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Despite the wide distribution and health importance of anisakids of the genus Contracaecum, epidemiological data on their occurrence in definitive bird hosts are scarce, particularly from certain parts of the world that represent important wintering sites or migration stopovers for different bird species. In the present study, Contracaecum spp. infecting six great white pelicans (Pelecanus onocrotalus) in Israel were identified using light and scanning electron microscopy and phylogenetic analyses of nuclear internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase II (cox2). A PCR-RFLP method was also developed and applied to screen large numbers of Contracaecum parasites. Most (415/455) worms recovered were C. micropapillatum, followed by C. gibsoni (31/455), C. quadripapillatum (8/455), and C. multipapillatum E (1/455). Contracaecum micropapillatum from Israel and C. bancrofti from Australia are distinguishable by cox2 but less well resolved with ITS sequences, and could not be distinguished morphologically. Worms with cox2 matching C. gibsoni had ITS matching specimens identified as C. multipapillatum A. To the authors' knowledge, this represents the first of such studies in Israel and provides useful data on the ecology and distribution of different Contracaecum species of health and economic interest.

Nematodes belonging to the genus

The diffusion of new eating habits and the increase of fish products demand have been raising concerns in the European countries, including Italy, about the transmission risk of zoonotic fish parasites to consumers, leading to the current EU "Hygiene Package" Regulations. In relation to the mandatory freezing treatment for “products intended to be consumed raw, or marinated, salted and any other treated fishery products, if the treatment is insufficient to kill the viable parasite”, an exception is included in the Reg. (EU) No 1276/2011 for farmed Atlantic salmon (Salmo salar), for which the risk was considered negligible according to EFSA Opinion (2010), but not for other fish species farmed in Europe. With the aim to collect the necessary epidemiological information on the presence/absence of zoonotic parasites in EU aquaculture, in the framework of the EU H2020 project ParaFishControl (www.parafishcontrol.eu), an extensive parasitological survey has been carried out on the main farmed fish species in Italy and other countries such as Spain, Greece, Denmark, Norway and Hungary. The present work reports the results from Italy. From 2016 to 2018 a total of 4728 farmed fish have been examined from 6 freshwater and 5 marine farms located in Italy: 1594 rainbow trout (Oncorhynchus mykiss), 1571 European sea bass (Dicentrachus labrax) and 1563 gilthead sea bream (Sparus aurata) have been collected based on a random polietapic and stratified sampling plan with a confidence level of 99% and a margin of error (MoE) of 4-8%. Besides harvest quality fish, runts were also examined in order to consider even the most predisposed hosts to acquire parasites through the natural food web. Parasitological analyses to search for anisakid nematodes in marine fish and diphyllobothriid cestodes and Opisthorchioidea digeneans in freshwater fish were performed utilizing methodologies such as visual inspection and candling as provided by the EU regulation, integrated by UV-press method, muscle compression/artificial digestion followed by microscopic examination when required (e.g. for Opisthorchioidea). No zoonotic parasites were found in any of the examined fish, including runts. The data obtained from this extensive survey show the absence of zoonotic parasites in the fish examined and are comparable to those reported in previous studies, leading to consider as negligible the risk of infections due to zoonotic helminths in the most important fish species of Italian aquaculture. Similar results have been obtained for the examined farmed fish in other countries involved in ParaFishControl Project. The results allow to assess the risk of the presence of zoonotic parasites in farmed rainbow trout, European sea bass and gilthead sea bream as negligible, indicating that also these farmed fish species should be considered suitable to benefit from the exemption from freezing treatment provided by EU Regulation No 1276/2011 for Atlantic salmon. Furthermore, the monitoring approach employed could be adopted for planning surveillance activities in EU fish farming systems, as it appears implementable also by industry, to better guarantee the safety of aquaculture products.

Dermocystidium spp. (Mesomycetozoea: Dermocystidiaceae) comprises several species able to infect a wide range of freshwater but also marine fish.The lesions caused by Dermocystidium spp. in fish are chronic and characterized by visible cysts or nodules on skin and fins, but severe gills and systemic infections causing mortality have been described in several species. The present study described a primary ocular infection by Dermocystidium sp. in a koi carp (Cyprinus carpio var. koi). The fish presented an evident subconjunctival whitish exophytic tissue, surrounded by reddish edges, that protruded over both ocular bulbs. The fish was necropsied, and tissues (eyes, gills, heart, liver, spleen, stomach, intestine, brain) were fixed in 10% buffered formalin for histology. according to the spore size range (5.53–12.90 µm), the morphology of spores and the infected tissue, the present case appears to be consistent with an infection by Dermocystidium koi.Considering the economic value of koi carps and the possible direct transmission of the infection in optimal temperature condition, Dermocystidium spp. infection should be considered as an important disease also in ornamental species.

Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish, and represent a potential threat to mariculture due to their pathogenicity and ability to cause mortality in fish maintained in controlled conditions. The identification of Neobenedenia species and, consequently, the definition of their host specificity is often problematic due to their highly conserved morphology; therefore, in order to establish their specific identity, microscopic observation should be complemented with molecular analysis. The present work aims at characterizing Neobenedenia specimens infecting the skin of cage reared gilthead seabream Sparus aurata from Portugal. Parasite samples obtained from caged fish were processed for morphological analysis, through observation in light and scanning electron microscopy, and for molecular analysis, through amplification and sequencing of 28S rDNA and cytB, aimed at identifying them to the species level. Our results showed that the collected parasites belonged to the species Neobenedenia girellae; the susceptibility of S. aurata towards this pathogenic capsalid monogenean highlighted in the present work represents an important risk in the farming of this valuable fish species.

Occurrence of metacercariae of potentially zoonotic trematodes (Platyhelminthes: Digenea) in the musculature of common carp (Cyprinus carpio L. 1758) was monitored in four Hungarian aquacultures. Four geographically distinct fish farms (located in the Northwestern, Southwestern, Northeastern and Southeastern parts of Hungary) were selected for the investigation. From each farm, a total of 258 one-summer-old fingerlings were sampled and examined in the years 2016 and 2017. In addition, in 2017, we examined 60 market size specimens (30 two-summers and 30 three-summers) sampled from the most infected aquaculture in the Northeastern part of Hungary. The fish were euthanized and decapitated whereafter their musculature (fillets) was digested in a pepsin solution to isolate metacercariae from the tissue whereafter morphological and molecular analyses (PCR and sequencing of ITS region) were performed. Opisthorchiid metacercariae were not recovered but in one of the farms numerous metacercariae were detected in the musculature of carp. They were identified as cyathocotylid trematodes based on their morphological characteristics and by sequencing the ITS region. The infection levels proved to be remarkably different among the four fish farms. Carps from the Northeastern farm were infected by large numbers of cyathocotylid metacercariae, while 8 Posthodiplostomum cuticola metacercariae were detected in the Northwestern aquaculture. In the other two farms (Southwestern and Southeastern) no infection was recorded. The infected farm is located close to a protected natural wetland habitat populated by a rich fauna of aquatic birds (potential final hosts) and snails (first intermediate host) which may create a higher risk of infection in the neighbouring fish farms.

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6 pages, 1 table.-- This work is licensed under a Creative Commons Attribution 4.0 International License, The increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment. Aim: To evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon. Methods: From 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae. Results: No zoonotic parasites were found in any of the fish investigated. Conclusions: The risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation, This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 634429

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.

Specimens of common smooth-hound, Mustelus mustelus, fished as bycatch in the Northwestern Adriatic Sea, were subjected to a complete parasitological examination. Parasites were processed for morphological and morphometric analyses aimed at identifying them to the species level. Parasite identity was confirmed through amplification and sequencing of 28S rDNA. The nematode Acanthocheilus rotundatus was recovered from the stomach and the proximal intestine, while the cestode Calliobothrium verticillatum was collected from the spiral valve. Our results confirm the occurrence of C. verticillatum in M. mustelus from the Mediterranean Sea and provide the first sequence data of A. rotundatus from the same area. Updated knowledge on the parasite fauna of M. mustelus from Adriatic Sea contributes to elucidating aspects of the ecology, biology and health status of elasmobranchs from the Mediterranean Sea, providing useful information for their proper management and conservation.

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

Mediterranean marine aquaculture has suffered significant economic losses due to viral nervous necrosis (VNN) outbreaks mainly caused by different RGNNV betanodavirus strains. In recent years, the marine aquaculture sector has experienced the emergence of the RGNNV/SJNNV reassortant betanodavirus, harbouring the RNA1 segment of RGNNV genotype and the RNA2 segment of SJNNV genotype. So far, the reassortant strains caused massive mortality outbreaks in gilthead sea bream (Sparus aurata) larvae sparing the European sea bass (Dicentrarchus labrax). In this study, multiple mortality outbreaks occurred in one Italian marine hatchery involving both European sea bass and gilthead sea bream at different life stages were investigated through a complete microbiological and molecular analysis. Gilthead sea bream larvae and juveniles have recorded the highest mortality rates, however, both European sea bass and gilthead sea bream incurred a RGNNV/SJNNV reassortant betanodavirus persistent infection, able to act as asymptomatic carriers and viral source for susceptible fish. These new epidemiological data on nervous necrosis virus (NNV) reassortant infection provide precious advice on how to manage fish to reduce VNN spread in Mediterranean aquaculture. Evidence of interspecies transmission of RGNNV/SJNNV reassortant strains and the persistent infection in both European sea bass and gilthead sea bream, point out the importance to enforce a wide surveillance and a strict biosecurity programme addressing both RGNNV and reassortant RGNNV/SJNNV betanodaviruses in Mediterranean European sea bass and gilthead sea bream farms. Furthermore, the presence assessment of betanoviruses in all newly-introduced fish batches in the farm, regardless of the species and a strict segregation between European sea bass and gilthead sea bream batches within farms can significantly reduce the risk of NNV transmission. Finally, surviving fish can act as carrier fish, and thereby must be segregated from other batches and protected from stress conditions that could trigger a new clinical phase.

In the Afrotropic region, the genus Clinostomum is represented by four accepted and four unnamed species distinguished using molecular data. Here, we describe one of the four unnamed species as Clinostomum ukolii n. sp. based on metacercariae from siluriform fishes (Synodontis batensoda, Schilbe intermedius) collected in Nigeria and South Africa. The new species is distinguished by molecular data (39 new sequences of partial cytochrome c oxidase I≥ 6.7% divergent from those of other species) and morphological differences from named and unnamed species in the same region. Metacercariae of C. ukolii n. sp. can be distinguished based on size, tegumental spines, and various aspects of the genital complex, including its position, lobation of the anterior testis, and the disposition and shape of the cirrus pouch. Although descriptions of new species of digeneans are typically based on the morphology of adults, we argue that in cases where data are available from metacercariae from regionally known species, new species can be described based on metacercariae, particularly when supported by molecular data, as here. Moreover, sub-adult reproductive structures can be clearly visualized in metacercaria of Clinostomum. Considering metacercariae as potential types for new species could advance clinostome systematics more rapidly, because metacercariae are encountered much more often than adults in avian definitive hosts.

Tra le diverse problematiche sanitarie causate dal consumo di prodotti ittici, nel presente capitolo vengono trattate le zoonosi parassitarie e le intossicazioni leagte al consumo di prodotti ittici e di molluschi bivalvi. Mentre in alcuni casi è possibile individuare a livello geografico aree di endemia (es. opistorchiasi/clonorchiasi, ciguatera), in altri casi gli agenti eziologici presentano diffusione cosmopolita (es. Anisakis spp.) e l’emergenza di casi clinici appare legata a fattori di natura diversa quali le abitudini alimentari e la mancata applicazione di trattamenti di inattivazione degli agenti patogeni o, come nel caso della sindrome sgombroide, a non idonee condizioni di conservazione ed igiene dei prodotti ittici. La crescente movimentazione di prodotti ittici (l’italia è il sesto importatore a livello mondiale), i rilevanti flussi demografici ed i cambiamenti climatici rendono comunque difficile un approccio sistemico a queste problematiche, richiedendo un continuo aggiornamento epidemiologico.

Ascocotyle (Phagicola) trentinii n. sp. is described based on adults from experimentally infected ducklings (Anas platyrhynchos domesticus) fed with metacercariae from the visceral serosa of the Mediterranean banded killifish, Aphanius fasciatus (Cyprinodontiformes: Aphaniidae), from coastal lagoons in northeastern Italy (Emilia-Romagna Region). The new species is placed into the subgenus Phagicola because of the presence of a single row of circumoral spines, vitelline follicles being confined between the ventral sucker and testes, and uterine loops not reaching anterior to the ventral sucker. Ascocotyle (P.) trentinii n. sp. differs from other members of the subgenus Phagicola, as well as other species of Ascocotyle, by the number (27–33) of circumoral spines which are 13.5–17 μm long and 3.5–5 μm wide, and by the morphology of a gonotyl which is composed of about 8 large refractile pockets. The occurrence of metacercariae in A. fasciatus indicates that the life cycle of the new species is completed in brackish water lagoons. It is the fourth species of Ascocotyle described in Europe and may be endemic to the Mediterranean region because its second (fish) intermediate host is endemic to this region.

Raphidascarididae are among the most abundant and widespread parasitic nematodes in the marine environment. The life-cycle of most raphidascaridid species is poorly known and information about their distribution and host range is lacking in many geographical areas, as is the taxonomy of several species. A study of larval and adult stages of Hysterothylacium fabri (Rudolphi, 1819) Deardorff and Overstreet, 1980 (Nematoda: Raphidascarididae) infecting the striped goatfish Mullus surmuletus Linnaeus, 1758 (Mullidae) and the Mediterranean stargazer Uranoscopus scaber Linnaeus, 1759 (Uranoscopidae) from the Ionian Sea (central Mediterranean) has been carried out by combining light and scanning electron microscopy observations and molecular analyses through polymerase chain reaction-restriction fragment length polymorphism and sequencing of the ITS rDNA gene. Results indicate that U. scaber and M. surmuletus represent suitable definitive and intermediate/paratenic hosts of H. fabri, respectively, in the Mediterranean and highlight the importance of combining genetic and morphological data to study the taxonomy and epidemiology of parasites widely distributed in different fish species and aquatic ecosystems.