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RAZVOJ POLUKVANTITATIVNOG BODOVNOG SUSTAVA ZA PROCJENU ZDRAVLJA ŠKRGA TIJEKOM SPARIKOTILOZE SA SPARICOTYLE CHRYSOPHRII (SPARUS AURATA)

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

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.

The Mediterranean aquaculture has suffered significant economic losses due to viral nervous necrosis mainly caused by RGNNV betanodavirus genotype primarily involving sea bass (Dicentrarchus labrax). Recently, a RGNNV/SJNNV reassortant betanodavirus, harbouring the RNA1 segment of RGNNV genotype and the RNA2 segment of SJNNV genotype, emerged. So far, the reassortant strain has caused a negative economic impact mainly on sea bream (Sparus aurata) hatcheries sparing the sea bass farming sector.

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

Turbot (Psetta maxima) is currently one of the most promising species for European mariculture but although most of the critical aspects threatening production have been optimized, transmissible diseases may still represent a limiting factor. In Autumn 2014 two batches of 5 turbot (mean weight 305 g) caged in northern Adriatic Sea and showing melanosis, exophthalmos and skin/fin lesions were subjected to bacteriological and parasitological examination. Visceral organs of twenty fish from the same two cages were fixed directly on the field in 10% buffered formalin. Necropsy showed in all specimens pale-yellow liver and intestinal distension with abundant mucous contents. From the fish owing to one batch ascites was also observed and bacteriological exam allowed to isolate Vibrio scophthalmi. Parasitological exam did not show presence of parasites in any organs except for intestine where developmental stages of not identifiable parasites were observed. In order to identify them, portions of intestine from all fish were subjected to DNA extraction followed by PCR of the 18S rDNA and sequencing. BLAST analysis of the sequences permitted to ascribe the parasites to the genus Cryptosporidium. At histology the presence of a mild-to-severe infection by cryptosporidia was detected in the intestine of all fish from both cages. Extracytoplasmic stages of parasites adhering to the luminal surface of the epithelial cells were observed, generally with the presence of intra-epithelial sporogonic stages located deeply within the enterocytes layer. In some areas, several oocysts were present within vacuolated areas in the mucosa, with diffuse necrosis, epithelial sloughing off and loss of the enterocytes brush border ; lamina propria and submucosa were oedematous. Inflammatory response involving mainly lymphocytes and, in heavy infections, rodlet cells was noticed. In some fish also the stomach was affected. Severe pathological changes were mainly observed in presence of numerous intra-epithelial oocysts. The lesions here described are similar to those already reported in farmed turbot in Spain. In our case the source of infection could not be assessed since fish had been introduced into the cages from abroad several months before. The role of wild carrier fish cannot be ruled out.