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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.

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

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.

The increase of global demand of aquaculture products as compensation for the lowering of fishery sustainability, has shown a parallel awareness by the consumers on the importance of the safety and quality of fish products. Among these, salmon industry has reached a leading position demonstrating the negligible risk of presence of zoonotic helminths such as anisakid nematodes in farmed salmon. Despite the massive production of data in literature on parasitological surveys carried out on fresh salmon, no data are published on processed farmed salmon such as smoked products. In 2016, 270 fillets of smoked farmed Atlantic salmon (Salmo salar) and 13 smoked fillets from wild sockeye salmon (Oncorhynchus nerka) have been analyzed by visual inspection and UV-press method searching for the presence of anisakid nematodes. No parasites were detected in fillets from farmed Atlantic salmon, while 10 out of 13 fillets from wild salmon were positive for Anisakis simplex s.s. larvae. This first survey on the possible presence of anisakid nematodes in processed smoked salmon confirms that this risk in farmed Atlantic salmon products has to be considered negligible.

Meagre Argyrosomus regius is a potential candidate for Mediterranean aquaculture diversification, although several health issues still represent important bottlenecks for its sustainable production, including systemic granulomatosis (SG). To evaluate the SG progression in meagre during a 10 mo period of cage ongrowing, a histopathological investigation was carried out on 108 meagre fed 3 different diets (commercial pellets, hydrated commercial pellets and defrosted sardines). Histological sections of the gills and visceral organs were examined and lesions referable to SG scored from 1-3 according to the severity of the granulomatosis. The kidney and liver were the most affected organs, showing the highest percentage of positivity for granulomas and severity of lesions along the whole observation period. Using a statistical mixed model (GLMM) followed by odds ratio analysis, an effect of diet and temperature was found: the severity of liver and digestive tract SG scores decreased in the Cage 3 group (defrosted sardines) and with increasing temperature (p < 0.05, negative estimates, odds ratio

Mycobacterium pseudoshottsii, a slow-growing mycobacterium closely related to M. marinum, has been isolated only in wild fish in the United States and in Japanese fish farms to date. Here, we report cases of mortality in three farmed fish species (Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus) caused by M. pseudoshottsii in Italy. Samples underwent necropsy, histology, and culture with pathogen identification based on PCR and sequencing of housekeeping genes (16S rRNA, hsp65, rpoB). Multifocal to coalescing granulomatous and necrotizing inflammation with acid-fast bacilli were observed in the parenchymatous organs, from which M. pseudoshottsii was isolated and identified. Phylogenetic analysis confirmed the results of gene sequencing and allowed subdivision of the isolates into three distinct groups. M. pseudoshottsii poses a potential threat for Mediterranean aquaculture. Its origin in the area under study needs to be clarified, as well as the threat to the farmed fish species.

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.

The monogenean Gyrodactylus salaris Malmberg, 1957 is considered one of the most important parasites of wild salmonids in the European Community due to the heavy ecological and economical damage it has inflicted on Atlantic salmon (Salmo salar) parr populations. Rainbow trout (Oncorhynchus mykiss) is susceptible to G. salaris and can act as a suitable carrier host and, consequently, its trade in EU territory is restricted in relation to the status of ‘‘recognized free’’ zones. Despite the economic importance of rainbow trout farming in Italy, information on the Italian gyrodactylid fauna is lacking and prior to this study, G. salaris had not been officially reported. During a routine health examination of farmed rainbow trout stock throughout Central and Northern Italy in 2004–2005, five fish farms were found to be infected with G. salaris alongside three other gyrodactylids. Morphological and molecular characterisation confirmed the presence of G. salaris, Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999 and Gyrodactylus derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007, while Gyrodactylus truttae Glasser, 1974 was identified by morphological analysis only. The findings from this study extend the distribution of G. salaris within Europe and highlight the importance of the rainbow trout trade in its dissemination.

Comunicación presentada en 17th International Conference on Diseases of Fish and Shellfish, celebrada en Las Palmas, España, del 7 al 11 de septiembre de 2015, Turbot (Psetta maxima) is currently one of the most promising species for European mariculture but although most of the critical aspects threatening production have been optimized, transmissible diseases may still represent a limiting factor. In Autumn 2014 two batches of 5 turbot (mean weight 305g) caged in northern Adriatic Sea and showing melanosis, exophthalmos and skin/fin lesions were subjected to bacteriological and parasitological examination. In addition, visceral organs of twenty fish from the same two cages were fixed directly on the field in 10% buffered formalin. Necropsy showed in all specimens pale-yellow liver and intestinal distension with abundant mucous contents. From the fish owing to one batch ascites was also observed and Vibrio scophthalmi was isolated at the bacteriological exam. Routine parasitological examination did not reveal parasites in any organs except for the intestines, where developmental stages of unidentified parasites were observed. In order to identify the parasite, portions of intestine from all fish were subjected to DNA extraction followed by amplification of 18S rDNA portions. BLAST search of the sequences permitted to ascribe the parasites to the genus Cryptosporidium. In the histopathological study, a mild-to-severe infection by cryptosporidia was detected in the intestine of all fish from both cages. Epicytoplasmic stages of parasites lining the luminal surface of the epithelial cells were observed, generally with the presence of intra-epithelial sporogonic stages located deeply within the enterocytes layer. In some areas, oocysts were present within vacuolated areas in the mucosa, with diffuse necrosis, sloughing off the epithelium and loss of the enterocytes brush border; lamina propria and submucosa were oedematous. Inflammatory response involving mainly lymphocytes and, in heavy infections, rodlet cells was noticed. In some fish also the stomach was affected.Severe pathological changes were mainly observed in relation to the presence of numerous intra-epithelial oocysts. The histopathological findings here described are similar to those already reported in farmed turbot in Spain, due to Cryptosporidium scophthalmi. In our case the source of infection could not be assessed since fish had been introduced into the cages from abroad several months before. The role of wild carrier fish cannot be ruled out.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.

Over recent decades, parasitic diseases have been increasingly considered a sanitary and economic threat to Mediterranean aquaculture. In order to monitor the distribution of parasites in cultured marine fish from Italy and study their pathogenic effects on the host, a three-year survey based on parasitological and histopathological exams was carried out on 2141 subjects from eleven fish species and coming from different farming systems (extensive, intensive inland farms, inshore floating cages, offshore floating cages and submersible cages). A number of parasitic species was detected, mostly in European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata), mullets (Chelon labrosus, Mugil cephalus, Liza ramada) and sharpsnout sea bream (Diplodus puntazzo), with distribution patterns and prevalence values varying in relation to the farming system, in-season period and size category. The epidemiology and pathological effects of the parasites found during the survey are discussed.

The distribution of Sphaerospora dicentrarchi Sitja-Bobadilla et Alvarez-Pellitero, 1992 and S. testicularis Sitja-Bobadilla et Alvarez-Pellitero, 1990, myxozoan parasites of European seabass Dicentrarchus labrax (L.), was investigated in different farming systems in Italy. In total, 1406 fish were examined. High S. dicentrarchi prevalence was observed in all the farming systems involved in this survey (extensive farms: 51.5%; intensive farms: inland 59.6%, inshore floating cages 76.2%, offshore floating cages 41.6%) except for submersible cages (7.4%). S. testicularis was detected only in nine male fish from two intensive farms. The epidemiology and pathological effects of the parasites are discussed.