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Nematodes of the genus Dictyocaulus are the causative agents of parasitic bronchitis and pneumonia in several domestic and wild ungulates. Various species have been described in wild cervids, as the case of Dictyocaulus cervi in red deer, recently described as a separate species from Dictyocaulus eckerti. In Italy, information on dictyocaulosis in wildlife is limited and often outdated. In this work, 250 red deer were examined for the presence of Dictyocaulus spp. in two areas of the Italian Alps (n = 104 from Valle d’Aosta, n = 146 from Stelvio National Park), and the retrieved lungworms were molecularly characterized. Lungworms were identified in 23 and 32 animals from Valle d’Aosta and Stelvio National Park, respectively. The nematodes, morphologically identified as D. cervi, were characterized molecularly (18S rDNA, ITS2, and coxI). Consistently, almost all specimens were found to be phylogenetically related to D. cervi. Three individuals, detected from both study sites and assigned to an undescribed Dictyocaulus sp., clustered with Dictyocaulus specimens isolated from red deer and fallow deer in previous studies. Within each of D. cervi and the undescribed Dictyocaulus sp., the newly isolated nematodes phylogenetically clustered based on their geographical origin. This study revealed the presence of D. cervi in Italian red deer, and an undetermined Dictyocaulus sp. that should be more deeply investigated. The results suggest that further analyses should be focused on population genetics of cervids and their lungworms to assess how they evolved, or co-evolved, throughout time and space and to assess the potential of transmission towards farmed animals.

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes.

Oomycetes of the genus Saprolegnia are widespread in freshwater environment and are among the main path- ogens causing economic losses in salmonid aquaculture. Infections by mycotic agents in fish farming are generally considered to result from chronic stress and poor fish condition associated with water quality problems, adverse environmental conditions, frequent/rough/incorrect handling, concurrent infections, physiological changes associated with reproduction and immunocompromised animals. To identify risk factors for Saprolegnia infections in trout and Atlantic salmon farming, longitudinal studies were carried out in different Italian, Spanish, and Scottish fish farms. Prevalence of saprolegniosis and fish mortality were monitored over time and statistically analysed with respect to husbandry and environmental factors. Overall, statistical results by production cycle (trout vs salmon farming) and by country indicate that the prevalence of Saprolegnia may be influenced by peculiarities of the culture system and farming environment. Nevertheless, a specific set of parameters, including lower water temperature, and handling procedures increased Saprolegnia prevalence in all the considered farms. Particularly, in trout farms Saprolegnia infections represented an important contribution to mortality, and prevalence was influenced by water temperature and pH, and by fish density within the tanks. Similarly, temperature and water quality were the main factors influencing the prev- alence of Saprolegnia in Atlantic salmon farms. Moreover, molecular analyses confirmed the role of S. parasitica as the main pathogenic oomycete in trout and salmon farming in the considered countries. The identification of risk factors for introduction and increase of Saprolegnia infection in fish farms will allow the correct design of bio- security and pathogen control strategies

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.

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 fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

The genus Clinostomoides Dollfus, 1950 was erected to accommodate a single worm from Ardea goliath sampled in the Belgian Congo. The specimen was distinguished from other clinostomids by its large size and posterior genitalia. In the following years, metacercariae of Clinostomoides brieni, have been described in Clarias spp. in southern and western Africa. A few authors have referred to Clinostomum brieni, but all such usages appear to be lapsus calami, and the validity of Clinostomoides remains widely accepted. In this study our aim was: position C. brieni among the growing clinostomids molecular database, and redescribe the species with emphasis on characters that have emerged as important in recent work. We sequenced two nuclear (partial 18S and ITS) and one mitochondrial marker (partial cytochrome c oxidase I) and studied morphology in metacercariae from hosts and localities likely to harbour the type species (Clarias spp., Democratic Republic of the Congo, South Africa). Phylogenetic analysis shows C. brieni belongs within Clinostomum Leidy, 1856. We therefore transfer C. brieni to Clinostomum, amend the diagnosis for the genus Clinostomum and provide a critical analysis of other species in Clinostomoides, all of which we consider species inquirendae, as they rest on comparisons of different developmental stages.

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.

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms., European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429

Introduction: Marine Protected Areas (MPAs) represent a privileged observatory to monitor disease outbreaks in the marine environment. Recently, several episodes of mass mortality events (MME) of the noble pen shell Pinna nobilis occurred in different parts of the Mediterranean Sea. In the present work we report a MME occurred in 2018, involving a protected population of P. nobilis within the MPA of Porto Cesareo (Southern Italy, Ionian Sea) which had been previously characterized and monitored within the framework of the “Marine Strategy” Directive (2008/56/EC). Methodology: Data on abundance and size structure of the P. nobilis population had been collected since September 2017 through scuba diving visual census. Following a MME occurred in June 2018, one moribund specimen was sampled by professional divers of Porto Cesareo MPA within the reserve boundaries. The specimen was subjected to a complete diagnostic exam, including parasitological, histopathological and molecular analyses. Results: The mortality affected 100% of the protected population. The diagnostic exams allowed to detect the presence of Haplosporidium pinnae, identified through amplification and sequencing of partial 18S rDNA region, in the intestine and digestive gland. Histopathological analyses revealed the presence of haplosporidian-like protozoa in different life cycle stages within the digestive gland. The epithelium of digestive tubuli showed a diffused degeneration with extended infiltrate of brownish pigment referable to brown cells, which replaced almost completely the decaying glandular tissue. Conclusion: Haplosporidium pinnae has been already indicated as responsible for other MME of P. nobilis occurred in Western and Central Mediterranean Sea. The implementation of routine monitoring programs to assess the presence and distribution of transmissible agents in wild marine environment is an essential step to protect biodiversity. Such procedures are especially relevant within the context of a MPA, and should focus on the identification of sanitary risks that could impair conservation efforts. With respect to H. pinnae, further studies aimed at elucidating the life-cycle of the parasite and its causative role in recent MME of noble pen shell are of primary importance.

13 pages, 6 figures, 2 tables, Coccidian parasites of the genus Aggregata are known to parasitize cephalopods as definitive hosts, however one of the genus members, A. octopiana, has shown an unresolved phylogeny within the same definitive host, the common octopus (Octopus vulgaris). Our study represents a large-scale investigation aimed at characterizing morphological traits and phylogeny of A. octopiana isolated from O. vulgaris inhabiting three distinct geographic areas of the central Mediterranean: The Adriatic, Ionian and Tyrrhenian Seas. The morphology of sporogonic stages of the parasite in octopus tissues was assessed by light and electron microscopy; molecular characterization has been carried out using the 18S rRNA locus. Our results support the hypothesis that two morphologically and genetically different A. octopiana infect O. vulgaris in the investigated areas of the Mediterranean Sea. Additional nuclear and mitochondrial markers for Aggregata should provide further information and better resolution of its phylogeny, This work has been initiated through the networking initiatives of the COST Action FA1301 (CephsInAction) and thanks to the support of a COST FA1301-Short Term Scientific Mission (P. Tedesco, from Italy to Vigo, Spain). The study has been supported through RITMARE Flagship project (MIUR-SZN) to PT and GF. G. Catanese would like to thank INIA for the INIA/CCAA research contract program

Lopes, Vanesa... et al.-- 8 pages, 1 table, Cephalopods are the sole invertebrates included in the list of regulated species following the Directive 2010/63/EU. According to the Directive, achieving competence through adequate training is a requisite for people having a role in the different functions (article 23) as such carrying out procedures on animals, designing procedures and projects, taking care of animals, killing animals. Cephalopod Biology and Care Training Program is specifically designed to comply with the requirements of the “working document on the development of a common education and training framework to fulfil the requirements under the Directive 2010/63/EU”. The training event occurred at the ICM-CSIC in Barcelona (Spain) where people coming from Europe, America and Asia were instructed on how to cope with regulations for the use of cephalopod molluscs for scientific purposes. The training encompasses discussion on the guidelines for the use and care of animals and their welfare with particular reference to procedures that may be of interest for neuroscience. Intensive discussion has been carried out during the training sessions with focus on behavioural studies and paradigms, welfare assessment, levels of severity of scientific procedures, animal care, handling, transport, individual identification and marking, substance administration, anaesthesia, analgesia and humane killing, The COST Action FA1301 CephsInAction, and the Association for Cephalopod Research supported the Training School “Cephs Biology and Care (CBC-ASP2016)” and the participation of 10 Trainers and 14 Trainees. Sponsorships from Leica and Tecniplast has also been granted

Background Betanodaviruses are the causative agents of Viral Encephalopathy and Retinopathy (VER). To date, more than 50 species have proved to be susceptible and among them, those found in genus Epinephelus are highly represented. Clinical disease outbreaks are generally characterized by typical nervous signs and significant mortalities mainly associated with aquaculture activities, although some concerns for the impact of this infection in wild fish have been raised. In this study, the authors present the first documented report describing an outbreak of VER in wild species in the Mediterranean basin. Case presentation In late summer - early winter 2011 (September-December), significant mortalities affecting wild Dusky grouper (Epinephelus marginatus), Golden grouper (Epinephelus costae) and European sea bass (Dicentrarchus labrax) were reported in the municipality of Santa Maria di Leuca (Northern Ionian Sea, Italy). The affected fish showed an abnormal swimming behavior and swollen abdomens. During this epizootic, five moribund fish showing clear neurological signs were captured and underwent laboratory investigations. Analytical results confirmed the diagnosis of VER in all the specimens. Genetic characterization classified all betanodavirus isolates as belonging to the RGNNV genotype, revealing a close genetic relationship with viral sequences obtained from diseased farmed fish reared in the same area in previous years. Conclusion The close relationship of the viral sequences between the isolates collected in wild affected fish and those isolated during clinical disease outbreaks in farmed fish in the same area in previous years suggests a persistent circulation of betanodaviruses and transmission between wild and farmed stocks. Further investigations are necessary to assess the risk of viral transmission between wild and farmed fish populations, particularly in marine protected areas where endangered species are present.

Gli oomiceti del genere Saprolegnia hanno un’ampia diffusione in ambiente dulciacquicolo e causano importanti perdite economiche nell’allevamento di specie ittiche d’acqua dolce, in particolare salmonidi. Obiettivo della presente ricerca è stato quello di indagare i fattori di rischio in grado di favorire l’insorgenza della saprolegniosi in troticoltura mediante uno studio epidemiologico longitudinale. Lo studio è stato condotto in 10 troticolture (5 in Italia e 5 in Spagna); gli allevamenti sono stati selezionati sulla base della presenza documentata di fattori gestionali considerati rilevanti in riferimento alla saprolegniosi (spremitura dei riproduttori, vaccinazione, selezione, frequenti cambi di vasca, ecc.). Le specie considerate nello studio sono state la trota iridea Onchorhynchus mykiss, la trota marmorata Salmo marmoratus e la trota fario Salmo trutta. In ciascun allevamento sono state condotte da Febbraio 2017 a Gennaio 2018 visite periodiche durante le quali le vasche oggetto di studio sono state controllate periodicamente per valutare la presenza di lesioni riferibili a saprolegniosi e sono state raccolte informazioni relative a fattori gestionali di diverso tipo, presenza di infezioni concomitanti e mortalità. Durante ciascuna visita sono stati inoltre registrati i paramentri ambientali (pH, temperatura e ossigeno disciolto). Il monitoraggio in continuo della temperatura dell'acqua è stato effettuato posizionando un data logger all’interno di ciascuna vasca selezionata. Le specie di Saprolegnia circolanti sono state caratterizzate mediante identificazione molecolare dei ceppi isolati da trote campionate in ciascun allevamento nel corso della prima visita. I dati raccolti sono stati analizzati statisticamente con l’obiettivo di correlare prevalenza di saprolegniosi e mortalità a diverse pratiche gestionali e fattori ambientali. Tutti gli isolati da trote con segni clinici appartenevano alla specie Saprolegnia parasitica. I risultati delle analisi statistiche indicano che la saprolegniosi rappresenta un’importante causa di mortalità nelle troticolture italiane e spagnole oggetto di studio. Con riferimento ai parametri ambientali, è stato evidenziato un contributo importante della temperatura e del pH dell’acqua. In particolare, si è confermato come la prevalenza di Saprolegnia sia mediamente più alta durante i mesi freddi (temperatura dell’acqua inferiore a 10°C) evidenziando l’influenza positiva di valori di pH alcalini. Riguardo agli aspetti gestionali, diverse tipologie di “handling” sono risultate fattori di rischio importanti. In uno degli allevamenti monitorati, è stato rilevato un incremento dei valori di prevalenza per saprolegniosi in seguito a vaccinazione intraperitoneale anche a temperature dell’acqua relativamente elevate (14°C). Inoltre, è stato osservato come frequenti cambi di vasca siano associati a valori di prevalenza più elevati anche quando, in seguito allo spostamento, la densità all’interno delle vasche viene ridotta. L’identificazione dei fattori di rischio per saprolegniosi è uno step fondamentale per una corretta progettazione ed implementazione di misure di biosicurezza e strategie di gestione finalizzate al controllo di questa parassitosi negli allevamenti di salmonidi.