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© 2021 by the authors., Enterospora nucleophila is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (Sparus aurata), leading to an emaciative syndrome. Thus far, the only available information about this infection comes from natural outbreaks in farmed fish. The aim of the present study was to determine whether E. nucleophila could be transmitted horizontally using naturally infected fish as donors, and to establish an experimental in vivo procedure to study this host–parasite model without depending on natural infections. Naïve fish were exposed to the infection by cohabitation, effluent, or intubated either orally or anally with intestinal scrapings of donor fish in four different trials. We succeeded in detecting parasite in naïve fish in all the challenges, but the infection level and the disease signs were always milder than in donor fish. The parasite was found in peripheral blood of naïve fish at 4 weeks post-challenge (wpc) in oral and effluent routes, and up to 12 wpc in the anal transmission trial. Molecular diagnosis detected E. nucleophila in other organs besides intestine, such as gills, liver, stomach or heart, although the intensity was not as high as in the target tissue. The infection tended to disappear through time in all the challenge routes assayed, except in the anal infection route., This work has been carried out with financial support from the European Union and the Spanish MINECO under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. A.P.-S. was contracted under ParaFishControl project. This publication reflects only the authors’ view, and the European Commission cannot be held responsible for any use which may be made of the information contained herein. M.C.P. was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/10.13039/501100011033 and ACOND/2020 Generalitat Valenciana), and R.P. was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I). Both contracts have been co-funded by the European Social Fund (ESF).We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Simple Summary Enterospora nucleophila is a microsporidian parasite infecting gilthead sea bream (Sparus aurata) that has been associated with increased mortality, growth arrestment and emaciation in aquaculture settings. The investigations on this parasite depend on material from spontaneous outbreaks in fish farms, which hampers advancements in infection research and control. In the present study, different ways of horizontal transmission were performed in order to establish an in vivo transmission model. Parasite transmission was achieved with all the assayed routes, but with milder clinical signs than in natural infections. Abstract Enterospora nucleophila is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (Sparus aurata), leading to an emaciative syndrome. Thus far, the only available information about this infection comes from natural outbreaks in farmed fish. The aim of the present study was to determine whether E. nucleophila could be transmitted horizontally using naturally infected fish as donors, and to establish an experimental in vivo procedure to study this host–parasite model without depending on natural infections. Naïve fish were exposed to the infection by cohabitation, effluent, or intubated either orally or anally with intestinal scrapings of donor fish in four different trials. We succeeded in detecting parasite in naïve fish in all the challenges, but the infection level and the disease signs were always milder than in donor fish. The parasite was found in peripheral blood of naïve fish at 4 weeks post-challenge (wpc) in oral and effluent routes, and up to 12 wpc in the anal transmission trial. Molecular diagnosis detected E. nucleophila in other organs besides intestine, such as gills, liver, stomach or heart, although the intensity was not as high as in the target tissue. The infection tended to disappear through time in all the challenge routes assayed, except in the anal infection route.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species., This work has been carried out with financial support from the European Union and the Spanish MINECO under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project, RDP was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I), and MCP under CSIC PIE Project No. 201740E013. NHA was the recipient of a fellowship from the Egyptian Cultural Office in Spain.

Trabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrado en Gran Canaria (España), del 16 al 20 de junio de 2019, Endocrine disruptors in general and oestrogenic compounds in particular have been widely investigated in view of their effects on several physiological processes and, therefore, their ecotoxicologic relevance. The modulation of the immune system by oestrogens has increasingly sparked interest in the research community, that previously had been mainly centered on the reproductive effects of these hormones. In fact, since the industrialization an increasing variety of endocrine disruptors, such as oestrogenic endocrine disruptors, are retrieved in the environment. These oestrogenic endocrine disrupting chemicals (EEDCs) have also been suggested to increase the prevalence of autoimmune diseases and cancer. With regard to the high degree of similarities in the immune system of jawed vertebrates as well as the conserved immunomodulatory roles of oestrogen, environmental EEDCs possibly have the capacity to alter immune system functions of teleost fish, which may impair their capacity to fight infectious diseases and eventually may contribute, together with overfishing, to wild stock losses. Importantly, the most deleterious effects of EEDCs, both in mammals and teleosts, appear to arise when the exposure occurs during specific periods of the immune system ontogenesis, commonly referred as critical windows of exposure. However, in mammals and especially in teleost fish, these stages of the immune system development as well as the EEDC-action remain to be fully identified and characterised. The concept and the importance of developmental immunotoxicity is presented by addressing the mechanisms of oestrogenic regulation and the mode of action of EEDCs from an immunological perspective. Emphasis is given to the critical windows of development of the immune system during which EEDCs may alter the immune system development and function with long-term consequences on immunocompetence. Results from different classes of vertebrates are compiled, highlighting studies on teleost fish and their relevance for the human immune system. Additionally, new results on the effects of environmentally relevant concentration of exogenous estradiol exposure during European sea bass (Dicentrarchus labrax) development will be presented with regard to oestrogen's ability to trigger effects on immunocompetence, contributing to fill the gaps on vertebrate immunotoxicology.

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1β, high il10 and low hsp90α expression)., This study was supported by EU H2020 program and by the Spanish Ministry of Economy and Competitiveness through ParaFishControl (634429) and AGL-2013-48560-R research projects, respectively. This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. MCP was contracted under CSIC PIE project no. 201740E013 and IE was recipient of APOSTD/2016/037 grant by the “Generalitat Valenciana”.

[Backgroud] Enteromyxum leei is a myxozoan parasite that produces a slow-progressing intestinal disease. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestine. The aim of the present study was to gain insights into fish T cell responses in the gilthead sea bream-E. leei infection model using a PCR-array with 30 signature molecules for different leukocyte responses in head kidney, spleen, anterior and posterior intestine., [Results] The PCR-array results suggest that E. leei induced migration of T cells from head kidney to intestines where TH1, CTL and TH17 profiles were activated and kept in balance by the upregulation of regulatory cytokines. These results were partially validated by the use of cross-reacting antibodies and BrdU immunostaining to monitor proliferation. Zap70 immunostaining supported the increased number of T cells in the anterior intestine detected by gene expression, but double staining with BrdU did not show active proliferation of this cell type at a local level, supporting the migration from lymphohaematopoietic tissues to the site of infection. Global analyses of the expression profiles revealed a clear separation between infected and exposed, but non-infected fish, more evident in the target organ. Exposed, non-infected animals showed an intermediate phenotype closer to the control fish., [Conclusions] These results evidence a clear modulation of the T cell response of gilthead sea bream upon E. leei infection. The effects occurred both at local and systemic levels, but the response was stronger and more specific at the site of infection, the intestine. Altogether, this research poses a promising basis to understand the response against this important parasite and establish effective preventive or palliative measures., This work has been carried out with financial support from the Spanish MINECO under project AGL2013-48560-R. Additional funding was provided by the European Union, through the Horizon H2020 research and innovation programme under grant agreement 634429 (ParaFishControl). This publication reflects only the authors’ view and the European Union cannot be held responsible for any use that may be made of the information contained herein. Further support was provided by Generalitat Valenciana (PROMETEOII/2014/085). MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the “Generalitat Valenciana”. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish‐to‐fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities., This work has been carried out with financial support from the European Commission, Horizon 2020 Framework Programme H2020, Societal Challenges under grant agreement 634429 (ParaFishControl). This publication reflects only the authors' view, and the European Union cannot be held responsible for any use that may be made of the information contained therein. IE was contracted under APOSTD/2016/037 grant by the “Generalitat Valenciana,” and MCP, under Consejo Superior de Investigaciones Científicas CSIC PIE project no. 201740E013.