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Trabajo presentado en la 18th International Conference on Diseases of Fish and Shellfish, celebrada en Belfast, del 4 al 8 de septiembre de 2017, The microsporidium Enterospora nucleophila is the causative agent of a parasitic emaciative disease in gilthead sea bream (Sparus aurata). This syndrome appears mainly during the winter season in 1. year juvenile gilthead seabream (GSB) as an outstanding growth retardation and size segregation, with serious mortality in some stocks. Diagnosis of E. nucleophila infection is quite challenging due to the minute size of the spores, which develop mainly within the nuclei of enterocytes and rodlet cells in the intestinal epithelium. A remarkable hypercellularity and nuclei alterations in infected cells are commonly observed in the histopathological analysis of infected intestines, but spores are often absent or missed, even in clinical infections. This work describes the optimization and application of a battery of methods for the diagnosis of E. nucleophila infections in GSB. The methods include optimised protocols for fluorescent stain with Calcofluor on tissue sections and smears, a quantitative PCR test, and in-situ hybridization (ISH) techniques. The procedures were applied to a large (>200) panel of clinical cases in order to compare their performance, validate routine qPCR tests, and obtain insights into the pathogenesis of GSB enterosporosis. The work generates validated methods for the diagnosis of E. nucleophila in clinical samples of GSB, which will be valuable for a better understanding of the epidemiology and pathogenesis of this disease. As a follow-up of the analytical and clinical validation covered in this work, a panel of samples will be submitted to diagnostic laboratories participating in an inter-proficiency test as a part of the H2020 project “ParaFishControl”., This study was partially supported by EU H2020 program through the ParaFishControl project (634429) and by AGL-2013-48560-R project from the Spanish Ministry of Economy and Competitiveness

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms., This work has been carried out with financial support from the Spanish MINECO under project AGL2013‐48560‐C2‐2‐R. Additional funding was provided by the European Union, through the Horizon 2020 research and innovation programme grant agreement 634429 (ParaFishControl).