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

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

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: Enteromyxum leei (Myxozoa) is an important pathogen of gilthead seabream (GSB), Sparus aurata and other Sparidae fish cultured in the Mediterranean. In GSB, E. leei induces chronic enteritis followed by anorexia, cachexia, and even death. It can cause significant growth delay and mortality in marine cage farms as well as land-based mariculture systems. Water temperature and water recirculation are critical risk factors in the contagion and onset of enteromyxosis (Palenzuela et al., 2006, Sitjà-Bobadilla and Palenzuela 2012). However, the relevance of other risks factors like farm management practices, and the epidemiological situation in gilthead sea bream farms, have not been thoroughly determined. In this study, a risk assessment questionnaire and cross-sectional epidemiological study was designed to get insights in the distribution and incidence of E. leei in more than 50 Mediterranean and Atlantic GSB farms throughout Western Europe., [Methods]: A questionnaire for GSB cage farms was designed to ascertain information about farm background and self-awareness of risk relative to E. leei infection, as well as details on the facilities, production and farm management routines. In addition, the farms were invited to participate in a cross-sectional targeted epidemiological survey to test the presence of the parasite in their stock. The participant farms were recruited by local expert pathologists and consultants acting as nodes in each geographical region, and the samples and questionnaires were coded and studied blindly. For those farms participating in the biological sampling, a protocol was submitted to sample one batch of fish at harvest time. The sampling was designed to detect prevalence higher than 9.5% with a 95% confidence. The intestinal rectal ampoules (portion with maximum predictive value for E. leei detection in GSB) were taken from at least 30 randomly selected fish during routine harvest from one or several cages. For the validation of the survey protocol, a land-based farm which is enzootic for the parasite was enrolled in the study and two different GSB lots were sampled as positive controls. The samples were fixed in 80-90 º Ethanol and paired into 15 tubes, which were shipped to the central diagnosis laboratory at IATS-CSIC. The routine procedures for diagnosis of E. leei consist of a SYBR-green qPCR test with primers specific for the parasite against a standard curve containing known numbers of copies of the target gene. Upon reception at the laboratory the samples were individually processed and homogenised. DNA was extracted from an aliquot using a robotic system, its quantity and quality were evaluated, and samples were tested by qPCR at two different dilutions. They were considered positive when the cycle threshold (Ct) was, [Results and Discussion]: A total of 45 cage farms from Croatia (12), Greece (12), Italy (13), Spain (7) and Malta (1) completed the questionnaires. Among them, 32% declared to have previous records of the parasite: 11 of them at some point in the last 5 years, 3 in the most recent production cycle, and 4 having infections recurrently. Among the farms with previous records, the disease was considered not relevant in 7, controllable in 7, and problematic in 4 sites. A total of 40 cage farms sent a total of 1330 fish biological samples which were tested by qPCR. The results show that the infection persists unnoticed at many farms (Table 1), Famers were most aware of the presence of the infection in Greek waters. On the contrary, Croatian farmers were unanimously unaware of the presence of the parasite although it was detected at most of the tested sites. Prevalence of infection was generally lower in sites where the infection was not believed to be present, suggesting that low level infections, although widespread, do not cause any noticeable problems at many farms. All countries sampled had the infection, and the only countries with farms in which the infection was not detected were Croatia and Italy. Prevalence was highest on average in Greece (Table 2). The statistical analysis considered prevalence against farm level factors as well as batch level factors. For most variables no significant effect was observed. At a farm level, farms removing mortalities more frequently, and those with stronger water exchange have a lower prevalence of infection. The number of hatcheries supplying the farm and the size of fish at harvest showed the strongest positive association with prevalence. Larger farms with higher numbers of cages, cage sizes and annual production tended to have higher prevalence. At the batch level, fish which had experienced highest minimum temperatures and those introduced to the cages at larger sizes had higher prevalence, whereas shorter times on the farm and higher specific growth rates were associated with lower prevalence.

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

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

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

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.

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