also present in the digestive tract, liver, heart, gills and skin. Similarly to non-fish species, monocytes/macrophages appeared to be the main producers of this cytokine; nevertheless, the presence of immunoreactive rodlet cells in different tissues was also reported. The nature and distribution of the labeled cells appeared to be related with a strategic localization for defense response to antigenic challenge. The relative abundance of TNFα-positive cells in the lymphohematopoietic organs also suggests that this cytokine may have a broader role in the normal physiology of those organs. The immunohistochemical technique allowed the in-situ characterization of TNFα expression, representing a valid tool to investigate the immune response of turbot.

RNA-seq analysis reveals significant transcriptome changes in turbot (Scophthalmus maximus) suffering severe enteromyxosis.

BACKGROUND: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis. RESULTS: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism. CONCLUSIONS: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.

Tenacibaculum maritimum infection: pathology and immunohistochemistry in experimentally challenged turbot (Psetta maxima L.).

Tenacibaculum maritimum is the causative agent of tenacibaculosis, a bacterial disease with a worldwide distribution, which causes important losses in the turbot aquaculture. Despite the importance of this bacterium, little is known about pathogenesis of the tenacibaculosis, pattern of lesions and the portal of entry of T. maritimum. Turbots (Psetta maxima) were experimentally infected with T. maritimum using subcutaneous and intraperitoneal routes of inoculation and samples of skin and internal organs were taken throughout the assay. Fish inoculated by both infection routes suffered a septicaemia but only the subcutaneous inoculation reproduces the disease signs described in natural outbreaks. Bacterial antigen was detected by immunohistochemistry in the internal organs 3 h after infection in fish inoculated by subcutaneous route and 6 h after the inoculation of fish by intraperitoneal route. In summary, both routes of inoculation are able to cause an infection and bacteraemia in the fish. However, subcutaneous inoculation route reproduces the disease in a faster and more reliable way than the intraperitoneal route. Moreover, bacterium spreads along the internal organs easily, but needs a gateway to penetrate in the organism and this portal of entry could be skin.