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Negli anni ’80-90 del secolo scorso sono stati segnalati i primi casi di infezioni da cocchi Gram positivi in pesci d’allevamento nei paesi che si affacciano nel bacino del Mediterraneo; la lattococcosi e la vagococcosi sono le patologie che hanno avuto una maggiore diffusione sul territorio europeo, anche se con una prevalenza ed un grado di diffusibilità e patogenicità alquanto diverse. I salmonidi rappresentano il gruppo dei pesci allevati che maggiormente vengono colpiti da questi germi. Sebbene queste affermazioni siano esatte, storicamente si conosceva già una patologia ascrivibile al gruppo dei cocchi gram positivi, che colpiva generalmente in modo sporadico e con una mortalità molto ridotta, soprattutto i grandi riproduttori, stabulati in allevamenti con acque fredde nelle zone pedemontane: la carnobatteriosi, sostenuta da Carnobacterium piscicola ora denominato C. maltaromaticus. Si conoscono 9 specie di germi appartenenti a questo genere, ma solo due di questi sono relativamente frequenti in ambiente acquatico (C. maltaromaticus e C. divergens); le altre specie sono presenti in ambienti più freddi, nei sedimenti polari, nel permafrost alpino e nell’intestino di pesci artici. Le due specie sopra denominate sono di isolamento relativamente comune soprattutto a livello della flora intestinale di pesci allevati in acque fredde, presenti in tutto il continente europeo, Italia compresa, ma sono stati isolati anche in pesci tropicali d’acqua dolce. Negli ultimi decenni non sono stati segnalati ritrovamenti importanti, causa di patologie nei salmonidi allevati in montagna, perdendo l’abitudine alla loro ricerca ed alla valutazione del possibile ruolo patogeno. L’effetto patogeno sui pesci è stato ampiamente descritto, con presenza di quadro setticemico, peritonite, esoftalmo, accumulo di liquido ascitico in cavità addominale e presenza di emorragie nei diversi organi interni. Questa segnalazione vuole descrivere l’isolamento di germi appartenenti al genere Carnobacterium dall’umor vitreo dell’occhio di salmerini di fonte (Salvelinus fontinalis) pescati in un lago di alta quota senza sintomatologia in atto. Nell’ambito del progetto ALPLA, nell’agosto 2018 sono stati pescati 73 salmerini di fonte dal lago della Balma sottano, sito a circa 2.100 s.l.m. nelle Alpi Cozie, nel comune di Coazze (TO). Tutti i pesci sono stati analizzati, effettuando un esame colturale direttamente da rene, cervello e occhio; il prelievo dall’occhio ha previsto dapprima una disinfezione esterna della superficie mediante un batuffolo di cotone intriso di alcool denaturato, passandolo direttamente sulla cornea; successivamente, con ansa di platino incandescente si è proceduto all’incisione della stessa e successivamente si è eseguito il prelievo direttamente dall’umor vitreo seminando su piastre di Agar sangue. Tutti i soggetti non hanno presentato crescita batterica sui prelievi condotti da rene e cervello, mentre 25 pesci sono risultati positivi al prelievo dall’occhio, con crescita di colonie rigogliose ed in purezza. La successiva identificazione biochimica e tramite Maldi-Tof delle colonie di tutti i soggetti risultati positivi, ha portato all’identificazione di Carnobacterium maltaromaticus (12 isolati) e C. divergens (13 isolati). Questo isolamento chiaramente non è legato ad una condizione patologica, in quanto non era presente né mortalità nella popolazione, né tantomeno una sintomatologia evidente, ma porta ad ipotizzare come questi potenziali patogeni possano rimanere in una sorta di latenza in un organo (cosiddetto “effetto cavallo di Troia”) per poi eventualmente esplicare azione patogena durante episodi di stress che abbassino le difese immunitarie dei soggetti colpiti. Questa possibilità dovrebbe essere maggiormente studiata per verificare tale ipotesi e portare a conoscenza nuovi aspetti sulla patogenesi di tali germi.

La malattia nodulare branchiale (NGD) rappresenta uno dei maggiori problemi sanitari negli allevamenti italiani di trota iridea (Oncorhynchus mykiss). Episodi di NGD sono stati descritti in passato in salmerini alpini (Salvelinus alpinus) e salmoni reali (Oncorhynchus tshawytscha) allevati in acqua dolce. A marzo 2017, durante un monitoraggio di NGD in trote iridee nella Provincia di Trento, sono stati osservati i primi casi di malattia nodulare branchiale nella trota fario. Le trote fario infette provenivano da due allevamenti dove erano contemporaneamente presenti episodi di NGD nelle trote iridee. I pesci, di taglia compresa tra i 20-25 g (vasca 1) e 40-45 g (vasca 2), venivano allevati in vasche a fondo naturale alimentate con acqua di pozzo e sorgente. La temperatura dell’acqua variava da 5,5 a 7,2°C nella vasca 1 e da 8,9 a 11,2°C nella vasca 2. Da ogni vasca sono stati prelevati mensilmente 25 pesci a marzo, aprile e maggio 2017 per un totale di 150 soggetti. Le trote sono state sottoposte ad esami necroscopici ed analisi microscopiche. Le branchie sono state sezionate e fissate in formalina tamponata al 10% per l’esame istologico. In seguito i campioni sono stati processati, inclusi in paraffina, sezionati a 4 μm e colorati con soluzione di Giemsa. Le trote fario osservate in vasca non mostravano né grave sintomatologia respiratoria né alte mortalità tipiche di NGD nelle trote iridee. In 53 pesci su 150 sono state osservate branchie dall’aspetto rigonfio e pallido con presenza di noduli biancastri lungo gli apici dei filamenti. Microscopicamente le branchie colpite mostravano reazioni proliferative a carico dei filamenti con fusione delle lamelle, specialmente nelle porzioni distali. Le lesioni descritte, sia a livello macroscopico che microscopico, erano tipiche delle infezioni da NGD. L’esame microscopico ha permesso di osservare trofozoiti di amebe (di diametro di circa 15 x 20 μm) lungo la superficie dei filamenti iperplastici. Le analisi istologiche hanno evidenziato iperplasia ed ipertrofia dell’epitelio branchiale e delle cellule mucipare, necrosi e presenza di amebe lungo la superficie dei filamenti in 32 trote su 150 campionate. Nonostante l’assenza di gravi sintomatologie respiratorie e di alte mortalità, le alterazioni proliferative delle branchie associate alla presenza di amebe hanno mostrato come anche la trota fario possa essere interessata dalla malattia nodulare branchiale. Ulteriori studi appaiono necessari per chiarire gli aspetti eziopatogenetici della NGD in questa specie ed il suo coinvolgimento nella diffusione della patologia nelle troticolture italiane.

Introduction: Nodular gill disease (NGD) represents one of the most serious pathology affecting freshwater farmed rainbow trout (Oncorhynchus mykiss) in Italy. This disease has also been observed in arctic char (Salvelinus alpinus) and Chinook salmon (Oncorhynchus tshawytscha) cultured in freshwater environment. The first case of branchitis associated with amoebic infection is detected in farmed brown trout (Salmo trutta L.) in March 2017, during an investigation of NGD in rainbow trout in the province of Trento (Northern Italy). Methodology: In a NGD affected rainbow trout farming an outbreak of low mortality occurred in brown trout in one earth tank supplied by spring water with temperature of 6°C. The average weight of the fish was 20-25g. Twenty five fish from the affected tank were collected for necropsy, microscopical and parasitological analysis. Gills were dissected and fixed in 10% neutral buffered formalin solution for histological examination. The samples were dehydrated, embedded in paraffin wax, sectioned (4 μm) and stained with Hematoxylin Eosin and Giemsa solution. Results: The fish did not exhibit the typical respiratory symptoms described in NGD of the rainbow trout. The gill tissue appeared pale and swollen at the necropsy with whitish nodules in 9 out of 25 sampled trout. Microscopically numerous Gyrodactylus spp. were observed on the skin. The affected gills showed a proliferative reaction of some filaments, especially in the distal part. These lesions were typical of NGD infection. The microscopic examination of gill tissue revealed the presence of amoebic organisms (approximately 15 x 20 μm). The histology showed multi-focal epithelial hyperplasia of the gills causing lamellar fusion, mucous cells hypertrophy, cellular necrosis and presence of limited number of amoebae along the surface of the affected filaments in 6 out of 25 collected gills. Conclusions: Despite absence of respiratory symptoms and high mortality rate, gill proliferative lesions associated with amoebae shows that brown trout may be affected by nodular gill disease. Studies are in progress to clarify pathogenesis, biological and environmental determinants of NGD in brown trout and to define the involvement of this species in the spread of the disease in Italian trout farms.

L’anisakiasi è una zoonosi parassitaria di origine alimentare sostenuta da larve al terzo stadio (L3) di nematodi appartenenti alla famiglia Anisakidae ed in particolare al genere Anisakis. Soprattutto in seguito alla recente emergenza di sindromi allergiche da Anisakis, l’EFSA nel 2010 ha pubblicato un parere scientifico sui rischi correlati alla presenza di parassiti zoonotici in prodotti ittici, considerando sicura la pratica d’allevamento del salmone atlantico (Salmo salar) in quanto, se allevati partendo da avannotti prodotti in sistemi chiusi e alimentati sempre con mangime inerte, questi animali presenterebbero un rischio di infestazione da larve di Anisakis trascurabile. Su questa base, il Reg. UE n. 1276/2011, che modifica l’allegato III del Reg. CE n. 853\2004, puntualizza la possibile deroga al trattamento di congelamento, obbligatorio per i prodotti della pesca destinati ad essere consumati sotto forma di preparazioni a rischio (crudi, marinati, affumicati a freddo, ecc.), per i prodotti ittici derivanti da allevamento quando vengono rispettati i criteri definiti dall’EFSA analogamente al salmone atlantico. Appare quindi d’importanza strategica fornire dati scientifici estesi ed attendibili sull’assenza di parassiti zoonotici nei prodotti della maricoltura mediterranea non solo ai fini di una loro migliore qualificazione commerciale ma anche per una più facile applicabilità di tale deroga da parte delle autorità competenti. Nell’ambito delle attività di ricerca previste dal progetto europeo H2020 ParaFishControl “Advanced Tools and Research Strategies for Parasite Control in European farmed fish”, da marzo 2016 a giugno 2017 è stato condotto un esteso monitoraggio parassitologico in spigole (Dicentrarchus labrax) ed orate (Sparus aurata) allevate in Italia. Le attività di campionamento hanno coinvolto 4 allevamenti nazionali, di cui 3 in gabbie galleggianti ed uno in vasche a terra, prelevando almeno 258 esemplari per specie/allevamento suddivisi in 4 campionamenti a cadenza stagionale per un totale di 1040 spigole e 1090 orate. La numerosità campionaria è stata calcolata in modo da ottenere almeno il 95% di probabilità di evidenziare un esemplare positivo con prevalenza >2%. Per la ricerca di larve di nematodi Anisakidae sono stati associati: esame visivo diretto e mediante transilluminatore di visceri e porzioni muscolari, metodo della UV-press dei filetti e digestione con soluzione cloro- peptica del pacchetto viscerale. Tutti gli esemplari di spigola e orata esaminati sono risultati negativi per la presenza di stadi larvali di Anisakis spp. In un unico esemplare di spigola proveniente da un allevamento in gabbia è stata evidenziata la presenza di una larva di Hysterothylacium fabri (nematode non zoonotico) incistata sulla superficie del fegato ed identificata a livello di specie mediante PCR-RFLP. Alla luce della trasmissione di questo parassita a pesci predatori per via trofica, similmente ai nematodi Anisakidae zoonotici, nel prossimo futuro verranno condotte indagini mirate ad identificare i fattori di rischio che hanno condotto all’infestazione da H. fabri in allevamento, seppur in un unico esemplare, ed individuare gli eventuali punti critici da monitorare. I risultati ottenuti da questa indagine indicano comunque come nelle spigole e nelle orate allevate in Italia il rischio d’infestazione da Anisakis sia da ritenersi trascurabile, aprendo la via ad azioni di qualificazione dei prodotti della maricoltura mediterranea.

Meagre (Argyrosomus regius) represents a promising fish species for the diversification in European aquaculture, on the basis of its rapid growth performances. Nevertheless, there are still several bottlenecks for optimization of meagre production, among which the Systemic Granulomatous Syndrome (SGS) is considered a widespread emerging threat. In order to evaluate the impact of SGS on the health of farmed meagre in Croatia, a histopathological investigation has been carried out. A total of 98 meagres were monthly sampled during the period July 2015-May 2016 from three cages with different feeding protocols: commercial pellet, hydrated commercial pellet and frozen sardines. From each sampled fish, portions of gills, stomach and intestine, liver, kidney, spleen and heart were fixed in 10% neutral buffered formalin and processed for histology. The SGS has been observed in the meagre from the 3 cages throughout the period under study. Few days after introduction of 6 g fish from a foreign hatchery, the SGS was already evident, with a disseminated granulomatosis in the kidney, mild granulomatosis in the liver and macrovacuolar hepatic steatosis. No significative differences in the fish from the three different cages have been observed, as well as a worsening of granulomatosis throughout the study period. In general, in all the examined fish the kidney and, to a lesser extent, the liver were the organs principally affected by SGS, with mild to severe granulomatosis, usually disseminated, in some case with coalescent granulomas. In the liver, the observation of macrovacuolar steatosis was constant. Based on histological observation and the few data from literature, SGS in meagre seems to be a pathological condition caused by concurrent factors, such as the presence of a Calcium/ Phosphorus imbalance (high CO2), worsened by the co-occurrence of a particularly susceptible life stage (0+ juvenile) and influenced by cold-water temperatures during winter period.When the ongrowing phase starts with fish showing an already established SGS during the nursery/hatchery phase, it greatly hampers health and productivity of meagre during the following production cycle. An improvement of knowledge on the optimization of diet in farmed meagre and related health issues is necessary to overtake one of the main bottlenecks that still limit the productivity of meagre.

Introduction: In the latest years the nodular gill disease (NGD) is one of the most serious health problem in rainbow trout cultured in Northern Italy. This disease caused by amoebae has been reported to affect salmonids farmed in freshwater environment of Northern Europe and North America. Relatively little is known about epidemiology and pathogenesis of NGD. Methodology: To clarify the possible risk factors related to the occurrence of NGD, a study area (province of Trento - Northern Italy) was identified where an epidemiological investigation at farm level was planned. Data required were collected during onsite visits by a veterinarian. A standardized questionnaire, implemented and tested during the first steps of the project, is being used for data collection. Information on disease status, species stocked, farm activities undertaken, risk factors related to NGD introduction and spread are included in the questionnaire together with data on management practices and biosecurity measures applied at farm level. Anamnestic and clinical information related to the health status of each farm are reported in the questionnaire. To assess distances from case farms identified as hazards and upstream-downstream proximity to other farms, data on the location of farms, stored in a regional web GIS, will be available. A database will be implemented in order to validate, store and analyze the data collected during the project. Data collection started in November 2017 and is still in progress. To understand the pathogenesis of NGD three tanks, respectively from three trout farms located in the province of Trento, were monitored every fifteen days from September 2016 to March 2017. Ten fish from each tank (five samples at the inlet and five at the outlet) were taken during each monitoring phase and subjected to necropsy and to microscopic and histological examination of the gills. Expected results: The epidemiological investigation provides information about the real diffusion of NGD in the province of Trento and identify the factors that may favor the development of disease. The study of pathogenesis could reveal the timing and mode of NGD development.

La saprolegniosi è considerata un’infezione ubiquitaria all’interno dell’ecosistema dulciacquicolo, con diffusione sia nei fiumi che negli allevamenti (Willoughby & Pickering, 1977). L’agente eziologico, Saprolegnia spp., è considerato un patogeno opportunista facoltativo e molto spesso le condizioni di allevamento possono contribuire a creare situazioni favorenti l’instaurarsi dell’infezione nella popolazione. Allo scopo di valutare il potenziale trasferimento di Saprolegnia spp. dai pesci allevati a quelli selvatici e viceversa è stata effettuata un’indagine in 2 allevamenti di trota iridea (in provincia di Vicenza e di Bolzano), in 2 allevamenti di trota fario e marmorata (in provincia di Udine e Pordenone) e nei sistemi idrici tributari ed effluenti dagli stessi allevamenti. Sono stati effettuati due campionamenti: il primo in autunno/inverno e il secondo in inverno-primavera, periodi di maggiore emergenza della saprolegniosi negli allevamenti di salmonidi. Nel corso di ciascun campionamento, da ogni allevamento sono stati prelevate 10 trote, preferibilmente con lesioni riferibili a Saprolegniosi; quando possibile altrettanti pesci, con o senza lesioni, sono stati prelevati nelle acque libere sia monte che a valle di ciascun allevamento. In ciascun sito di campionamento sono inoltre state collocate 5 trappole di rete metallica contenenti ciascuna 7 porzioni di semi di canapa precedentemente sterilizzate (Seymour, 1970; Johnson et al., 2002), che sono stati lasciate in situ per almeno 10 giorni e poi raccolte. Due frammenti di micelio, per quanto riguarda i pesci con lesioni, un pezzetto di pinna dorsale e di branchia per i pesci senza lesioni, e tutti i semi di canapa recuperati dalle trappole sono stati seminati su glucose-yeast extract agar (Min et al., 1994) addizionato di penicillina (6 mg/l) e acido ossolinico (10/mg/l) (Alderman & Polglase, 1986) (GY+P+OX). Gli isolati ottenuti sono stati inviati al CSIC-RJB, Spagna, per le analisi molecolari volte alla loro identificazione a livello di specie (Sandoval-Sierra et al., 2014). Da 81 pesci campionati negli allevamenti sono stati ottenuti 61 isolati, mentre dai 27 pesci selvatici catturati a monte e dai 45 a valle, quasi tutti senza lesioni, si sono ottenuti rispettivamente 9 e 17 isolati. La specie di oomiceti riscontrata con maggior frequenza è stata Saprolegnia parasitica (78,6%), mentre altre specie sono state trovate più raramente. Dai 463 semi di canapa messe in coltura sono stati ottenuti 415 isolati, con Saprolegnia ferax quale specie più frequente (31,3%). Sebbene la composizione delle specie riscontrata in allevamento, nelle acque a monte e a valle dello stesso e nei pesci senza lesioni, sia risultata simile, nei pesci con lesioni si è osservata una maggiore prevalenza di S. parasitica. In un allevamento della provincia di Bolzano dove la saprolegniosi non veniva ritenuta un problema sanitario, S. parasitica è stata isolata solo da un pesce selvatico senza lesioni catturato a valle dell’impianto, in una zona dove venivano introdotti salmonidi per ripopolamento. Questa pratica potrebbe quindi rappresentare un rischio per la trasmissione di Saprolegnia alle popolazioni selvatiche, come già ipotizzato in altri studi. La genotipizzazione dei ceppi isolati, ancora in corso, sarà utile a chiarire meglio la circolazione di Saprolegnia spp. negli ambienti in studio, permettendo di definire le interazioni esistenti tra popolazioni allevate e selvatiche nella trasmissione di questo oomicete. Il lavoro ha comunque evidenziato come il metodo delle trappole con semi di canapa possa essere utile nell’ambito di studi epidemiologici sulla diffusione di Saprolegnia spp., in quanto permette di evitare o limitare tecniche invasive sui pesci e superare i problemi di campionamento dei pesci selvatici.

The humphead wrasse, Cheilinus undulatus, also known as Napoleon wrasse or Maori, is one of the giant coral reef fish of the Indo-Pacific Area. In public aquaria C. undulatus is frequently exhibited for its huge body size, longevity and as a conservation ambassador of reef ecosystems. The knowledge about diseases of this precious and rare fish species is very limited and only a few disorders have been reported in literature. In May 2008 one humphead wrasse was introduced to the Cattolica Aquarium, Italy and exhibited in a 56,000 l tank together with a mixed population of teleosts, sharks and rays. In February 2017 the fish, weighing 15 kg, died and no clinical signs were observed in the days before its death. The fish was immediately necropsied and standard parasitological, bacteriological and histological exams were carried out in order to investigate causes of death. At necropsy, fish showed slightly pale gill and the presence of a severe fibrinous peritonitis. In the posterior part of the kidney, a diffuse area of necrosis with suppurative-like aspect was also observed. No gross lesions were noticed in other organs and no parasites have been found both macro and microscopically. After 24 h of incubation at 25±1°C, from all the inoculated organs (kidney, spleen, liver and brain) a number of morphologically similar colonies grew on TSA+1.5% NaCl and TCBS plates respectively. The isolates were identified as Vibrio harveyi and antibiotic sensitivity test was performed by Kirby-Bauer disk diffusion method. Histological exams showed the presence of bacterial aggregatesassociated to necrosis in most of the internal organs, especially in the kidney. Macrovacuolar liver steatosis was also observed. Furthermore several elongated fungal hyphae were found only in liver blood vessels. This case is to our knowledge the first report of systemic infection by Vibrio harveyi in a humphead wrasse and highlights the potential pathogenic role of these microorganisms in fish kept under confined condition.

Introduction: Several zoonotic helminths can be transmitted to humans by consumption of raw, undercooked, marinated or cold smoked fish products, representing a relevant public health concern. Although up to now in Italy the presence of zoonotic helminths has been documented only in wild fish populations, extensive epidemiological surveys are needed in order to assess the possible zoonotic risks linked to consumption of national aquaculture products. At this purpose, a wide parasitological survey has being undertaken on rainbow trout Oncorhynchus mykiss (RBT), gilthead seabream Sparus aurata (GSB) and European seabass Dicentrarchus labrax (ESB) farmed in Italy. Methodology: From spring 2016 to spring 2017 a total of 2347 fish have been examined. In particular, 738 RBT from 4 Italian freshwater trout farms (two farms located in the area endemic for diphyllobothriasis in Northwestern Italy, 1 in the area endemic for opisthorchiasis in Central Italy and 1 in a highly productive area of Northeastern Italy) were subjected to methods aimed to search for larval stages of diphyllobothriid cestodes (visual inspection and candling) and opisthorchiid digeneans (muscular compression/artificial digestion followed by microscopic examination). Furthermore, 781 GSB and 828 ESB from 4 marine farms (3 cage systems and 1 inland farm located in Tyrrhenian and Adriatic seas) were examined for anisakid larvae by UV-press method and artificial digestion. A seasonal periodicity has been applied, sampling 65 specimens/fish species/farm/season in order to reach a statistically significant amount of fish at the end of the survey (258 fish/farm). Results: No zoonotic parasites have been found in all the examined fish at the level of confidence of 99% with a margin of error of 5%. Conclusions: The results so far obtained during this survey are encouraging and seem to confirm that the risks linked to zoonotic helminths in Italian aquacultured fish species are null or negligible, when good farming practices are applied along the production chain.

Introduction: Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Compounds of proven activity against Saprolegnia spp. (i.e. malachite green) are potentially hazardous to human health and the environment, and their use is forbidden by European regulations. Therefore, the demand for new treatments pushes towards the selection of more safe and environmentally friendly products. In the present work, in vitro activity of two pure compounds (Oxalic acid; Sodium percarbonate) and two commercial products (Actidrox®, De Marco, Italy; Virkon® S, Dupont) was tested on different strains of Saprolegnia. Malachite green was used as reference compound. Methodology: Preliminary trials were performed using two protocols available in the literature by Alderman: one screening method in Agar aimed at assessing the minimum inhibitory concentration (MIC) and one hour bath in aqueous solution of mycelium growing on polycarbonate membrane, to assess the minimum lethal concentration (MLC). Two field strains of Saprolegnia spp. isolated from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) and one reference strain of S. parasitica (CBS 223.65 furnished by CSICRJB) have been tested in triplicate per each concentration. Results: Our results show that oxalic acid and Virkon® are effective in inhibiting the growth of the mycelium, although at concentrations too high to be applied in the field (MIC 1000 mg/ L). Actidrox® showed a different activity between the two methods (MIC 5000 mg/ L; MLC 500 mg/ L), possibly due to its mechanism of action, that requires presence of water. Tested concentrations of Sodium percarbonate were effective only in slowing down the mycelium growth. Conclusions: Further in vitro trials will be necessary, considering a wider range of promising compounds. The combination of the two methods (inoculation in agar and contact in aqueous solution) represents a good investigation approach for screening the activity of different molecules and products against Saprolegnia spp.

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

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.

A parasitological survey has been carried out in wild and farmed salmonids from northern Italy in order to clarify the current diffusion of the swimbladder nematode Cystidicola farionis (Nematoda: Cystidicolidae), reported since 2006 in wild salmonids from Adige river in the Bolzano province. 314 315 P ARASSITI DELLA FAUNA ACQUATICA P ARASSITI DELLA FAUNA ACQUATICA POSTER 06 POSTER 06 XXVIII Congresso Nazionale SoIPa, Roma 24-27 giugno 2014 XXVIII Congresso Nazionale SoIPa, Roma 24-27 giugno 2014 P06.3 EPIDEMIOLOGY AND LIFE CYCLE OF CYSTIDICOLA FARIONIS (NEMATODA: CYSTIDICOLIDAE), PARASITE OF SALMONIDS SPREADING IN ITALY Cavazza G. [1] , Gustinelli A. * [1] , Menconi V. [1] , Caffara M. [1] , Fioravanti M.L. [1] [1] Department of Veterinary Medical Sciences Alma Mater Studiorum University of Bologna ~ Bologna ~ Italy INTRODUCTION: A parasitological survey has been carried out in wild and farmed salmonids from northern Italy in order to clarify the current diffusion of the swimbladder nematode Cystidicola farionis (Nematoda: Cystidicolidae), reported since 2006 in wild salmonids from Adige river in the Bolzano province. From July 2012 to September 2013 a total of 103 trouts have been examined, in particular 99 rainbow trout (Oncorhynchus mykiss), 6 wild from Adige river in Bolzano province, 63 and 30 rainbow trout from a farm in Vicenza province (F1) and a farm (F2) located in Trento province, respectively; and 4 wild brown trout (Salmo trutta fario) from Adige river in Bolzano province. All the fish were weighted, measured and then subjected to parasitological examination, with more attention to swim bladder. Parasites were isolated, rinsed in deionized water, fixed in 70% ethanol and clarified by Amman’s lactophenol for morphological studies. Prevalence values (%), Mean Intensity (MI) and Abundance (A) have been calculated according to Bush et al., 1997, J.Parasitol., 83: 575-583. Sixty-four (64.6%) out of 99 rainbow trout were positive for nematodes (MI=13.4,A=8.64); 35 (55.5%) out of 63 fish (MI=12.6, A=7) from F1, 26 (86.6%) out of 30 (MI=14.7, A=12.7) from F2 and 3 (50%) out of 6 (MI=53, A=26,5) from the wild. Only 1 (25%) out of the 4 wild brown trout were parasitized by 31 nematodes. All the parasites were identified as Cystidicola farionis. In F1, 4 samples of parasite amphipods described as intermediate host of C. farionis, have been collected upstream and within the tanks by a plankton net. Amphipods were transported alive to the laboratory and then examined for the presence of larval nematodes at the dissection microscope. The larvae were isolated, fixed in 5% buffered formalin and submitted to morphological study. A total of 4505 amphipods, all identified as Echinogammarus spp., have been examined. One hundred-four larvae referable mainly to third stage, L3 of C. farionis were found (MI=1.22, ranging from 1 to 5 larvae/amphipod) in 85 (1.9%) amphipods. A slightly higher positivity (2.1%) for C. farionis larvae has been observed inside the farm compared to upstream (1.7%), and the highest prevalence value (8.4%) has been observed in the month of October. In Europe the occurrence of C. farionis is documented in several northern and central European countries, including the European part of Russia, but its report in Italy is quite recent (Gustinelli et al., 2008, Ittiopatologia, 5:87-97). On the basis of this study C. farionis spread from Adige river to other water systems involving also trout farms with a potential impact on their commercial value. Infected farms show common features such as a not-intensive management, with particular reference to low biomass density and integrative feeding, leading the fish to feed on natural preys as amphipods and allowing the development of C. farionis life cycle. The increasing presence of C. farionis in Italian salmonid species confirms its stable introduction in both freshwater wild and farming environments and give a strong warning on the hazard of the introduction of not-monitored fish and their exotic parasites that could become a risk factor on national scale.

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.

The sub-Alpine lakes of Switzerland, Italy and France have long been reported as an endemic area of diphyllobothriosis, a parasitic zoonosis aused by Dibothriocephalus latus. With this study, we explored the hypothesis for a relationship between the prevalence of D. latus in Perca fluviatilis and the Escherichia coli load in lake water. To do this, we identified eleven sampling sites in three areas (north, centre and south) of Lake Iseo (north Italy) to determine E. coli load and the prevalence of D. latus in P. fluviatilis. Prevalence and 95% confidence interval (CI) of D. latus infestation ranged from 0% (95% CI: 0.71–0.0) in Sarnico (southern area) to 20% (95% CI: 33.0–11.2) in Pisogne (northern area). There were significant differences in prevalence be-tween the sites (χ2= 31.12; p- value= .0006) and in E. coli load (Kruskal–Wallis test; p- value= .0005). There was decreasing gradient of E. coli load and prevalence of D. latus infestation from north to south. A significant positive correlation (r= .881; p- value= .003) was found between E. coli load and prevalence of D. latus. Also, linear regression showed a significant relationship between E. coli load and prevalence of infestation (R2= .775). Our findings offer an explanation for the link between E. coliload in water and D. latus prevalence. The potential factors in this link are the ef-ficiency of the local wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.KEYWORDSItaly, Lake Iseo, Perca fluviatilis, prevalence of infestation, wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.

Simple Summary Copepods of the genus Pennella are parasites of marine aquatic organisms (e.g., cephalopods, pelagic fish, cetaceans). They can infest fish of economic interest, including tuna and swordfish. The infestation of Pennella sp. in fish creates problems of food safety and fish marketing. Generally, these copepods penetrate the host’s muscle, without damaging internal organs. Here, we report on Pennella sp. infestation in swordfish heart muscle. The severity of the cardiac lesions we observed suggests that parasites of the genus Pennella pose a concern for food safety as well as animal health. Abstract The genus Pennella comprises hematophagous parasites of marine aquatic species, including cephalopods, marine mammals, and pelagic fish. Nine species have been officially included in the genus Pennella plus another six species inquirendae. They are most often found in the host’s musculature, without penetrating internal organs. For the present study, 83 hearts from swordfish (Xiphias gladius) caught in the Mediterranean Sea were sampled and immediately fixed in formalin for histopathological analysis. In total, 10 (12.05%) hearts were found to be parasitized by copepods of the genus Pennella. Macroscopically, there was mild-to-severe fibrinous pericarditis with atrial wall thickening and multiple parasitic nodules. Histologically, the parasitic nodules were surrounded by an inflammatory-necrotizing reaction. Parasitic infestation by Pennella spp. is common in pelagic fish and in swordfish, in particular. Here, however, we report atypical cardiac localization. A future area of focus is the evaluation of cardiac Pennella spp. infestation by histopathology and genetic identification of the parasites.

Cystidicola farionis is a nematode of the swim bladder that parasitizes Salmonidae and Osmeridae families, with a range of diffusion in Europe, Asia, and North America. During a fish health monitoring performed in July 2017 in a trout farm located in northwest Italy, a prevalence of 70% for C. farionis was recorded in rainbow trout from a unique tank. In order to clarify the first occurrence of this parasite in northwest Italy, a total of 180 specimens were analysed in different seasons to obtain prevalence, mean intensity, and mean abundance of infestation. Moreover, amphipods were also sampled during each fish sampling campaign. Only fish sampled in August 2017 were positive for C. farionis, since fish were then sold and replaced with a new batch. All isolated nematodes were identified by morphological criteria as subadults (L4), but not sexually matured. All examined amphipods (genus Gammarus) resulted negative for the presence of the parasite. The most probable explanation of this report was due to the movement of a single fish batch from northeast Italy, previously described as an endemic area for this parasite. The absence of the parasite in the amphipods and consequently in other fish batches from other tanks are due to the lifetime cycle of the parasite that had no time to infect new hosts.

The nematode Eustrongylides excisus is a parasite of freshwater fish- and fish-eating birds, with known differences on prevalence values among fish species. Thus, the present study aims to explore the hypothesis that the feeding behavior and the size of fish belonging to different trophic levels could explain such differences. For that, 14 sampling sites were selected to perform a fish parasitological survey on Lake Garda (Italy) during spring-summer 2020. Amplification of nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences of nematodes morphologically ascribable to the genus Eustrongylides allowed to identify them as E. excisus. From the five studied fish species (Perca fluviatilis, Lepomis gibbous, Coregonus lavaretus, Alosa fallax lacustris and Micropterus salmoides), only three presented the parasite E. excisus: P. fluviatilis, L. gibbous and M. salmoides, with significant differences in prevalence values among species (p = 0.002). Additionally, there were differences in prevalence values within the same fish species captured from different sampling sites. Findings showed that mainly piscivorous fish were positive for E. excisus and how the prevalence was highest in M. salmoides. As regard the fish size, a negative correlation between body size and E. excisus was found in P. fluviatilis due to the feeding habit of juvenile perch which feed mainly zooplankton and benthic invertebrates (i.e., oligochaetes, which are the first intermediate hosts of E. excisus). The study findings advance novel knowledge in the field of pathogens of zoonotic importance in the aquatic environment.

With this study we investigated the accumulation of potentially toxic elements (As, Cd, Cr, Cu, Hg, Pb, Zn), six indicators (28, 52, 101, 138, 153, 180) of non-dioxin-like polychlorinated biphenyls (Σ6 NDL-PCBs), polycyclic aromatic hydrocarbons (PAHs), and microplastics in S. quadrata (edible part) collected from two sampling sites (1 and 2) from the Arno River Basin (Central Italy). A risk assessment of the implications for human health was also performed. Levels of potentially toxic elements in gastropods from site 2 were slightly higher and the Σ6 NDL-PCB concentration was significantly higher (7.32ngg−1vs. 3.07ngg−1) compared to site 1 due to higher anthropogenic pressures. The concentration of chrysene, benzo[b]fluoranthene, and benzo(a)pyrene was below the limit of quantification (0.5μgkg−1). Benzo[a]anthracene was detected in gastropods from both sites (0.5 ± 0.02μgkg−1 and 0.7 ± 0.02μgkg−1 from site 1 and 2, respectively). The microplastics frequency (mainly polyethylene terephthalate) differed significantly between the sites (site 1, 0.8 ± 1.30; site 2, 1 ± 0.37 items/specimen). All contaminant levels were compliant with international regulatory limits and guidelines. Incremental lifetime cancer risk (ILCR) values for As, Cd, Cr, and Pb were far below the safety values of 1 × 10–4. Similarly, the ILCR values from the Monte Carlo simulation model were all within the safety region of 1 × 10–4 and 1 × 10–6. Findings from the health risk assessment indicated no adverse effects for human health from any of the contaminants analysed here, except for microplastics for which no limits or legislation are currently in force.

Carnobacteria are common bacteria in cold and temperate environments; they are also reported during fish mortality events. In a previous study, carnobacteria were isolated from the eyes of healthy wild salmonids from a high-mountain lake. To better understand these findings, salmonids were captured from three high-mountain lakes (Lower and Upper Balma Lake, Rouen Lake; northwest Italy) during August 2019 and subjected to bacteriological and histological examination. Although all were healthy, 8.7% (Lower Balma Lake), 24% (Upper Balma Lake), and 32.6% (Rouen Lake) were positive for carnobacteria colonization of the eyes. A Trojan-horse effect was hypothesized to explain carnobacteria isolation in the eye. This immune-escaping macrophage-mediated mechanism has been identified in other Gram-positive bacteria. Biochemical, molecular, and phylogenetic analysis were carried out on isolated bacteria (Carnobacterium maltaromaticum and C. divergens). Based on previous references for carnobacteria isolated from fish, C. maltaromaticum strains were tested for the pisA precursor gene of the bacteriocin piscicolin 126. Carnobacterium maltaromaticum strains were found to display genotypic heterogeneity and a low percentage of pisA positive amplification. Features of geomorphology, geographic isolation, and microbiota common to the three lakes are thought to be possibly related to our findings. Moreover, terrestrial insects collected from the lake shoreline and the stomach contents were screened for the presence of carnobacteria. The salmonids in these high-mountain environments feed mainly on terrestrial insects, which are considered possible vectors for carnobacteria that might catabolize the exoskeleton chitin. All insects tested negative for carnobacteria, but as a small number of samples were analyzed, their role as possible vectors of infection cannot be excluded. Further studies are needed to corroborate our research hypothesis.

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.

High-mountain lakes are pristine ecosystems characterized by extreme environmental conditions. The atmospheric transport of pollutants from lowlands may add further stress to organisms inhabiting these environments. We investigated the environmental stress pressure on brook trout (Salvelinus fontinalis) from a high-mountain lake in the Cottian Alps (Piedmont, northwest Italy). To do this, males and females of brook trout were sampled from Balma Lake in summer (August) and autumn (October) 2017 in order to assess the influence of trace elements accumulation and environmental parameters (physicochemical parameters and nutrient characteristics of water) on oxidative stress biomarkers. Bioaccumulation of Al, As, Cd, Cr, Cu, Fe, Hg, Pb, Ni, Se, and Zn and metallothionein levels were measured in muscle tissue of males and females. Liver, gills, kidney, and spleen tissue samples were analyzed for superoxide dismutase, catalase, total glutathione peroxidase, selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione S-transferase activity. Analysis of environmental parameters showed changes in biomarker levels with seasonal variations. Water temperature was significantly higher in summer than autumn (Wilcoxon test; p = .0078), while pH was significantly higher in autumn than in summer (Wilcoxon test; p = .0078). Sex-related differences in oxidative stress biomarkers in tissues were unremarkable, whereas seasonal variability of oxidative stress biomarkers was observed, with major differences occurred for liver in summer and for gills, kidney, spleen and muscle in autumn. Positive correlations between environmental parameters and biomarkers were noted. Major fluctuations in water temperature, pH, Cu, Pb and Hg produced changes in biomarker levels; however, increased food intake during the ice-free season was probably the main factor that influenced changes in oxidative stress biomarker levels in brook trout in this extreme ecosystem.

Plastic pollution has become a pervasive environmental problem on a global scale, from the ocean depths to the aquatic ecosystems of the Tibetan Plateau. To date, data on plastic and microplastic occurrence in pristine ecosystems like high-mountain lakes are lacking. In this study, plastic (>5000 μm) and microplastic (10–5000 μm) levels were measured in snow at the end of the winter season (April 2020), and in water, sediment, and biological samples collected monthly (June–October 2019) during the ice-free season from the Dimon Lake, a high-mountain lake in the Carnic Alps, northeast Italy. Biological samples consisted of chironomids (Diptera, Chironomidae; n = 150) and stomach contents of Cottus gobio (n = 40). Analysis of the water, sediment, and biological samples revealed the absence of plastic and microplastics larger than 10 μm, whereas the snow samples contained microplastics of polyethylene terephthalate (PET) albeit at very low levels (0.11 ± 0.19 L-1). These results show that while the lake ecosystem could be considered unpolluted by microplastics, abundant snow precipitation in winter can trap microplastic particles that deposit on the ground. The very low levels of PET microparticles recorded in the snow samples suggest the need for further research to better understand the source of microplastic pollution in this environmental matrix.

This study aimed to assess the biological and sanitary conditions of alien fish in a high-mountain lake (Balma Lake) located in the Cottian Alps. A single fish sampling session (August 2018) using gillnets collected 90 specimens of brook trout (Salvelinus fontinalis). Sex and age were determined (59 females and 31 males, age class 0+ to 4+). Regression analysis showed no difference in total weight and total length between males and females (ANCOVA: F = 0.453, p = 0.954). The mean condition factor (Kmean) decreased with increasing age for males and females. Terrestrial insects were the main prey found in the fish stomachs. The parasitological exam was negative, and the bacteriological exam was positive for Carnobacterium maltaromaticum and C. divergens in 33% of specimens. The total mercury, cadmium, and lead concentration in muscle tissue was within the maximum limit established by the European Commission for human consumption. The brook trout population was found to be well structured, these findings may help local administrations in the implementation of eradication measures.

Nodular gill disease (NGD) is a serious parasitic condition associated with amoebic infection affecting farmed rainbow trout (Oncorhynchus mykiss) in several countries worldwide. Outbreaks of NGD have been recently observed in several rainbow trout farms in Italy, with clinical signs strongly indicative of respiratory distress and high mortality registered mainly during winter. From December 2013 to April 2015 a total of 494 rainbow trout were collected from 13 Italian farms and subjected to anatomo-pathological and histological exams. Gross gill lesions consisted in excessive mucus production, swelling, whitish nodules in the distal parts of filaments and presence of alternate discolored and congested areas. In 261 (52.8%) fish histology allowed to identify the presence of amoebae as showed also by the parasitological exam of fresh gill samples. Histological lesions, in presence of amoebae, consisted in strong hyperplasia of the gill epithelial cells producing extensive lamellar fusion with obliteration of interlamellar spaces. In more severe cases mucous cell hypertrophy, lamellar edema, necrosis and exfoliation of epithelial cells were observed. Among the epithelial cells, inflammatory infiltrate with numerous lymphocytes, macrophages and eosinophilic granule cells/mast cells was detected. Further analyses are in progress in order to identify the species of amoebae involved as causative agent of NGD in Italy.

Il problema delle infestazioni parassitarie di origine ittica ha un duplice aspetto, da una parte i riflessi sul valore merceologico del prodotto ittico e dell’altra la salute del consumatore, anche se nel nostro Paese le abitudini alimentari non contemplano spesso il consumo di pesce crudo, se non limitatamente a prodotti tipici locali e ad usanze territoriali. Relativamente al trattamento per l’uccisione di parassiti vitali in prodotti della pesca destinati al consumo umano, il Reg. UE n. 1276/2011 ha indicato la possibilità di deroga ai trattamenti di congelamento previsti per i prodotti che vanno consumati crudi o sottoposti a trattamenti che non garantiscono l’uccisione dei parassiti vivi. Tale deroga può essere rilasciata solo per prodotti che derivano dall’acquacoltura e che soddisfano particolari requisiti, quali essere stati allevati in un ambiente privo di parassiti vivi, oppure in seguito a verifica, mediante procedure approvate dall’autorità competente, che non vi sia un rischio sanitario per la presenza di parassiti vivi. Si tratta di indicazioni che si riferiscono al parere scientifico emesso dall’EFSA nel 2010 sulla valutazione del rischio correlato alla presenza di parassiti nei prodotti ittici. Nel proprio parere l’EFSA raccomanda inoltre di condurre studi epidemiologici approfonditi in tutte le specie ittiche allevate. Proprio in riferimento a questa raccomandazione, è in corso un progetto di ricerca sulle zoonosi parassitarie trasmesse dai pesci dulciacquicoli; in tale ambito è stata effettuata un’indagine preliminare in un allevamento di trota iridea situato lungo l’asta del fiume Ticino, nel tratto emissario dal lago Maggiore; questo primo approccio è stato condotto al fine di ottenere, da un lato dati di prevalenza relativi alla eventuale presenza di parassiti e dall'altro uno strumento da utilizzare con la GDO per dimostrare che il problema possa considerarsi trascurabile. Presso l’allevamento preso in esame coesistono 2 fonti di approvvigionamento idrico con un diverso livello di rischio nei confronti dell’infestazione da Diphyllobothrium: acque di sola risorgiva e acque di provenienza superficiale. Il ciclo produttivo, dopo una prima fase di allevamento degli avannotti in vasche le cui acque sono di risorgiva (o di pozzo), prevede lo stazionamento degli animali in raceways, alimentati con acqua superficiale; queste vasche rappresentano il punto a maggior rischio per la presenza eventuale di infestazione, dal momento che il corpo idrico che alimenta l’allevamento, proviene da un canale di derivazione del Ticino, emissario del lago Maggiore, dove la parassitosi è presente nella fauna ittica con diverse prevalenze a seconda dei diversi punti di prelievo (prevalenza 2-5%). Il prelievo è stato condotto nel punto di maggior rischio lungo la filiera produttiva e la numerosità campionaria è stata fissata a priori, con approccio conservativo, in modo da svelare la presenza del parassita nel caso in cui la sua prevalenza fosse pari almeno al 2% con un livello di sicurezza del 95%. Sono stati campionati 150 individui di taglia commerciale (350-500 g), suddivisi in 6 campagne di campionamento svolte tra l’estate 2014 e l’estate 2015, con cadenza bimestrale (25 esemplari per prelievo). L’esame parassitologico specifico effettuato mediante sfilettatura della porzione muscolare di entrambi i lati di ogni soggetto e visione mediante vetri compressori di tutti i muscoli non ha evidenziato in alcun esemplare la presenza di parassiti. Con questi risultati, si può quindi affermare, con una sicurezza pari al 95%, che la prevalenza del parassita nell’allevamento considerato non può comunque essere superiore al 2%. Questo approccio dovrebbe essere adottato in altri comparti produttivi posti in bacini idrografici afferenti ai grandi laghi alpini, in cui la presenza della difillobotriasi è ancora attuale, per raccogliere dati epidemiologici consistenti su tale parassitosi, in modo da rafforzare l’immagine della troticoltura nazionale, conferendo un plus valore ai prodotti dell’acquacoltura.

Le malattie parassitarie possono rappresentare un fattore limitante in maricoltura, determinando in alcuni casi perdite di tipo diretto e, nella maggior parte dei casi, perdite indirette dovute alle ripercussioni negative sullo stato di benessere e sulle performance produttive dell’ospite. Nel presente lavoro vengono presentati i risultati delle analisi parassitologiche effettuate nel periodo gennaio 2010-settembre 2015 su spigole (Dicentrarchus labrax) ed orate (Sparus aurata) provenienti da diversi allevamenti intensivi nazionali, allo scopo di fornire un quadro generale delle specie parassitarie reperibili in queste specie ittiche nei sistemi di maricoltura nazionale. Sono stati esaminati in totale 4435 pesci, di cui 2957 spigole e 1478 orate, di diverse classi di taglia e provenienti da differenti sistemi d’allevamento qui suddivisi per macrocategorie (impianti a terra: T e impianti in gabbia: G). Tutti gli esemplari sono stati sottoposti ad esame parassitologico completo secondo metodiche di laboratorio standard. Delle 2957 spigole esaminate, 1872 (63,3%) sono risultate positive per almeno una specie parassitaria, con numerosi casi di coinfezione da più taxa parassitari. Nella tabella “Parassiti spigola” vengono riportati i valori di prevalenza osservati, suddivisi per macrocategorie di sistemi di allevamento; non sono riportati i parassiti riscontrati con percentuali di positività

During 2013 two outbreaks of massive infestation by metacercariae in wild chub Squalius cephalus and farmed black bullhead Ictalurus melas were observed. The metacercariae were about 1 mm, whitish and scattered in muscle. The morphological study allowed identifying the parasites as Hysteromorpha triloba, a cosmopolitan Diplostomidae previously reported in Italy only at the adult stage in Ciconiiformes. Histological examination of skeletal muscle of infected chub revealed a massive presence of metacercariae within large elliptical cavities containing amorphous basophilic substance. At opposite ends of the cavities a mild inflammatory reaction with fibroblasts, lymphocytes, macrophages and rodlet cells was observed. Muscular fibres around the metacercariae presented hyaline degeneration and in some sections appeared replaced by granulation tissue. In black bullhead inflammatory reaction toward H. triloba appeared more severe than observed in chub; metacercariae were enclosed in roundish cysts characterized by a thick wall of granulation tissue, fibroblasts, epithelioid cells and macrophages. The differences observed regarding the tissue reactivity and pathogenic effects of H. triloba metacercariae in chub and black bullhead may be related to a different degree of adaptation of the parasite to autochthonous (the former) and allochthonous (the latter) host. The macroscopic evidence of metacercariae of H. triloba in the muscle, even in the absence of zoonotic implications, could adversely affect the marketability of parasitized fish species.

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.

European eels live most of their lives in freshwater until spawning migration to the Sargasso Sea. During seawater adaptation, eels modify their physiology, and their digestive system adapts to the new environment, drinking salt water to compensate for the continuous water loss. In that period, eels stop feeding until spawning. Thus, the eel represents a unique model to understand the adaptive changes of the enteric nervous system (ENS) to modified salinity and starvation. To this purpose, we assessed and compared the enteric neuronal density in the cranial portion of the intestine of freshwater eels (control), lagoon eels captured in brackish water before their migration to the Sargasso Sea (T0), and starved seawater eels hormonally induced to sexual maturity (T18; 18 weeks of starvation and treatment with standardized carp pituitary extract). Furthermore, we analyzed the modification of intestinal neuronal density of hormonally untreated eels during prolonged starvation (10 weeks) in seawater and freshwater. The density of myenteric (MP) and submucosal plexus (SMP) HuC/D-immunoreactive (Hu-IR) neurons was assessed in wholemount preparations and cryosections. The number of MP and SMP HuC/D-IR neurons progressively increased from the freshwater to the salty water habitat (control > T0 > T18; P < 0.05). Compared with freshwater eels, the number of MP and SMP HuC/ D-IR neurons significantly increased (P < 0.05) in the intestine of starved untreated salt water eels. In conclusion, high salinity evokes enteric neuroplasticity as indicated by the increasing number of HuC/D-IR MP and SMP neurons, a mechanism likely contributing to maintaining the body homeostasis of this fish in extreme conditions.