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As many other farming activities, aquatic animals and particularly finfish can be affected by several infectious and non-infectious diseases. The specific diseases and pathogens or other causes triggering the development of these diseases, since there are usually multifactorial, can be related to a number of different factors: characteristics of the infectious agents, affected fish species or genetic susceptibility, environment and environmental conditions, geographical parameters, and farming systems amongst others.

Despite the technological progress applied in recent years to the various production phases of Mediterranean Marine Fish Farming (MMFF), from reproduction to nutrition, several welfare and health issues of farmed fish still represent relevant limiting factors for sustainable production. In particular, the attempts to increase and diversify MMFF productions have been often accompanied by the emergence or reoccurrence of transmissible diseases strongly conditioned by management and environmental factors with consequent significant economic losses and repercussions on fish welfare and health parameters. The experience gained in the attempt to control diseases has amply demonstrated that the treatment (when applicable) is often not conclusive, highlighting that the “disease event” is the result of many interacting factors linked to the pathogen, the host and the environment (intended as production system and surrounding ecosystem), which underly the occurrence of disease and influence morbidity and mortality rates. It is evident that an integrated approach is required to prevent and control diseases of farmed fish, which means adopting a holistic view to take into consideration the integrated manner all the different factors involved in the development of a disease and in fish health management. Based on this concept, the project PerformFISH has developed in WP3 an integrated network of research activities aiming to provide the Mediterranean finfish aquaculture with effective tools to prevent, mitigate and control the most relevant diseases of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata).

Despite the technological progress applied in recent years to the various production phases of Mediterranean Marine Fish Farming (MMFF), from reproduction to nutrition, several welfare and health issues of farmed fish still represent relevant limiting factors for sustainable production. In particular, the attempts to increase and diversify MMFF productions have been often accompanied by the emergence or reoccurrence of transmissible diseases strongly conditioned by management and environmental factors with consequent significant economic losses and repercussions on fish welfare and health parameters. The experience gained in the attempt to control diseases has amply demonstrated that the treatment (when applicable) is often not conclusive, highlighting that the “disease event” is the result of many interacting factors linked to the pathogen, the host and the environment (intended as production system and surrounding ecosystem), which underly the occurrence of disease and influence morbidity and mortality rates. It is evident that an integrated approach is required to prevent and control diseases of farmed fish, which means adopting a holistic view to take into consideration the integrated manner all the different factors involved in the development of a disease and in fish health management. Based on this concept, the project PerformFISH has developed in WP3 an integrated network of research activities aiming to provide the Mediterranean finfish aquaculture with effective tools to prevent, mitigate and control the most relevant diseases of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata).

As many other farming activities, aquatic animals and particularly finfish can be affected by several infectious and non-infectious diseases. The specific diseases and pathogens or other causes triggering the development of these diseases, since there are usually multifactorial, can be related to a number of different factors: characteristics of the infectious agents, affected fish species or genetic susceptibility, environment and environmental conditions, geographical parameters, and farming systems amongst others.

Nematodes belonging to the genus Contracaecum (family: Anisakidae) are heteroxenous parasites with a complex life cycle. Contracaecum larvae infecting farmed fish and fishery products are economically important causing market rejection in massive infection and may have zoonotic potential. In Israel, Contracaecum larvae have been described morphologically in several fish species; however, none of these descriptions were supported by molecular tools. In 2019–2020, hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and red drum (Sciaenops ocellatus), farmed in polyculture were found to be heavily infected with nematodes referable to Contracaecum larvae. Prevalence of infection in hybrid tilapia and red drum was 53.8% and 40.9%, respectively. A combined (morphological and molecular) approach revealed that both infected fish species were parasitized by the same species of Contracaecum, although larvae in hybrid tilapia were localized in the pericardial cavity whereas in red drum, they were observed in the abdominal cavity. Genetic analysis of internal transcribed spacer rDNA and cox2 mtDNA showed high similarity to unidentified Contracaecum larvae detected in several fish species in Ethiopia, Egypt and Kenya. In this study, molecular and morphological analyses place the possible new species in the C. multipapillatum complex and was provisionally named C. multipapillatum E. Further analyses combining morphological and molecular approaches are required on adult specimens collected from piscivorous birds living in the same area to support the identification of a potentially new species.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms., This work has been carried out with financial support from the Spanish MINECO under project AGL2013‐48560‐C2‐2‐R. Additional funding was provided by the European Union, through the Horizon 2020 research and innovation programme grant agreement 634429 (ParaFishControl).

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

The present study aimed to describe clinical signs of cryptosporidiosis in neonatal foals hospitalized in an Equine Perinatology Unit and to compare the clinical signs between Cryptosporidium parvum and Cryptosporidium horse genotype infection. The study was divided into two parts. In the retrospective study, nine foals infected by C. parvum were considered. In the prospective study, 70 foals, less than 15 days old, were prospectively included. Historical and clinical data were recorded, and in the prospective study, multiple fecal samples were collected. C. parvum (n = 13) and Cryptosporidium horse genotype (n = 7) were isolated. In four foals, there was a mixed infection with both the Cryptosporidium. Diarrhea, when present, showed similar duration and characteristics. Sixteen foals showed decreased abdominal sounds and colic pain before evidence of diarrhea. Nineteen foals had hyperthermia at least once. Although survival rates were similar between C. parvum (77%), C. horse genotype (100%), and cryptosporidial mixed infection (100%), foals affected by C. parvum presented anorexia (P

Occurrence of metacercariae of potentially zoonotic trematodes (Platyhelminthes: Digenea) in the musculature of common carp (Cyprinus carpio L. 1758) was monitored in four Hungarian aquacultures. Four geographically distinct fish farms (located in the Northwestern, Southwestern, Northeastern and Southeastern parts of Hungary) were selected for the investigation. From each farm, a total of 258 one-summer-old fingerlings were sampled and examined in the years 2016 and 2017. In addition, in 2017, we examined 60 market size specimens (30 two-summers and 30 three-summers) sampled from the most infected aquaculture in the Northeastern part of Hungary. The fish were euthanized and decapitated whereafter their musculature (fillets) was digested in a pepsin solution to isolate metacercariae from the tissue whereafter morphological and molecular analyses (PCR and sequencing of ITS region) were performed. Opisthorchiid metacercariae were not recovered but in one of the farms numerous metacercariae were detected in the musculature of carp. They were identified as cyathocotylid trematodes based on their morphological characteristics and by sequencing the ITS region. The infection levels proved to be remarkably different among the four fish farms. Carps from the Northeastern farm were infected by large numbers of cyathocotylid metacercariae, while 8 Posthodiplostomum cuticola metacercariae were detected in the Northwestern aquaculture. In the other two farms (Southwestern and Southeastern) no infection was recorded. The infected farm is located close to a protected natural wetland habitat populated by a rich fauna of aquatic birds (potential final hosts) and snails (first intermediate host) which may create a higher risk of infection in the neighbouring fish farms.

6 pages, 1 table.-- This work is licensed under a Creative Commons Attribution 4.0 International License, The increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment. Aim: To evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon. Methods: From 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae. Results: No zoonotic parasites were found in any of the fish investigated. Conclusions: The risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation, This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 634429

In the Afrotropic region, the genus Clinostomum is represented by four accepted and four unnamed species distinguished using molecular data. Here, we describe one of the four unnamed species as Clinostomum ukolii n. sp. based on metacercariae from siluriform fishes (Synodontis batensoda, Schilbe intermedius) collected in Nigeria and South Africa. The new species is distinguished by molecular data (39 new sequences of partial cytochrome c oxidase I≥ 6.7% divergent from those of other species) and morphological differences from named and unnamed species in the same region. Metacercariae of C. ukolii n. sp. can be distinguished based on size, tegumental spines, and various aspects of the genital complex, including its position, lobation of the anterior testis, and the disposition and shape of the cirrus pouch. Although descriptions of new species of digeneans are typically based on the morphology of adults, we argue that in cases where data are available from metacercariae from regionally known species, new species can be described based on metacercariae, particularly when supported by molecular data, as here. Moreover, sub-adult reproductive structures can be clearly visualized in metacercaria of Clinostomum. Considering metacercariae as potential types for new species could advance clinostome systematics more rapidly, because metacercariae are encountered much more often than adults in avian definitive hosts.

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.

Raphidascarididae are among the most abundant and widespread parasitic nematodes in the marine environment. The life-cycle of most raphidascaridid species is poorly known and information about their distribution and host range is lacking in many geographical areas, as is the taxonomy of several species. A study of larval and adult stages of Hysterothylacium fabri (Rudolphi, 1819) Deardorff and Overstreet, 1980 (Nematoda: Raphidascarididae) infecting the striped goatfish Mullus surmuletus Linnaeus, 1758 (Mullidae) and the Mediterranean stargazer Uranoscopus scaber Linnaeus, 1759 (Uranoscopidae) from the Ionian Sea (central Mediterranean) has been carried out by combining light and scanning electron microscopy observations and molecular analyses through polymerase chain reaction-restriction fragment length polymorphism and sequencing of the ITS rDNA gene. Results indicate that U. scaber and M. surmuletus represent suitable definitive and intermediate/paratenic hosts of H. fabri, respectively, in the Mediterranean and highlight the importance of combining genetic and morphological data to study the taxonomy and epidemiology of parasites widely distributed in different fish species and aquatic ecosystems.

Higher systematics within the Digenea, Carus 1863 have been relatively stable since a phylogenetic analysis of partial nuclear ribosomal markers (rDNA) led to the erection of the Diplostomida Olson, Cribb, Tkach, Bray, and Littlewood, 2003. However, recent mitochondrial (mt) genome phylogenies suggest this order might be paraphyletic. These analyses show members of two diplostomidan superfamilies are more closely related to the Plagiorchiida La Rue, 1957 than to other members of the Diplostomida. A recent phylogeny based on partial cytochrome c oxidase I also indicates one of the groups implicated, the Diplostomoidea Poirier, 1886, is non-monophyletic. To determine if these results were robust to additional taxon sampling, we analyzed mt genomes from seven diplostomoids in three families. To choose between phylogenetic alternatives based on mt genomes and the prior rDNA-based topology, we analyzed hundreds of ultra-conserved genomic elements assembled from shotgun sequencing. The Diplostomida was paraphyletic in the mt genome phylogeny but supported in the ultra-conserved genomic element phylogeny. We speculate this mitonuclear discordance is related to ancient, rapid radiation in the Digenea. Both ultra-conserved genomic elements and mt genomes support the monophyly of the Diplostomoidea and show congruent relationships within it. The Cyathocotylidae Muhling, 1898 are early diverging descendants of a paraphyletic clade of Diplostomidae Poirier, 1886, in which are nested members of the Strigeidae Railliet, 1919; the results support prior suggestions that the Crassiphialinae Sudarikov, 1960 will rise to the family level. Morphological traits of diplostomoid metacercariae appear to be more useful for differentiating clades than those of adults. We describe a new species of Cotylurus Szidat, 1928, resurrect a species of Hysteromorpha Lutz, 1931, and find support for a species of Alaria Schrank, 1788 of contested validity. Complete rDNA operons from seven diplostomoid species are provided as a resource for future studies.

SUMMARYThe genus Clinostomum Leidy, 1856 (Digenea: Clinostomidae) has been reported in all ecozones of the world and a clear separation between the species of the ‘Old World’ and ‘New World’ has been recognized based on molecular studies. Recent works on Afrotropical species include redescriptions of C. cutaneum and C. phalacrocoracis, while C. tilapiae has yet to be studied using modern taxonomic approaches. In the present research, morphological redescription of C. tilapiae metacercariae from a new host, Synodontis batensoda sampled at Anambra River Basin, Nigeria, together with molecular analysis of nuclear internal transcribed spacer rDNA and cytochrome c oxidase 1 mtDNA are reported. We also provide morphological and molecular data from four further putative species of Clinostomum (morphotypes 1–4) from different areas of Africa, as well as the first report of C. phalacrocoracis in South Africa.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method.

The presence of Cryptosporidium in institutions such as veterinary teaching hospitals, where students and staff are in frequent contact with animals, could represent a serious public health risk. In this study the detection and quantification of the Cryptosporidium oocysts present on the environmental surfaces of an Equine Perinatology Unit (EPU) were investigated. During 3 foaling seasons 175 samples obtained by swabbing an area of the floor and walls of boxes and utility rooms of EPU with sterile gauze, in 3 different moments. Samples were collected at the end of foaling season (July), after washing procedures (September) and after washing and disinfecting procedures, at the beginning of a new foaling season (December). All the samples were subjected to nested-PCR, followed by genotyping and sub-typing methods and to qPCR, allowing the oocyst quantification. Cryptosporidium spp. was detected in 14 samples, of which 11 were from walls and three were from floors. The highest number of oocysts was found in a sample collected from the floor of one utility room used for setting up therapies and treatments. In most cases, oocyst numbers, estimated by qPCR, were reduced or eliminated after washing and disinfecting procedures. The genotyping and sub-typing methods allowed identification of 2 subtypes of C. parvum (IIaA15G2R1 and IIdA23G1) and 1 of Cryptosporidium horse genotype (VIaA15G4) that were described in foals hospitalized at the EPU in the same years. The results of the present study show that qPCR can be used to evaluate Cryptosporidium contamination of environmental surfaces of a veterinary teaching hospital and the efficacy of the disinfection procedures.

In recent years there has been a re-emergence of diphyllobothriasis by Diphyllobothrium latum (Cestoda: Diphyllobothriidae) in Italy, France and Switzerland, where in the past this fish-borne zoonosis was widespread and then virtually disappeared. A change in eating habits such as the consumption of raw/undercooked freshwater fish, has led to an increased risk for consumers of ingesting infective larvae of D. latum. A survey on the factors responsible for the re-emergence of human diphyllobothriasis in Italy was carried out from March 2013 to December 2014. The aim of this study was to assess the diffusion of D. latum plerocercoids in the fish populations of the sub-alpine lakes of Maggiore, Como, Iseo and Garda, updating the scarce historical data and assessing a preliminary “risk level” of the lacustrine environments and fish species under investigation. A total of 2228 fish belonging to 5 species, 690 from Lake Maggiore, 500 from Lake Como, 655 from Lake Iseo and 383 from Lake Garda were submitted to parasitological examination. The presence of D. latum plerocercoid larvae was detected in 6.6%, 25.4% and 7.6% of perch (Perca fluviatilis) from Lakes Maggiore, Como and Iseo respectively. The parasite was also present in pike (Esox lucius) with prevalence values ranging from 71.4 to 84.2% and in 3.6–3.8% of burbot (Lota lota) from Lakes Iseo and Como. Fish from Lake Garda were negative as well as sampled whitefish (Coregonus lavaretus) and shad (Alosa fallax lacustris). The results of this survey showed a widespread presence of D. latum plerocercoid larvae in Maggiore, Como and Iseo fish populations. Urban fecal contamination of water is still a key issue to be resolved, together with the improvement of communication with consumers regarding the best dietary habits and the most effective processes of parasite inactivation, required for the consumption of raw/undercooked fish caught in high-risk areas.

Introduction: The diffusion of new eating habits and the increase of fish products demand lead to a raising risk for consumers inherent food-borne parasitic zoonoses. In this scenario the scientific community and food security authorities are called to assess the possible risks linked to consumption of products from fishery and aquaculture and to set up strategies aimed to their management. Thus, the collection of epidemiological information on the presence of zoonotic parasites in fish products through extensive epidemiological surveys is necessary. Concerning aquaculture, according to EFSA (2010) and Regulation (EU) 1276/2011 only for the Atlantic salmon the risk of transmission of parasites to man can be considered negligible. At this purpose, in the framework of the EU funded project ParaFishControl a wide parasitological survey has been carried out on the main farmed fish species in Italy and other countries such as Spain, Greece, Denmark, Norway and Hungary. The present work reports the results from Italy. Methodology: From 2016 to 2018 a total of 4728 farmed fish have been examined from 5 marine and 5 freshwater farms located in Italy: 1563 gilthead sea bream (GSB), 1571 European sea bass (ESB) and 1594 rainbow trout (RT) have been sampled. Besides harvest quality fish, runts were also examined. Parasitological analyses to search for anisakid nematodes, diphyllobothriid cestodes and Opisthorchioidea digeneans were performed utilizing different methodologies such as visual inspection and candling as provided by the EU regulation, implemented by UV-press method, muscular compression/artificial digestion followed by microscopic examination when required. Results: No zoonotic parasites were found in any of the examined fish, including runts. Only one L4 specimen of the nematode Hysterothylacium fabri has been found on the surface of the liver in one ESB. Conclusion: The results of this survey are very encouraging and allow to assess the risk of the presence of zoonotic parasites in farmed GSB, ESB and RT as negligible, similarly to Atlantic salmon.

AIM OF THE STUDY: Winter disease (WD) is a multifactorial disease found primarily in sea caged gilthead sea bream (Sparus aurata) along the Mediterranean coast.1,2 An emaciative syndrome has been recently observed in Spain and Enterospora nucleophila, a new microsporidian species of the family Enterocytozoonidae has been described; these intracellular, non-forming xenoma microsporidia, opportunistic in nature exploit a w eakened host immune status, as it could happen in WD.6 Teleost intestine contains Mast Cells (MCs), w hose functional properties are similar to those of mammalian mast cells;5 recruitment of MCs to sites of persistent inflammation is a general response in parasites-affected fish. An increased number of the MCs is also reported in WD-affected fish.3,4 In December 2013 a disease outbreak in sea caged gilthead seabream in Italy, affecting 0+ year fish occurred. The aim of this study w as to evaluate the histopathological changes related to these tw o conditions, to characterize the MCs by immunohistochemistry and the microsporidia by PCR. MATERIAL AND METHODS: From tw enty gilthead sea bream intestinal tracts w ere fixed in buffered formalin at the sample site and sent to DIMEVET. Routine histological sections w ere obtained; Luna stain w as also performed. Immunohistochemistry w ith CD117 antibody (1:100, Dako) w as also carried out. Intestinal tissue w as also subjected to molecular analysis; a fragment of the 18S rDNA w as amplified and then sequenced. RESULTS: Hindgut show ed a moderate dilatation of the lumen in association w ith w hitish casts, similar to the milk-like mucous casts reported in WD outbreaks.1,2 A severe mucosal atrophy w ith total folds flattening w as present; w ithin lamina propria and submucosa a mild to moderate MCs hyperplasia and mild mucous cells hyperplasia w ere observed. Multifocally, w ithin enterocytes and rodlet cells, the nucleus and/or cytoplasm contained microsporidian spores, more evident w ith Luna stain. The sequences obtained from intestines show ed 99.9% identity w ith E. nucleophila. Within perivisceral exocrine pancreatic acini, focal necrosis and MCs infiltration have been observed, as reported by other authors during WD outbreaks. CONCLUSIONS: MCs are interpreted as ‘‘standing force’’ in particular tissues consistently exposed to pathogens, in contrast to a ‘‘mobilization force’’ that has been an advantage in those being exposed to noxious agents only occasionally.7 The severe mucosal flattening could be interpreted as an effect of a chronic insult, not only related to the microsporidia infection; other concurrent predisposing factors as those reported in WD could be then considered into the development of an overt pathology. BIBLIOGRAPHY: 1Birincioglu et al, 2013; 2Colorni, 2011; 3Contessi et al,2006; 4Ibarz et al, 2010; 5Lauriano et al.,2012;6 Palenzuela et al.,2014; 7Reite and Evensen,2006.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently 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. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method’s detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.