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Viral nervous necrosis (VNN) otherwise known as viral encephalopathy and retinopathy (VER), is the most threatening infectious disease in Mediterranean aquaculture. VNN is caused by the nervous necrosis virus (NNV) a bisegmented ssRNA+ virus included in the genus Betanodavirus, family Nodaviridae. NNV genome consists of two molecules named RNA1 and RNA2, which encodes for the RNA-dependent RNA polymerase and the coat protein, respectively. Four genotypes of NNV have been so far described: RGNNV, SJNNV, BFNNV, TPNNV (Sahul Hameed et al., 2019). Furthermore, reassortant strains have emerged from the reassortment between the RGNNV and the SJNNV genotypes and named RGNNV/SJNNV and SJNNV/RGNNV according to the RNA1 and RNA2 origin. Currently, several NNV strains are co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant (Bandin and Souto, 2020; Volpe et al., 2020). So far, identification of NNV genotype and reassortant strains is based on amplification, sequencing and phylogenetic analysis of both viral genome RNA molecules requiring time and expertise. As viral identification is an essential information for the management of the disease in the field (Toffan et al., 2021), the development of an easy and affordable method to genotype betanodaviruses was developed in the framework of the H2020 Peformfish Project, an industry-driven project targeting industry defined priorities.&nbsp

Introduction Monogenean outbreaks threaten the health of farmed fish stocks and severely affect aquaculture production. The meagre Argyrosomus regius is a promising species for diversification in Mediterranean fish farming: in the wild, this species is known to host a variety of ectoparasites, however reports of infection and pathology in farmed fish are rather infrequent. The present investigation reports the occurrence of an outbreak of monogenean infection in A. regius broodstock, with identification of the parasite species involved and associated histopathological findings. Methodology During a mortality outbreak registered in a meagre broodstock batch, parasites were isolated from gills and skin and preserved in 70% ethanol and 10% buffered formalin. Gill samples were also fixed in 10% buffered formalin for histology. The parasites were subjected to morphological analysis in light and scanning electron microscopy, and to molecular analysis amplifying and sequencing the complete ITS and partial 28S rDNA. Results and Conclusions Two monogenean species were identified, based on their morphology: the calceostomatid Ktariella polyorchis, reported for the first time in farmed A. regius, and the diplectanid Diplectanum sciaenae, already reported from A. regius broodstock in Spain. K. polyorchis were mainly found on the skin, mainly along the dorsal part of the body and tail, and in the oral cavity, while D. sciaenae were recovered from the gills. At histological level, the presence of D. scienae was associated to severe gill damage with diffuse epithelial hyperplasia and sloughing off in the lamellae, focal necrosis, inflammatory infiltration, hyperemia, telangiectasias and hemorrhages. Histological lesions were similar to those observed in previous reports and confirmed D. scienae as health-threatening parasite of meagre. Our findings suggest a role of both K. polyorchis and D. sciaenae as pathogenic parasites of A. regius farmed in the Mediterranean area, although at the moment they have been reported only from broodstock. In addition, we provide the first sequence data for these two monogenean species, as a further diagnostic tool for their correct taxonomical identification in possible future outbreaks.

submitted to the 20th International Conference on Diseases of Fish and Shellfish. The abstract explains the authors' set up ofa rapid identification method for nervous necrosis virus (NNV) variants circulating in Southern Europe, as conducted through the project PerformFISH.

INTRODUCTION. Monogeneans of the Family Calceostomatidae Parona & Perugia, 1890 parasitize different species of Sciaenids worldwide: among these, the calceostomatid Ktariella polyorchis Vala & Euzet, 1977 has been described infecting the gills of wild meagre Argyrosomus regius (Asso, 1801) caught off Tunisia and Egypt. The present investigation reports for the first time the occurrence of K. polyorchis in broodstock of A. regius from a Croatian farm. MATERIALS AND METHODS. At the farm, gills samples and skin scrapings were collected from heavily infected fish and preserved in 70% ethanol and 10% buffered formalin. Ethanol-fixed parasites were subjected to morphological analysis following clarification in Amman’s lactophenol. Before clearing the parasites, a section of the body without diagnostic characters was excised with a sterile scalpel and processed for molecular analysis, amplifying and sequencing the complete ITS and partial 28S rDNA. For observation in Scanning Electron Microscopy (SEM), formalin-fixed specimens were dehydrated in a graded ethanol series, critical point dried and sputter coated with gold-palladium. RESULTS AND CONCLUSIONS. Although being reported mainly from the gills, in our samples K. polyorchis were mainly found on the fins and skin, while the gills were infected with another monogenean species, the diplectanid Diplectanum sciaenae Van Beneden & Hesse, 1863; this finding may indicate a possible displacement of K. polyorchis in concurrent heavy infections with D. sciaenae. Originally assigned to the genus Calceostoma Van Beneden, 1958 based on the morphology of the anterior region and of the armature in the haptor, K. polyorchis was subsequently assigned to a separate genus based on the morphology of the male reproductive system. Our study adds further morphological information, providing the first detailed description of the parasite surface by SEM analysis, and the first molecular data, useful in future taxonomical and phylogenetic studies.

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.

Antibacterial therapy is still in many cases the only way to control bacterial disease outbreaks, with relevant economic issues. Nevertheless, this necessity should also be well balanced with other relevant aspects such as suitability, efficacy and refinement of the treatments but also with consumer and environmental welfare. With this aim, the literature pertaining to the use of antibacterials (i.e. oxytetracycline, oxolinic acid, flumequine and potentiated sulphonamides) in Mediterranean farmed European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) was reviewed and addressed. Knowledge of drug pharmacokinetics along with the related legislation is also presented. The main criteria, technical aspects and constraints affecting the design of an appropriate antibacterial therapy are also discussed. An evaluation of available bibliography revealed the existence of considerable information on several registered antibacterials, while it is limited for others. Typically, minimum inhibitory concentrations (MIC) have been used as a reference for antibacterial selection. However, the methodologies used for MIC assessment require refinement and more sophisticated data such as epidemiological cut‐off breakpoint values. Due to the characteristics of farming systems, antibacterials are mostly delivered through medicated feeds. The large number of production units and number of fish per unit, together with a limited timeframe margin for efficient therapy, makes Mediterranean gilthead seabream and European seabass, one of the best examples where the metaphylactic concept has to be considered in aquatic medicine. The information presented in this review should guide future action taken to fulfil research gaps and promote effective and prudent antibacterial practices.

The European eel (Anguilla anguilla) is a catadromous species evaluated as Critically Endangered (CR) by IUCN based on the significant population decline occurred in the last 30 years. The threats on this species are numerous, among these, the presence of the nematode Anguillicoloides crassus and the infection caused by rhabdovirus EVEX (Eel virus European X) represent the major causes of reduction efficiency in eel’s swimming performances, compromising the migration capacity of the species. For the purpose to carry out an experimental test regarding the artificial spawning of 56 this species, some specimens of A. anguilla were captured from some watercourses in Sardegna Region. A few days later, some morbidity event occurred with the appearance of clinical signs as lethargy and hemorrhagic skin lesions of lateral line, fins and head. For this reason, between 2016 and 2017, 53 individuals of A. anguilla were sampled from different watercourses of Sardegna Region. In particular, 20 from Rio Pramaera (Municipality of Lotzorai, OG), 13 from Rio Barca (Municipality of Alghero, SS) and 20 from Flumedosa River (Municipality of Villaputzu, CA) in order to verify the possible presence of the virus in wild populations. The virological analysis (cell lines and PCR end point) were negative for all samples. Nevertheless, it is important to carried out a health monitoring on wild population to investigate the etiological role and the distribution of the virus in wild population of eels.

The rising global temperature and sea-level have led to concern about the increase of parasites at our latitudes. Ecological studies on parasitism is forcibly restricted to short-time intervals (months/years). The Holocene fossil record can offer a quantitative archive of ecological responses to geological short (102-103 yr) but societally relevant past climate transitions. This study investigates infestation frequency (prevalence) of trematode flatworms attributed to Gymnophallids through the last 9ky in modern and Holocene brackish settings. These parasites typically display a complex lifecycle with three hosts. It is usually in the second host that gymnophallids encyst in a latent stage of metacercaria and induce the active growth of oval pits on the interior of the shell. These pits are preserved in the fossil record, thus providing a proxy for past parasitic dynamics. We focused on the brackish bivalve Abra segmentum due to its abundance in Holocene brackish settings. A sampling campaign was performed in the lagoons of Piallassa Baiona, Saline di Cervia (Emilia Romagna) and Lesina (Apulia). As for Emilia Romagna sites, ~200 specimens have been collected from June to July 2017. In Lesina, ~100 specimens were collected in the winter of 2016 and ~200 specimens in the following summer. Each specimen was examined to detect the presence of sporocyst, cercariae and metacercariae of digenean trematodes. The soft tissues of infected clams have been preserved in ethanol 70% and photographed with a digital camera at different magnification (4x and 10x) for morphological and molecular identification. The shells of all bivalves (modern and fossil) were observed with a stereomicroscope to detect the presence of parasite traces. In modern brackish setting parasite prevalence, when restricted to samples with only metacercariae, shows a low spatial variability. The mean prevalence is comparable among Lesina and Saline di Cervia (21%±5%, and 20%±5%, respectively) and lower in Piallassa Baiona (7%±3%). At seasonal scale, Lesina samples collected in the winter show a reduced prevalence respect to the summer ones, attaining a maximum value of 6%. On the contrary, across Holocene brackish deposits single sample infestation, range from 6% to 70% and display a significant temporal variation along core (that is, outside the 95% confidence bound estimated via randomization). Significantly elevated infestations are recovered proximity to previously documented flooding surfaces. Whereas, non-significantly prevalence estimates (i.e., within 95% confidence bound) all occurred within the sedimentary units bounded by surfaces that testify past floods. In addition, measurements of the pits on the living bivalve shells are smaller and morphologically less pronounced (shallow) than the fossil counterparts. Finally, the average infestation (pits) in the brackish deposits during phases of rapid sea level rise (flooding surfaces), is significantly higher (~57% ± 4%) than the highest prevalence recorded in modern settings (~32% ± 4%). This result hints a possible association between significantly elevated prevalence and centennial scale flooding events and support the link between sea-level rise and increasing parasite activity. The recognition of the link between parasite prevalence and past sea-level rises provides us with an important reference framework for assessing near-future parasite related threats ignited by global warming.

A complete morphological description, supported by molecular data, of Clinostomum detruncatum metacercariae is reported in this study. The metacercariae were found infecting Synbranchus marmoratus from the Paraná River, municipality of Guaíra, Paraná State, Brazil. The taenioidean uterus is the main morphological characteristic of this species. Both Neighbour-Joining (NJ) and Maximum Likelihood (ML) tree is showed 2 clear separate clusters grouping together the species from the Palearctic region (Clinostomum complanatum, Clinostomum cutaneum, Clinostomum phalacrocoracis, Clinostomum philippinense) and from the Nearctic/Neotropical regions (Clinostomum marginatum, Clinostomum tataxumui, C. detruncatum, Clinostomum sp. 7). The latter clade is divided into 2 subclades grouping species from North America and Mexico (C. marginatum and C. tataxumui), and from Brazil (C. detruncatum and Clinostomum sp. 7).

Scopo primario del presente studio è stato quello di determinare la diffusione di larve di nematodi Anisakidae del genere Anisakis in specie ittiche, in primis alici e sardine, pescate in diversi areali del Mar Adriatico centro-meridionale, comparando i risultati con i dati disponibili per gli areali adriatici settentrionali e valutando le eventuali correlazioni con fattori di tipo ambientale ed ecologico. I risultati ottenuti nel corso di questo studio hanno mostrato, su 2.332 pesci esaminati in totale e rappresentati da diverse specie ittiche di interesse commerciale provenienti da areali di pesca del mar Adriatico centrale e meridionale (Ancona, S. Benedetto del Tronto, Manfredonia, Molfetta e Lecce), una prevalenza totale di 8,1% per larve di nematodi Anisakidae appartenenti al genere Anisakis, mentre il 27,2% è risultato positivo per larve di Hysterothylacium sp. Le prevalenze riscontrate per Anisakis spp. nelle diverse specie ittiche hanno presentato valori differenti: 1,9% nelle sardine (Sardina pilchardus), 8,6% nelle alici (Engraulis encrasicolus), 17,1% nei naselli (Merluccius merluccius), 44,4% nei suri (Trachurus trachurus), 60,7% nei lanzardi (Scomber colias) e 66,6% negli sgombri (S. scombrus), con rilevanti differenze in relazione all’areale di pesca soprattutto quando comparati ai dati esistenti per le popolazioni ittiche provenienti da areali settentrionali del Mar Adriatico. Le differenze rilevate sono state messe in relazione con le caratteristiche idrologiche, morfologiche ed ecologiche che contraddistinguono i diversi areali adriatici e lo suddividono in due diversi ecosistemi, le cui proprietà incidono in larga misura sulla composizione della comunità faunistica, che include i principali ospiti di Anisakis e si riflette anche sulla sua distribuzione nel tempo e nello spazio. Il Mar Adriatico può essere infatti diviso in una porzione settentrionale definita come un'area costiera neritica ed eutrofica per i fondali poco profondi e i numerosi fiumi dell'Italia settentrionale che immettono abbondante acqua dolce ricca di nutrimento al suo interno, ed una porzione centro-meridionale identificata come un ecosistema di tipo oceanico ed oligotrofico, in quanto i fondali sono molto profondi e la ridotta presenza dei fiumi rende le acque scarse di nutrimento, ma con concentrazione salina elevata. Queste caratteristiche influenzano fortemente la distribuzione e l'abbondanza di molti degli ospiti coinvolti nel ciclo biologico di Anisakis, in particolare quelli intermedi (crostacei eufasiacei) e definitivi (cetacei), portando ad importanti differenze anche nella diffusione degli stadi larvali del parassita nelle popolazioni ittiche marine. Main aim of this study was to determine the distribution of larvae of Anisakidae nematodes owing to the genus Anisakis in fish, especially sardines and anchovies, caught in different central-southern fishing grounds of the Adriatic Sea, comparing the results with the data available for the northern Adriatic areas and evaluating possible correlations with environmental and ecological factors. The results obtained from this survey showed, out of 2332 fish examined that were composed of several species of commercial interest caught in fishing grounds of the central-southern Adriatic Sea (Ancona, S. Benedetto del Tronto (AP), Manfredonia (FG), Molfetta (BA) and Lecce), an overall prevalence of 8,1% for larvae of Anisakidae nematodes owing to the Anisakis genus, while 27,2% was positive for Hysterothylacium sp. larvae. Prevalence values of Anisakis sp. were different in the several fish species examined: 1,9% in sardines (Sardina pilchardus), 8,6% in anchovies (Engraulis encrasicolus), 17,1% in European hakes (Merluccius merluccius), 44,4% in Atlantic horse mackerels (Trachurus trachurus), 60,7% in Atlantic chub mackerels (Scomber colias) and 66,6% in Atlantic mackerels (S. scombrus), with notable differences among fishing grounds in particular when compared to the data available for fish populations from northern Adriatic Sea. The differences have been linked with the hydrological, morphological and ecological features that distinguish the different parts of the Adriatic Sea and divide it into two main ecosystems, whose properties affect at a larger extent on the composition of faunal communities including the main hosts of Anisakis and reflect on its distribution in time and space. In fact, the Adriatic Sea can be divided in a northern portion defined as a coastal neritic and eutrophic area for its shallow waters and for the presence of many Italian rivers that enter freshwater rich in nutrients, and a central-southern portion which is as an ecosystem oceanic and oligotrophic characterized by higher depth and salinity, and reduced nutrient loads from rivers. These features strongly influence the distribution and abundance of many of the hosts involved in the biological cycle of Anisakis, particularly intermediate hosts (euphasiacean crustaceans) and definitive hosts (cetaceans), leading to significant differences also in the distribution of the larval stages of the parasite in marine fish populations.

The authors would like to make a clarification regarding thestatement “To our knowledge, no zoonotic transmission fromhorses has been reported before”, following the recognition of atleast three previous papers (Choen and Snowden, 1996; Konkleet al., 1997; Okhuysen et al., 1999) in which cases of cryptosporid-iosis in veterinary students after contact with foals had been cited.In particular:Choen and Snowden (1996), in a “continuing education article”about Cryptosporidial diarrhea in foals, reported: “Recently, veteri-nary students exposed to foals with cryptosporidial diarrhea at ourcollege developed parasitologically confirmed intestinal cryptosporid-iosis”.Konkle et al. (1997) described nosocomial transmission of Cryp-tosporidium in a veterinary hospital and stated that the source ofinfection had been a calf that spread Cryptosporidium parvum infec-tion to a pony, an arabian foal (probably the source of infection forthe vet students) and a llama.Okhuysen et al. (1999), in a study dealing with the virulence ofthree distinct C. parvum isolates, reported: “The third isolate, desig-nated TAMU, was collected from a veterinary student who was exposedduring necropsy of an infected foal”.In all the above-mentioned papers, the transmission from foalsto humans was not explicitly defined in the abstract and was notconsidered in the discussion. Furthermore, PubMed search usingthe keywords ‘Cryptosporidium’ and ‘foals’ failed to identify thesepapers The authors thank the reader that pointed out the omission.Other studies applying sub-genotyping, although not describ-ing cases of zoonoses, have indicated that C. parvum causing foaldiarrhea is often indistinguishable from the parasites isolatedfrom local human cryptosporidiosis cases (Grinberg et al., 2008,2009) suggesting that infected foals represent a potential source ofzoonotic cryptosporidiosis.The authors deem necessary to draw the readership’s attentionto these papers in order to remove any possible doubt about thepublic health hazard posed by infected foals.

The present paper shows the preliminary data about the identification of Eustrongylides sp. larvae found in fish of the Trasimeno lake (Umbria) during 2015-2016. 100 specimens have been examined, 65 perches and 35 bleak. Visual examination has revealed larvae of nematodes in the musculature of 11 (16.9%) perches and 5 (14.3%) bleak, with an average infestation intensity of 1 and 2 parasites/host respectively. 10 larvae were subjected to morphological study, which allowed the identification as L4 larvae of Eustrongylides sp. Molecular analyses are underway in order to identify the species of the nematodes. Since the potential zoonotic role of Eustrongylides sp. and the growing tendency to consumption of raw or undercooked fish, is extremely interesting to study the epidemiology of the parasite not only in Trasimeno, but also in other lakes, in order to understand the mechanisms of transmission, provide apposite monitoring measures and apply appropriate sanitary measures.

La Malattia Proliferativa Renale (MPR) o Proliferative Kidney Disease (PKD) è un’importante malattia parassitaria dei salmonidi sostenuta da Tetracapsuloides bryosalmonae (Myxozoa, Malacosporea) che viene trasmessa ai pesci mediante spore rilasciate in acqua da briozoi dulciacquicoli. Al momento si ritiene che solo Salmo trutta, Salvelinus spp. e ceppi nord-americani di trota iridea (Oncorhynchus mykiss) rientrino attivamente nel ciclo biologico di T. bryosalmonae, sviluppando ed eliminando spore infettanti per i briozoi, mentre le trote iridee di ceppo europeo ed altri salmonidi del genere Salmo e Oncorhynchus non sarebbero idonei a produrre spore infettanti e rappresenterebbero ospiti a fondo cieco nei quali la malattia si sviluppa in modo molto più grave. Alcuni studi recenti hanno però posto in relazione l’infezione da T. bryosalmonae con il declino delle popolazioni selvatiche di salmone atlantico (Salmo salar) in Norvegia e di trota fario in Svizzera ed in Danimarca. In seguito all’osservazione di una forte diminuzione delle popolazioni di salmonidi selvatici nei fiumi dell’Alto -Adige si è deciso quindi di intraprendere un monitoraggio volto a comprendere l’eventuale ruolo della MPR nel determinismo di tale fenomeno. Le ricerche sono state condotte da aprile a novembre 2013 e sono stati effettuati 4 campionamenti mediante elettrostorditore nei mesi di aprile, giugno, luglio e settembre per ognuno dei fiumi Adige, Passirio, Isarco e Rienza. Sono stati inoltre sottoposti a campionamento tre allevamenti di trote situati in bacini idrografici siti nella Provincia di Bolzano. Nel corso della ricerca sono state esaminate in totale 238 trote selvatiche, di cui 65 S. trutta fario, 70 S. trutta marmorata, 102 soggetti ibridi ed un unico esemplare di trota iridea pescato nel fiume Passirio. Per quanto riguarda gli allevamenti sono stati esaminati 154 pesci, di cui 140 trote marmorate e 14 trote fario. Ogni esemplare campionato veniva sottoposto ad esame anatomo-patologico e da ogni soggetto venivano eseguite sul campo impronte di rene e milza successivamente colorate con la metodica di May-Grünwald-Giemsa; porzioni di rene, milza, fegato, branchie, cervello ed intestino venivano fissate in formalina tamponata al 10% per la conduzione dell’esame istologico. Una porzione di rene posteriore veniva inoltre prelevata da ogni pesce per la diagnosi molecolare mediante PCR. Su tutti gli esemplari esaminati non si sono mai evidenziate lesioni anatomo-patologiche riferibili a MPR. L’esame microscopico delle impronte di rene e milza colorate con MGG e l’esame istologico non hanno evidenziato presenza di stadi extrasporogonici o sporogonici di T. bryosalmonae né lesioni riferibili alla malattia. Le analisi molecolari hanno permesso di evidenziare positività per T. bryosalmonae in 11 (4,6%) delle 238 trote selvatiche esaminate, in particolare in 4 esemplari (2 ibridi, una trota fario ed una trota marmorata) prelevati dal Basso Adige (6,8% sul totale di 59 trote campionate da questo fiume) ed in 7 esemplari (3 trote fario, 3 ibridi e 1 trota marmorata) prelevati dal Basso Isarco (11,7% sul totale di 60 trote campionate da questo fiume). Tutti i salmonidi campionati dai fiumi Passirio e Rienza sono risultati negativi. Per quanto riguarda gli allevamenti, in un solo impianto sono risultate positive 6 (42,9%) delle 14 trote fario esaminate nel mese di novembre. Il riscontro di positività solo mediante PCR potrebbe essere correlata a cariche parassitarie molto basse o ad infezioni precoci. Alla luce dei risultati ottenuti, anche in considerazione delle temperature rilevate nel corso dell’indagine, sempre

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 305 g) caged in northern Adriatic Sea and showing melanosis, exophthalmos and skin/fin lesions were subjected to bacteriological and parasitological examination. 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 bacteriological exam allowed to isolate Vibrio scophthalmi. Parasitological exam did not show presence of parasites in any organs except for intestine where developmental stages of not identifiable parasites were observed. In order to identify them, portions of intestine from all fish were subjected to DNA extraction followed by PCR of the 18S rDNA and sequencing. BLAST analysis of the sequences permitted to ascribe the parasites to the genus Cryptosporidium. At histology the presence of a mild-to-severe infection by cryptosporidia was detected in the intestine of all fish from both cages. Extracytoplasmic stages of parasites adhering to 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, several oocysts were present within vacuolated areas in the mucosa, with diffuse necrosis, epithelial sloughing off 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 presence of numerous intra-epithelial oocysts. The lesions here described are similar to those already reported in farmed turbot in Spain. 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.

Fish-borne parasitic zoonoses have aroused an increasing interest in Italy over recent years for the changing of culinary habits towards an increasing consumption of raw/undercooked/marinated fish, in association with the expansion of international commercial trade in fishery products, globalization and migration flows across world, all factors which have driven to an increased risk of acquiring fish- borne zoonoses. Several zoonotic helminths can be transmitted through consumption of raw and/or undercooked fish products to humans, since fishes actively participate to biological cycles of heteroxenous parasites that involve man as definitive or accidental host, causing different degrees of pathology. Although currently WHO include, with regard to zoonotic fish helminthes, only Opisthorchiidae (Opisthorchis spp. and Clonorchis spp.) in the priority list of the foodborne parasites “that could produce a substantive burden of disease” (Torgerson et al., 2014, Trends Parasitol., 30: 20-26) it is undeniable that other fish parasites such as Anisakid nematodes and Diphyllobothriid cestodes should be taken into account when considering the zoonotic risks linked to European fish products consumption. In fact, according to EFSA Scientific Opinion on risk assessment of parasites in fishery products (European Food Safety Authority, 2010, EFSA J., 8, 1543) all wild fish should be considered at risk of containing any viable zoonotic parasites if these products are to be eaten raw or almost raw, pointing out the need to carry out epidemiological surveys on presence/diffusion of zoonotic parasites in all fishery grounds. Respect to infections due to Anisakid larvae in marine fish, until recently in Italy a lower attention has been devoted to the study of zoonotic helminths in freshwater fish populations, such as in primis the cestode Diphyllobothrium latum and the digenean Opisthorchis felineus. The recrudescence of cases of human Diphyllobothriasis registered in Switzerland, France and Italy during the last decades (Scholz et al., 2009, Clin.Microbiol.Rev., 22: 146-160) and the recent outbreaks of human Opisthorchiasis observed in Italy, with more than 200 autochthonous human cases reported from 2004 up to 2011 (Pozio et al., 2013, Acta Tropica, 126: 54-62), have determined a renewed interest in epidemiology of infections due to zoonotic parasites in freshwater fish. Concerning Diphyllobothriasis, recent surveys pointed out a widespread presence of D. latum plerocercoids in perch (P. fluviatilis) populations of Como Lake and, to a lesser extent, in perch from Iseo and Maggiore Lakes, confirming this species as elective second intermediate host of D. latum in Italy. The occurrence of D. latum plerocercoid larvae was also found in pike (Esox lucius) and burbot (Lota lota), main paratenic hosts of D. latum, coming from the same sub-alpine lakes, while fish from Garda lake did not show D. latum infections (Gustinelli et al., 2013, Atti XIX Conv. SIPI, Siracusa, 75). With regard to Opisthorchiasis, although several species of cyprinids have been described as suitable second intermediate hosts of O. felineus from different endemic countries, so far in Italy only tench (Tinca tinca) has been found positive from Bolsena, Bracciano and Vico lakes (De Liberato et al., 2011, Vet.Parasitol., 177: 67-71; Gustinelli et al., 2011, 15th Conf. EAFP, Split, HR, 424), always with high prevalence and intensities. The consumption of raw perch (“perch carpaccio”) and marinated tench in the positive areas has been identified as the main food source of viable infective stages of D. latum and O. felineus respectively. In the case of O. felineus, it has to be pointed out that the microscopical size of metacercariae make their detection impossible through the visual inspection procedures commonly applied for the purpose of detecting parasites in fishery products, requiring more destructive and time consuming techniques. Anyway, both for Diphyllobothrium plerocercoids and Opisthorchiid metacercariae, morphology is not sufficient for identification at species level needing the support of molecular methods. The analysis of risk factors influencing the epidemiology of these zoonotic helminths indicate the lack of efficient urban wastewater depuration plants around lake shores as the main aspect influencing the maintenance of D. latum cycle, being humans the elective definitive hosts, together with the influence of abiotic parameters such as temperature and oxygen on development of Diphyllobothrium first stages (Wicht et al., 2009, Bull.Eur.Ass.Fish Pathol., 29: 58-65). Concerning O. felineus, the presence of parasitised stray cats, main reservoirs of the infection, and of Bythinia spp. gastropods, suitable first intermediate hosts, seem to be the key factors in the subsistence of this zoonotic helminth described in Italy since XIX century in dogs and cats (Rivolta, 1884, Giorn. Anat. Fisiol. Patol. An., 16: 20–28), although the role of fox and other wild animals as reservoirs and the possible involvement of cyprinids other than tench as second intermediate hosts have to be better studied. Since the maintenance of the complex biological cycles of these zoonotic helminths is influenced by several biotic and abiotic factors, investigating the interplay of these factors may be crucial in next future not only for a better understanding of parasite transmission patterns, but also for predictive studies on presence/ maintenance of zoonotic parasites in different fish species in order to map the risks linked to consumption of freshwater fishery products. At this purpose, further surveys aimed at investigate the possible occurrence of larval stages of other zoonotic helminths such as the Opisthorchiid Metorchis sp. and the Heterophyid Metagonimus sp. in Italian freshwater fish populations should be carried out in order to increase the knowledge on epidemiology of fish-borne zoonotic parasites and to prevent outbreaks of new zoonoses. Furthermore, although so far in Italy the presence of zoonotic helminths has been documented only in wild fish populations, extensive epidemiological surveys should be conducted on parasites of public health importance in farmed fish species, as required by EFSA and by current EU food hygiene regulations, in order to confirm as null or negligible the zoonotic risk linked to consumption of national aquaculture products.

In order to study the first case of emaciative syndrome due to an enteric microsporidian infection in gilthead sea bream farmed in sea cages in Italy, a parasitological and histopathological study have been carried out. Enterosporora nucleophila is a microsporidian parasite of the family Enterocytozoonidae responsible for an emaciative syndrome in farmed gilthead sea bream (Sparus aurata), recently described in Spain. In December 2013 a disease outbreak occurred in sea caged gilthead seabream in Italy, affecting 0+ year fish with very low mortality rate and showing clinical signs such as lethargy, anorexia and pronounced weight loss up to cachexia. Twenty gilthead sea bream (mean weight 32 g) were sent by the farmer to the DIMEVET for diagnostic purposes. All the fish were necropsied, and subjected to parasitological examination. Samples of intestine were fixed at the sampling site in 10% buffered formalin and processed for routine histology. Four μm sections were stained with H&E and Luna, according to AFIP manual, and evaluated for histopathology. Intestinal tissue was also subjected to molecular analysis; a fragment of the 18S rDNA was amplified by PCR and then sequenced. At necropsy the fish showed emaciation, skin and fin erosive lesions, gill necrosis and keratitis. Hindgut showed a moderate dilatation of the lumen in association with the presence of whitish casts. Results of parasitological examinations allowed to detect, besides gill monogeneans and gall bladder myxozoans at low intensities, the presence of many small clusters of minute spores referable to microsporidians in intestinal scrapings and wall fresh mounts. The sequences obtained from infected intestines showed 99.9% identity with E. nucleophila. In all cases a severe mucosal atrophy with total folds flattening was detected; in all histological sections a limited number of mast cells and lymphocytes was found within lamina propria and submucosa (mild enteritis) accompanied by a mild mucous cells hyperplasia. Multifocally within enterocytes and rodlet cells, the nucleus and/or cytoplasm contained central drop-like and clusters of microsporidian spores tightly packed, more evident with Luna stain. In recent years fish microsporidioses have become increasingly important among the factors threatening aquaculture, both for their economic impact and for lack of effective/authorized treatments. The newly described emaciative syndrome caused by E. nucleophila, microsporidian unique in infecting intestinal epithelium of gilthead seabream and showing, in addition to intracytoplasmic, an intranuclear development in this host, may represent an emerging pathology for the Mediterranean mariculture and should be urgently investigated and understood in terms of biology, epidemiology and pathogenesis. In the cases studied the severe mucosal flattening could be interpreted as an effect of a chronic insult, not only related to the microsporidia infection; other concurrent predisposing factors could be then considered into the development of this disease.

La malattia proliferativa branchiale da amebe risul ta uno dei maggiori problemi sanitari negli allevamenti marini di salmonidi in Tasmania ed Aust ralia ed è segnalata in Scozia, Nuova Zelanda, Stati Uniti, Canada, Francia, Spagna, Irlanda, Cile e Norvegia. Gravi episodi di mortalità in trote ir idee ( Oncorhynchus mykiss ) allevate in acqua dolce, conseguenti ad infezioni branchiali da amebe, sono descritti in Danimarca, Polonia, Germania, Stati Un iti e Canada. In quest’ultimo paese la malattia è descritta anche nel salmerino alpino ( Salvelinus alpinus ). Negli ultimi tre anni in alcune troticolture del Tr entino si è manifestata una nuova forma di malattia branchiale in esemplari giovanili ed adulti di trot a iridea, con elevati tassi di mortalità durante tu tto l’anno e con particolare incidenza nel periodo inve rnale. A partire da novembre 2013 fino all’estate 2014 sono stati effettuati prelievi in alcuni allev amenti del Comprensorio delle Valli Giudicarie e dell’alto bacino idrografico del fiume Brenta allo scopo di individuare l’eziologia della patologia. Campioni di trote sono stati sottoposti in laborato rio ad esame necroscopico, parassitologico, istologico e batteriologico. I soggetti con sintomatologia clinica all’esame nec roscopico mostravano branchie caratterizzate da un aspetto proliferativo con ipermucosità, alternanza di aree anemiche e congeste, presenza di noduli biancastri, margini dilatati e porzione degli apici dei filamenti ricurvi e collabiti fra loro. Sulla superficie branchiale detriti di sostanza organica venivano talvolta osservati frammisti a muco. Le lesioni riscontrate a livello anatomo-patologico so no state valutate secondo un indice di gravità. In alcuni campioni l’esame microscopico a fresco ha evidenziato nelle branchie di alcuni soggetti la massiva presenza di elementi rotondeggianti riferib ili ad amebe di dimensioni intorno a 20μm. La semina di porzioni di branchie su piastre di Trypti case Soy Agar preventivamente inoculate con un ceppo di Escherichia coli ha permesso di osservare dopo 24-48 ore numerose a mebe, spesso in replicazione, morfologicamente simili agli elementi riscontrati a fresco negli stessi campioni. Il clonaggio delle amebe non ha avuto esito positivo. All’esame istologico le branchie hanno mostrato mar cata proliferazione dell’epitelio di tutto o parte del filamento, maggiormente nella porzione distale. Si è osservata ipertrofia cellulare e fusione dell e lamelle. I filamenti nei casi più gravi risultavano totalmente coalescenti, talvolta interessando l’in tero arco branchiale. La porzione apicale del filamento iperplastico presentava marcata spongiosi, spesso accompagnata da sfaldamento cellulare. Organismi ri feribili ad amebe sono stati osservati adesi alla superficie dell’epitelio branchiale, più frequentem ente nella porzione apicale. In alcuni casi le ameb e sembravano essere in profondità nel tessuto epiteli ale con reazioni flogistiche diffuse. Frequentemente, nelle aree in proliferazione, si ri levano cavità interlamellari contenenti amebe. I parassiti venivano maggiormente evidenziati con il metodo di colorazione di Giemsa. L’esame batteriologico delle branchie ha messo in e videnza la scarsa correlazione tra presenza batterica e la sindrome branchiale in studio. A dif ferenza della malattia branchiale da amebe descritt a nei salmoni allevati in mare, le conoscenze su ques ta patologia nella trota iridea sono ancora scarse. Pertanto studi approfonditi appaiono necessari per chiarire gli aspetti ezio-patogenetici della malatt ia e per individuare gli eventuali co-fattori di natura ambientale, gestionale e biologica che favoriscono l’instaurarsi della malattia proliferativa branchiale da amebe.

The exposure to parasitic nematodes (Anisakidae) is usually through consumption of contaminated fish, eaten raw, undercooked, or prepared without treatment able to inactivate the larvae infectivity (eg. marinating or cold smoking). Human beings are only accidental hosts that do not allow the normal evolution of the larva. However Anisakids may affect the human health in two ways: by means of a severe intestinal immune response and by an allergic reaction to chemicals released by worms in fish flesh. The objective of our work was to assess the potential risk associated with the consumption of fish from the Ligurian Sea. The sampling was carried out in the Ligurian Sea from October 2010 to February 2013; 902 red mullets were analyzed. Nematodes of the genus Anisakis (subfamily Anisakinae) and Hysterothylacium (subfamily Raphidascarinae) were isolated. Thus, in some fish of the Mediterranean Sea, Anisakids may be a risk for the food safety.

Elasmobranches play a key-role as definitive hosts in the complex life cycle of several parasitic species being at the top of the food chain. Despite their biological importance, studies on the parasitofauna of sharks and rays are rather difficult to be carried out, as most of them are endangered species and specimens collection and examination are sporadic and mainly limited to biodiversity and environmental monitoring research projects. During 2013, in collaboration with the Lab of Marine Biology and Fishery, University of Bologna, located in Fano (Ancona province), Italy, a parasitological survey has been carried out on elasmobranchs caught in the Adriatic sea for stocks evaluation purposes. A total of 184 fish from georeferenced fishing stations and belonging to 10 species have been examined: 50 Galeus melastomus, 43 Scyliorhinus canicula, 34 Raja clavata, 27 Squalus acanthias, 11 Mustelus punctulatus, 8 Mustelus mustelus, 5 Raja miraletus, 3 Myliobatis Aquila, 1 Torpedo maculata, 1 Torpedo marmorata. All fish were weighed, measured and submitted to parasitological examination focused on the gastrointestinal tract. Parasites were cleaned in water and fixed in alcohol 70%, then mounted and clarified in Amman’s lactophenol for morphological identification. Prevalence (P) values, Mean Intensity (MI) and Abundance (A) have been calculated according to Bush et al,. 1997, J. Parasitol., 83: 575-583. All the individuals of the remaining species, Galeus melastomus, Myliobatis aquila, Torpedo maculata and Torpedo marmorata were negative for the presence of parasites. All the digenean trematodes have been referred to one species, Ptychogonimus megastomus (Rudolphi, 1819), already reported in M. mustelus and R. clavata but never described in Mustelus punctulatus. This represents to our knowledge a new host-record. Concerning cestodes, the specimens isolated belongs to two trypanorhinch species, in particolar Nybelinia lingualis in S. canicula and Eutetrarhynchus ruficollis in M. punctulatus, while the tapeworm isolated from S. canicula was not well preserved and impossible to be identified. Nematodes are the most represented endoparasites in our survey as they have been isolated from the intestine of all the six positive fish species. In S. canicula nematodes were identified as Proleptus obtusus, already reported in this fish species, while in R. clavata, S. acanthias, M. punctulatus, M. mustelus and R. miraletus, the parasites isolated seem referable to Pseudanisakis spp., but further morphological analyses need to be carried out to confirm this preliminary study. In addition in S. acanthias and M. mustelus, the presence of Hysterothylacium sp. and Acanthocheilus sp. has been respectively observed. The data collected during this survey give a preliminary sight on the parasitofauna present in elasmobranchs species not easy to be investigated and add new information on the host specificity of some parasitic species,such as Ptychogonimus megastomus, to our knowledge for the first time reported in M. punctulatus. Furthermore identification at species level and morphological data on nematodes such as Pseudanisakis spp., should contribute to improve the knowledge on this poorly known genus.

Etiology, biology, epidemiology and diagnosis of human dirofilariasis are described.

Recentemente in Francia e nelle zone mediterranee della Spagna sono stati segnalati gravi episodi di mortalità in orate allevate in gabbie galleggianti; i soggetti colpiti mostravano anoressia, crescita ritardata, emaciazione, cachessia, anemia generalizzata delle mucose e dei parenchimi. L’indagine istopatologica dei soggetti sintomatici ha rivelato una grave infezione sostenuta da microsporidi non formanti xenomi, Nucleospora-like, nel nucleo delle cellule epiteliali del tratto gastroenterico, nelle rodlet cells e in minor misura anche nella sottomucosa. L’evidenziazione di questi piccoli microsporidi intranucleari nelle sezioni istologiche risulta però complicata; per questo motivo è stata proposta da alcuni autori l’applicazione di una colorazione speciale, Luna, al fine di rendere più facile la loro individuazione. Lo scopo dello studio è stato quello di verificare la presenza/assenza di microsporidi Nucleospora-like in sezioni istologiche di intestino di orate allevate in Italia e nel Mediterraneo. Nel periodo ottobre-marzo 2012/2013 si è proceduto al reperimento dei soggetti di orata sia da allevamenti italiani (Emilia-Romagna, Toscana, Liguria, Lazio) che esteri (Croazia), e di casi di archivio del Dipartimento di Scienze Animali, Sezione di Biologia e Patologia Animale, Università di Udine provenienti da allevamenti italiani (Friuli-Venezia-Giulia, Liguria e Sicilia) ed esteri (Croazia e Tunisia). Gli animali campionati provenivano da allevamenti in gabbia a mare, vasche a terra, o da vallicoltura. Sono state ottenute sezioni istologiche di intestino di 4 µm di spessore, colorate con Ematossilina-eosina e, su 4 soggetti per ciascuna area geografica (totale 28 soggetti), è stata effettuata successivamente la colorazione speciale Luna. In 12 soggetti è stata osservata la presenza di strutture sferoidali intranucleari colorate in rosso intenso compatibili per morfologia con microsporidi Nucleospora-like. Nello specifico, le aree geografiche da cui i soggetti positivi provenivano erano Croazia, Liguria e Tunisia e in tutti i casi si trattava di allevamenti in gabbia a mare. Questo parassita è simile morfologicamente a Nucleospora salmonis, un microsporidio intranucleare dei salmonidi che si localizza nel tessuto linfoide renale causando una linfoblastosi cronica e una condizione simil-leucemica sia in animali allevati che selvatici. Nucleospora salmonis è stata segnalata per la prima volta in salmone reale (Oncorhynchus tshawytscha) ma è noto che è in grado di infettare un ampio range di salmonidi e altre specie ittiche marine. Un’altra specie, Nucleospora cyclopteri, è stata descritta invece in Icelandic lumpfish (Cyclopterus lumpus) selvatici, sempre a carico del tessuto linfoide interstiziale del rene. I risultati ottenuti in questa indagine sono stati confrontati con l’unico lavoro disponibile in letteratura che mostra immagini istologiche, permettendo di ipotizzare una possibile infezione da microsporidi Nucleospora-like. Ulteriori indagini di tipo molecolare (PCR e ibridazione in situ), sono necessarie al fine di confermare definitivamente l’appartenenza dei microsporidi reperiti al genere Nucleospora.

Mullus surmuletus is a species of high commercial interest and represents one of the main targets of artisanal fishing. This species lives in shallow coastal areas, over rocky to sandy bottoms, and recruits in Posidonia oceanica beds. Its particular feeding habit of scouring in the sediment in search of preys makes this fish a susceptible host for several parasites species. Several studies about the parasite fauna of M. surmuletus in Mediterranean sea have been carried out in the last decades, however there is a lack of knowledge about Ionian sea. In the present study, a total of 90 specimens of M. surmuletus were sampled from the Marine Protected Area of Porto Cesareo (Ionian sea) from January to June 2013. Fish were caught by trammel nets and collection sites geo referred. Each specimen was measured and weighted, and then dissected for parasitological examination mainly aimed to investigate the presence of anisakid nematodes. The collected anisakids were then identified, through morphological and molecular analyses, as Hysterothylacium fabri (Nematoda, Anisakidae). The occurrence of different developmental stages of H. fabri was observed in all (100%) of the examined specimens, with infection intensities ranging from 1 to 77 (Mean Intensity 22.86). Although H. fabri has already been reported from M. surmuletus in Mediterranean sea, our results show the highest prevalence so far observed in this host. Ecological and environmental determinants are discussed.