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Among others, the families Opisthorchiidae and Heterophyidae includes several genera causing fish-borne zoonoses and distributed also in European Countries and that are included in the ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish) H2020 EU project. Due to the small size of the metacercariae, the infective stage for human, these parasites cannot be detected visually in fish and monitoring requires expert application of time-consuming techniques. The aim of this was to develop a rapid and affordable molecular method based on multiplex PCR for simultaneous identification of metacercariae of the most common European Opisthorchiid and Heterophyid in fish or fish products.

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).

The genus Eustrongylides includes nematodes known as the etiological agent of the ‘‘big red worm disease.’’ The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant (Phalacrocorax carbo) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus, which has not yet been identified as causal agent of human cases.

Introduction: Enteric parasites affecting farmed gilthead sea bream (GSB) have become a serious threat for Mediterranean aquaculture in the last few years; among these parasites Enteromyxum leei, Enterospora nucleophila and Cryptosporidium molnari are undoubtedly the most concerning ones. While the enteric myxozoan E. leei has been broadly studied, the available data on the occurrence of E. nucleophila and C. molnari infections are still scarce. Aim of this work was to improve the knowledge about these latter two parasites in Mediterranean aquaculture by carrying out an epidemiological survey in GSB farmed in Italy and Croatia. Methodology: 308 GSB of which 40 from an Italian hatchery, 174 from three cage farms located in Italy and 94 GSB from one cage farm located in Croatia were tested with qPCR and PCR to assess the presence of E. nucleophila and C. molnari. Histology was also performed on infected GSB. Results: All the examined farms tested positive for both parasites: 60% of the fish examined from hatchery were positive for E. nucleophila, while 22.5% resulted positive for C. molnari. Concerning caged fish, E. nucleophila was found in 63.2% of the GSB coming from Italian farms and in 45.7% of fish from Croatia. C. molnari was detected in 3.4% of Italian GSB and in 2.1% of the Croatian ones. Histological lesions were consistent with those already reported in literature for these enteric parasitic infections. Conclusion: This study showed a diffuse presence and a high prevalence of E. nucleophila in Italian and Croatian farmed GSB. Although at lower prevalence, also C. molnari showed to occur in farmed GSB, especially in hatchery and juveniles. Thus, further investigations are required to establish their epidemiology, transmission routes and pathogenic role in farmed GSB along its production cycle in order to assess and manage the risks arousing from these emerging enteric parasites.