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Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.

Pinna nobilis populations, constituting the largest bivalve mollusk endemic to the Mediterranean, is characterized as critically endangered, threatened by extinction. Among the various factors proposed as etiological agents are the Haplosporidium pinnae and Mycobacterium sp. parasites. Nevertheless, devastation of the fan mussel populations is still far from clear. The current work is undertaken under a broader study aiming to evaluate the health status of Pinna nobilis population in Aegean Sea, after the mass mortalities that occurred in 2019. A significant objective was also (a) the investigation of the etiological agents of small-scale winter mortalities in the remaining populations after the devastating results of Haplosporidium pinnae and Mycobacterium sp. infections, as well as (b) the examination of the susceptibility of the identified bacterial strains in antibiotics for future laboratory experiments. Microbiological assays were used in order to detect the presence of potential bacterial pathogens in moribund animals in combination with molecular tools for their identification. Our results provide evidence that Vibrio bacterial species are directly implicated in the winter mortalities, particularly in cases where the haplosporidian parasite was absent. Additionally, this is the first report of Vibrio mediterranei and V. splendidus hosted by any bivalve on the Greek coastline.

The purpose of this work was to investigate the size-relative effectiveness of clove oil as an anaesthetic for rainbow trout and goldfish. In total, 128 rainbow trout (Oncorhynchus mykiss) (two groups of 20-23 and 30-33 cm mean fork length) and 160 goldfish (Carassius auratus) (four size groups of 1.5-2.5, 5-7, 11-15 and 20-25 cm) were anaesthetized at different clove oil concentrations of 50, 100, 150 mg·l -1 for trouts and 75, 100, 150 mg·l -1 for goldfish. Rainbow trout exhibited total loss of balance and no response to external stimuli with shorter induction time as dosage increased (120.5 s, 64.4 s and 44.3 s, respectively). Goldfish exhibited total loss of balance and no response to external stimuli after induction time that varied with dosage used and body size of fish. The small fish (1.5-7 cm) exhibited shorter induction time which ranged from 84.28 s at 75 mg·l-1 clove oil to 41.14 s at 150 mg·l-1 clove oil. The larger fish had a longer induction time inversely related to the dosage. Recovery time was longer than induction time in both species. Both species recovered within 6 min after anaesthesia at 150 mg·l-1 clove oil. Clove oil did not produce marked changes (P 0.05). For both fish species, clove oil was effective, producing minimum stress and zero mortalities, and can be recommended as an effective anaesthetic. Syzygium aromaticum, fish anaesthesia, animal welfare Rapid expansion of the aquaculture industry that occurred in previous decades prompted scientific debates on the potential suffering of fish being handled during common aquaculture procedures or during slaughtering. Research aimed at lessening the suffering of cultured fish is vital to meet the concern for farmed fish welfare (Ashley 2007). Handling stress and various manipulations in aquaculture can have a negative impact on fish health and their growth (Hoskonen and Pirhonen 2006). Anaesthetics are therefore applied to reduce these negative effects to the minimum. The dosage required to induce general anaesthesia varies according to the anaesthetic used and other factors such as water temperature, hardness, salinity, oxygen concentration, length of exposure, body weight, the ratio of gill area/body surface area and the species of fish. In general, small fish are more sensitive to anaesthesia than larger fish (Ross and Ross 1999). An ideal anaesthetic for fish should induce anaesthesia in less than 3 to 5 min, with total loss of balance and muscle tone, allowing an uneventful and rapid (i.e. less than 10 min) recovery with low tissue residues after recovery, thus being safe to users and consumers. The anaesthetic should be inexpensive and easy to use (Gilderhus and Marking 1987

The causal agent of viral encephalopathy and retinopathy in fish is an RNA virus known as nodavirus, a member of the Nodaviridae family. According to published data, sea bream (Sparus aurata L.) that are infected with the virus show no clinical signs and are considered an asympto- matic carrier. In this paper we report for the first time a clinical case of nodavirus in commercially reared sea bream. Fish with clinical signs underwent necropsy and routine microbiological, para- sitological, and histopathological examination. The virus was detected by RT-PCR and isolated in SSN-1 cell cultures. The results described in this study demonstrate the presence of a nodavirus agent that can infect sea bream in sea water, causing a disease similar to that observed in marine sea bass but with lower mortality and milder neurological signs. Histopathological lesions were also similar to those observed in sea bass infected with nodavirus.