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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.

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).

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, Passive immunization is used in humans for treating or preventing some infectious diseases, but it also constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Intracoelomically-injected antibodies can be detected in fish sera within the first 8 h and their half-life ranges from 7 to 22 days post-injection, depending on the species. The fish models studied so far used fast-acting pathogens such as virus, bacteria or ciliate parasites. The current work aimed to determine if passive immunization could help to prevent enteromyxosis in gilthead sea bream (GSB, Sparus aurata). Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that GSB that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific serum antibodies. In the current study, we evaluated whether injection with sera from resistant animals would protect naïve fish when challenged by effluent exposure to the parasite. Serum from a pool of resistant (R) and naïve (N) animals (intact or heat inactivated, 10 ¿l/g BW) was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). At 23 dpc, the different groups were allocated in separate tanks and the effluent exposure was terminated. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for hematology, circulating antibodies, histological and molecular diagnosis and gene expression. Groups injected with R sera had lower prevalence and intensity of infection than those with N sera, both in the intermediate and final samplings. At 100 dpc, the prevalence of infection in the PBS and N groups was 70%, whereas in R group it only reached 55%. Condition factor (CF) and specific growth rate (SGR), key parameters affected by enteromyxosis, were higher in R group. There was a significant correlation between prevalence of infection and SGR and CF. Immunohistochemistry and gene expression studies will reveal whether this partial protection was due to higher presence of specific antibodies or specific cell populations. These results show that, even with this long term disease, passive immunization can confer some degree of protection. The administration of specific antibodies during exposure, probably provided fish with time to activate the specific defenses before the parasite proliferated., This work was funded by EU H2020 program through ParaFishControl Project (634429) and Spanish MINECO project AGL2013-48560. M.C.P. was contracted under CSIC PIE project no. 201740E013 and FPDI-2013-15741 and I.E. under APOSTD/2016/037 grant by the “Generalitat Valenciana”.