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A survey of fresh great Mediterranean scallops (Pecten jacobaeus) and frozen queen scallops (Aequipecten opercularis) collected from the northern Adriatic Sea demonstrated the presence of nematode larvae that were morphologically and molecularly identified as Sulcascaris sulcata. The presence of this nematode in the northern Adriatic Sea has been described in the loggerhead sea turtle, which is the final host, but it was only recently described in the intermediate molluscan host. Affected scallops present rust-brown lesions in the adductor muscle. In this study, necroscopic and histopathological lesions are described in affected scallops. Furthermore, the detrimental effects of this parasite on the commercialization of scallops under the current European legislation concerning the hygiene of seafood products are evaluated. [ABSTRACT FROM AUTHOR]

Viral encephalopathy and retinopathy ( VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT- PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock-infected group. VERv presence was confirmed by rRT- PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide. [ABSTRACT FROM AUTHOR]

The article focuses on the first documented occurrence of viral encephalopathy and retinopathy (VER) outbreak in Salaria fluviatilis after its introduction into natural habitats in Catalonia, Spain. It says that the presence of the virus was due to cross-contamination during the shipping of the specimens to the laboratory. It was also hypothesized that the infection could be induced by the origin of the water and the cohabitation of other species such as Gobio lozanoi and Anguilla anguilla.

Gilthead sea bream (GSB) (Sparus aurata) have been generally considered to be resistant to viral encephalo-retinopathy. However, the recent increase of the number of outbreaks in sea bream hatcheries caused by nervous necrosis virus (NNV) called RGNNV/SJNNV, a reassortant virus, has encouraged researchers to investigate the disease pathogenesis in this species, with particular emphasis on fish age. For this purpose, experimental challenges of juveniles and larvae were carried out. Four experimental trials were performed by infecting GSB of different ages with the RGNNV/SJNNV virus: juveniles of 7 g of weight and larvae of 70, 35 and 21 days post hatch (dph). Virological, histological and immunohistochemical analysis, as well as qualitative and quantitative real time PCR at different time points post infection (pi) were performed. ELISA test for antibody detection was also implemented, where applicable. Overall results showed that the gilthead seabream larvae are susceptible to RGNNV/SJNNV infection irrespective to the age, as proved by the massive viral replication detected by quantitative RT-PCR and the massive presence of immunoprecipitates in the nervous tissues evidenced by immunohistochemistry. However, clear clinical signs and mortality were observed only in the youngest group of 21-dph larvae. The viral kinetics investigations led to think that the efficiency of RGNNV/SJNNV replication was higher in 21dph larvae compared to the older ones, which in turn correlated with the disease outcome. Most likely the latter was related to the developmental stage of the immune system. Noteworthy survivors were always tested negative for the presence of specific antibodies. The identification of the factors governing the host ability to control viral replication, or conversely those inhibiting the virus in older fish will be crucial to better understand the disease pathogenesis in this species. Notably, despite the age and the disease outcome of the infection, GSB remained persistently infected for a long time, even up to one year, becoming asymptomatic carriers. • Only sea bream larvae younger than 35 days post-hatch showed clinical signs and mortality after bath infection • Massive viral replication was detected in sea beam larvae of all ages despite the absence of clinical signs • Infected sea bream tested positive for RGNNV/SJNNV in the brain for about 1 year • No seroconversion was detected by ELISA test in survivors [ABSTRACT FROM AUTHOR]

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus ( Nodaviridae , Betanodavirus ). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin ( Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans ., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In order to establish the meaning of data generated in antimicrobial agent susceptibility tests, it is necessary to develop internationally harmonised interpretive criteria. Currently, such criteria have not been developed for data generated in studies of the susceptibility of the fish pathogen Yersinia ruckeri. This work generated the data that would be required to set epidemiological cut-off values for the susceptibility data of this species that had been generated using a standardised disc diffusion method that specified the use of Mueller Hinton agar and incubation at 22°C for 24-28 h. Using this method, sets of inhibition zones data for 4 antimicrobial agents were generated by 3 independent laboratories. The data from these laboratories were aggregated and analysed using the statistically based normalised resistance interpretation. For ampicillin, florfenicol, oxytetracycline and trimethoprim-sulfamethoxazole the cut-off values calculated by this analysis were ≥16, ≥23, ≥24 and ≥30 mm, respectively. Evidence is presented demonstrating that the data for these 4 agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for Y. ruckeri.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Among the numerous pathogenic nontuberculous mycobacteria (NTM), which may cause disease in both poikilothermic and homoeothermic organisms, members of the unique clade Mycobacterium ulcerans/Mycobacterium marinum (MuMC) may cause disease in both fish and humans. Here, we describe the emergence of Mycobacterium pseudoshottsii, one of the four MuMC members, in Israel. For many years, M. marinum was the dominant NTM that was diagnosed in Israel as a fish pathogen. To the best of our knowledge, this is the first isolation and genomic characterization of M. pseudoshottsii infecting edible fish from two different fish species farmed in offshore sea cages in the eastern Mediterranean as well as in a recirculating aquaculture system in Israel. We compared the M. pseudoshottsii whole-genome sequences to all available genomic sequences of MuMC in free, publicly accessible databases. IMPORTANCE Mycobacterium pseudoshottsii was first detected in 1997 in the USA, infecting wild striped bass (Morone saxatilis). Since then, several reports from different countries worldwide have shown its capacity to become established in new regions as well as its pathogenicity to saltwater and euryhaline finfish of different genera. Our phylogenetic analysis revealed that the Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) is divided into two main branches: one that includes M. marinum and M. pseudoshottsii , and the second, which includes other M. marinum isolates as well as two isolates of M. shottsii. Our results reinforce the proposition that the geographical distribution of M. pseudoshottsii is much more extensive than is commonly believed. The emergence of M. pseudoshottsii in different parts of the world and its pathogenic traits that affect finfish of different genera may be a cause for concern among fish farmers, researchers, and environmental organizations., Competing Interests: The authors declare no conflict of interest.

Biological invasions pose a serious threat to local flora and fauna and have negative impacts on ecosystems. Invasive parasites can also cause severe losses in aquaculture. In this article, we provide evidence of the recent spillover of an African parasite with a complex, three-host life cycle that has rapidly and successfully established itself in the Middle East, most likely due to the recent migration of its final hosts (great cormorant) from Africa. This case of parasite introduction into a country with intensive aquaculture is also important from an economic point of view, since large (up to 2 cm long) larvae of this parasite, the cyclophyllidean tapeworm Amirthalingamia macracantha (Cestoda) localised in the liver, can be pathogenic to their fish hosts, including farmed and wild fish, as shown by our histopathological examination of heavily infected fish. Since its first detection in Israel in November 2020, the parasite has spread rapidly and is currently found in both migratory (great cormorant, Phalacrocorax carbo ) and non-migratory birds (pygmy cormorant, Microcarbo pygmaeus ), as well as in fish intermediate hosts, including farmed tilapia in several farms in Israel and wild cichlids. There are numerous examples of the spillover of introduced parasites, including those that parasitise fish of commercial importance, but have a direct life cycle or use only a single intermediate host. Tilapines are the second most important group of farmed fish in the world after carps and are produced mainly in Southeast Asia, Central and South America. The global spread of great cormorants and the early evidence that pygmy cormorant may also harbour A. macracantha pose the risk of further spread of this invasive parasite to other countries and areas. In addition, global warming and reductions in foraging and resting areas near these waters may allow the parasite to complete its life cycle in new hosts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

The interaction of nanomaterials with pollutants in the marine environment might alter bioavailability, as well as toxicity, of both nanomaterials and pollutants, representing a risk, not only for marine organisms, but also for consumers through the marine food chain.The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO 2 NPs) in terms of bioaccumulation and toxicity on Mediterranean mussels ( Mytilus galloprovincialis ) exposed to six-indicator non-dioxin-like polychlorinated biphenyls (ndl-PCBs). Mussels were exposed to ndl-PCBs (20 µg/mL) (groups 3-4) or to a combination of ndl-PCBs (20 µg/mL) and TiO 2 NPs (100 µg/mL) (groups 5-6) for four consecutive days. TiO 2 NPs was detected in groups 5-6 (3247 ± 567 and 1620 ± 223 µg/kg respectively), but their presence did not affect ndl-PCBs bioaccumulation in mussels. In fact, in groups 3-4, the concentration of ndl-PCBs (ranging from 3818.4 ± 166.0-10,176 ± 664.3 µg/kg and 2712.7 ± 36.1-9498.0 ± 794.1 µg/kg respectively) was not statistically different from that of groups 5-6 (3048.6 ± 24.0-14,635.9 ± 1029.3 and 5726.0 ± 571.0-9931.2 ± 700.3 µg/kg respectively). Histological analyses showed alterations to the structure of the gill tissue with respect to the control groups, with more severe and diffuse dilatation of the central hemolymphatic vessels of the gill lamellae in groups 5-6 (treated with TiO 2 NPs and ndl-PCBs concurrently) compared to groups 3-4 (ndl-PCBs only). Finally, in mussels submitted to a seven-day depuration process, most TiO 2 NPs were eliminated, and NPs had a synergistic effect on ndl-PCBs elimination; as a matter of fact, in groups 5-6, the percentage of concentration was statically inferior to the one observed in groups 3-4. In any case, consumers might be exposed to TiO 2 NPs and ndl-PCBs (both concurrently and separately) if edible mussels, harvested in a contaminated environment, are consumed without a proper depuration process.

The accurate determination of the local temperature is one of the most important challenges in the field of nanotechnology and nanomedicine. For this purpose, different techniques and materials have been extensively studied in order to identify both the best-performing materials and the techniques with greatest sensitivity. In this study, the Raman technique was exploited for the determination of the local temperature as a non-contact technique and titania nanoparticles (NPs) were tested as nanothermometer Raman active material. Biocompatible titania NPs were synthesized following a combination of sol-gel and solvothermal green synthesis approaches, with the aim of obtaining pure anatase samples. In particular, the optimization of three different synthesis protocols allowed materials to be obtained with well-defined crystallite dimensions and good control over the final morphology and dispersibility. TiO 2 powders were characterized by X-ray diffraction (XRD) analyses and room-temperature Raman measurements, to confirm that the synthesized samples were single-phase anatase titania, and using SEM measurements, which clearly showed the nanometric dimension of the NPs. Stokes and anti-Stokes Raman measurements were collected, with the excitation laser at 514.5 nm (CW Ar/Kr ion laser), in the temperature range of 293-323 K, a range of interest for biological applications. The power of the laser was carefully chosen in order to avoid possible heating due to the laser irradiation. The data support the possibility of evaluating the local temperature and show that TiO 2 NPs possess high sensitivity and low uncertainty in the range of a few degrees as a Raman nanothermometer material.

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout.

Marine invertebrates such as rotifers or Artemia , frequently used for fish larvae feeding, can be a potential source of pathogens. It has been demonstrated that Artemia can act as a nervous necrosis virus (NNV)-vector to Senegalese sole larvae. Therefore, in this study, we aimed to clarify the role of rotifers in NNV transmission to sea bass larvae following an oral challenge. Our results showed that sea bass larvae fed on a single dose of rotifers retaining NNV displayed clinical signs, mortality, and viral replication similar to the immersion challenge, although the course of the infection was slightly different between the two infection routes. Furthermore, we also demonstrated that rotifers can internalize NNV particles due to their filtering nature and maintain virus viability since viral particles were detected by immunohistochemistry, immunofluorescence, and cell culture within the rotifer body. However, viral quantification data suggested that rotifers are not permissive to NNV replication. In conclusion, this research demonstrated NNV horizontal transmission through rotifers to sea bass larvae, highlighting the importance of establishing strict routine controls on live food to prevent the introduction of potential pathogens to hatcheries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vázquez-Salgado, Pascoli, Marsella, Biasini, Buratin, Pretto, Abbadi, Melchiotti, Bandín and Toffan.)

The redclaw crayfish (Cherax quadricarinatus) is a freshwater decapod crustacean, cultured in numerous countries worldwide for both food and ornamental purposes. Redclaw crayfish has become an important aquaculture species due to its physical and biological traits, relatively easy breeding, and a short growing-out period to reach commercial size. Bacterial infections are the second-most studied pathogens of freshwater crayfish. However, redclaw crayfish rickettsiosis, caused by Coxiella cheraxi, was reported in only a few scientific papers in the early 2000s, in Australia and Ecuador. Coxiella cheraxi is a rod-shaped intracellular bacterium that can cause mortality of 22%-80% in naturally infected crayfish. In experimental infections, mortality rates may be even higher (40%-90%). Coxiella cheraxi is closely related to Coxiella burnetii, the agent of Q-fever, which affects ruminants (goats, sheep, and cattle) and occasionally may cause zoonotic infections. According to the scientific knowledge available, C. cheraxi is a species-specific pathogen because it has been only detected in Cherax quadricarinatus and thus far, there is no evidence of a zoonotic potential. In this study, we describe an outbreak of rickettsiosis in a batch of redclaw crayfish imported to Israel from an Australian hatchery, observed 2 months after introduction in a quarantine facility. Initial mortality was evaluated through histopathology, revealing infection by rickettsia-like organisms (RLO) that were subsequently investigated by molecular analysis and transmission electron microscopy examination. Phylogenetic analysis revealed that the detected RLO were closely related to C. cheraxi from a single source (Australian strain TO98), available in free publicly accessible databases. After 5 months in quarantine, almost 99% of the crayfish population had died. Our findings raise valuable questions related to aquatic animal trade and the importance of mitigation measures, such as quarantine and routine diagnostic procedures, to limit the spread of infectious diseases., (© 2021 Wiley-VCH GmbH.)

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described., (© 2021 John Wiley & Sons Ltd.)

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass ( Dicentrarchus labrax ) in the Mediterranean. In the current studies, we tested a novel Pichia -produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia -like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae , the genera Mycoplasma and Francisella , and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens., (Copyright © 2020 Cano, Ryder, Webb, Jones, Brosnahan, Carrasco, Bodinier, Furones, Pretto, Carella, Chollet, Arzul, Cheslett, Collins, Lohrmann, Valdivia, Ward, Carballal, Villalba, Marigómez, Mortensen, Christison, Kevin, Bustos, Christie, Green and Feist.)

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii , Shewanella putrefaciens , Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae.

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population., (© 2020 John Wiley & Sons Ltd.)

The aim of this study was to evaluate the bioaccumulation of titanium dioxide nanoparticles (TiO 2 NPs) in edible mussels bred in polluted artificial seawater. An in vivo study was conducted by exposing mussels to different concentrations of TiO 2 NPs (0.25 mg/L and 2.5 mg/L) or ionic titanium (1.6 mg/L) for 4 days. Inductively coupled plasma mass spectrometry (ICP-MS) showed titanium presence in all groups proportional to exposure levels (concentration range: 209-1119 µg/kg). Single particle ICP-MS revealed NPs in both TiO 2 NP treated mussels (concentration range: 231-1778 µg/kg) and in ionic titanium treated mussels (concentration 1574 µg/kg), suggesting potential nanoparticle formation in vivo. These results were confirmed by transmission electron microscopy with energy dispersive X-ray detection. Nonetheless, mussels eliminated more than 70% of the TiO 2 NPs after 3 days' depuration. These results show the potential for consumer exposure to TiO 2 NPs when contaminated mussels are consumed without a proper depuration process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species., (© 2019 John Wiley & Sons Ltd.)

Stocking density is considered a stress factor for fish and is therefore one of the numerous concerns about fish welfare in an aquaculture context. Stress coping styles (SCS) are defined as a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context and appear to be promising for improving fish welfare in aquaculture. The aim of the present study was to describe the physiological and zootechnical performances of gilthead sea bream (Sparus aurata) at different stocking densities (low density, LD: 15 kg/m 3 and high density, HD: 30 kg/m 3 ), depending on individual SCS. To do so, the fish SCS were first screened by measuring boldness (prior to the experiment). Three consecutive samplings were performed over the experiment to measure several blood parameters, including hematocrit (Hct), red blood cell count (RBCC), hemoglobin (Hb), cortisol, adrenalin, noradrenalin, glucose, lactate, and lysozyme, to infer the consequence of the SCS profile on the welfare condition in response to stocking density. Finally, swimming activity was recorded in a subsample of individuals (9 BOLD and 9 SHY individuals per density), and BOLD individuals displayed higher swimming activity than SHY ones at HD, while the opposite pattern was observed at LD. According to principal component analysis, physiological parameters are linked to the SCS profile, mostly at the beginning of the experiment, while density effects on physiology remain during the entire experiment duration. In conclusion, regarding all the variables observed, fish SCS appeared to be promising criteria to select the most adaptive individuals relating to rearing conditions and therefore improve welfare., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The redclaw crayfish Cherax quadricarinatus (von Martens, 1868) is one of the most commonly exploited freshwater crayfish species worldwide. Redclaw crayfish are susceptible to a number of pathogens but none have been linked to widespread epizootics. Mycobacterial infections have been sporadically reported in crayfish. In the case described, histopathology and bacterial identification confirmed an opportunistic infection caused by Mycobacterium gordonae in a hatchery of C. quadricarinatus in Israel. Intranuclear inclusion bodies, recorded in cells of the tubular epithelium of the hepatopancreas by histopathology, indicate a co-infection with a viral agent, referable to C. quadricarinatus bacilliform virus (CqBV). To the best of our knowledge this is the first description of mycobacteriosis in redclaw crayfish.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.

This is the first description of a betanodavirus infection in the dusky grouper Epinephelus marginatus within the marine protected areas (MPAs) of the Balearic Islands. Histopathology techniques were employed to describe neurological lesions in infected fish. Abnormal swimming, mortality, and neurological lesions were detected in all analysed grouper individuals. Virus particles were observed by means of transmission electron microscopy. Reverse transcription of RNA1 and RNA2 followed by cDNA amplification and sequencing allowed viral classification. Phylogenetic analysis showed the isolates from wild E. marginatus of the Balearic Islands MPAs to be closely related to Dicentrarchus labrax and Mullus barbatus strains from Cyprus and Italy. Although vertical transmission from infected spawners has been described as the major route for nodavirus infection, we point out in this work that horizontal transmission among sub-clinical fishes after migration or commercial import for aquaculture production could play a major role in the spreading of the disease in MPAs.

The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993-2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities., (© 2018 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

The microsporidiosis of the endangered white-clawed crayfish Austropotamobius pallipes complex has generally been attributed to only one species, Thelohania contejeani, the agent of porcelain disease. Species identification was mostly assessed by macroscopic examination or microscopic evaluation of muscle samples rather than by molecular or ultrastructural analyses. A survey conducted on A. pallipes complex populations in Northern Italy highlighted the presence of two different microsporidia causing similar muscular lesions, T. contejeani and an undescribed octosporoblastic species Vairimorpha austropotamobii sp. nov. Mature spores and earlier developmental stages of V. austropotamobii sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, which gave rise to a rosette-shaped plasmodium, and eight uninucleate spores were produced within the persistent SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and an average of 3.9 × 2.2 µm in size. The polar filament was coiled 11-14 times, lateral to the posterior vacuole. The small subunit ribosomal RNA gene (SSU rRNA) and the large subunit RNA polymerase II gene (RPB1) of V. austropotamobii sp. nov. were sequenced and compared with other microsporidia. The highest sequence identity of SSU rRNA (99%) and RPB1 (74%) genes was with the amphipod parasite Nosema granulosis and subsequently with V. cheracis, which infects the Australian yabby Cherax destructor. In our work we discuss about the reasons for placing this new species in the genus Vairimorpha. In addition, we provide for T. contejeani a RPB1 gene sequence, supplemental sequences of SSU rRNA gene and ultrastructural details of its sporogony in the host A. pallipes complex., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: Fish kills are events of strong emotional impact on the population because of the frequent suspicion that they can be the result of serious pollution accidents. As a matter of fact, they are often due to natural occurrences, such as low levels of dissolved oxygen in the water, but in many cases the causes remain unknown. Fish are particularly sensitive to pesticides and pyrethroids are reported to be the most ecotoxicologically active in the aquatic environment. Nevertheless, the reported cases of massive wild fish mortalities due to these toxicants are very few. This paper describes a fish kill episode occurred in the Padua Province (Veneto Region - North Eastern Italy) which involved several fish species and for which it was possible to identify the cause in the presence of pyrethroids in the water., Case Presentation: When a whitish liquid coming from the rainwater drain of an industrial area was seen to be spilling into a drainage channel, a fish massive mortality was noticed and investigated. The collected water samples showed the presence of relevant concentrations of cypermethrin, permethrin, deltamethrin and tetramethrin. Analyses on the fish tissues revealed the presence of cypermethrin and permethrin at a concentration range of 476-2834μg/kg and 346-2826μg/kg on a lipid basis, respectively., Discussion: According to the results of the performed analyses, we can reasonably state that the described episode had been caused by the exposure of biota to high concentrations of pyrethroids. The present case report significantly contributes to the limited literature available on pesticides-related fish kills. Moreover, it highlights the importance of sharing protocols for fish kill management at a national level, as this would help to better define the roles of the different institutions involved and to improve the investigation and the reporting of these events., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Viral nervous necrosis (VNN) certainly represents the biggest challenge for the sustainability and the development of aquaculture. A large number of economically relevant fish species have proven to be susceptible to the disease. Conversely, gilthead sea bream has generally been considered resistant to VNN, although it has been possible to isolate the virus from apparently healthy sea bream and sporadically from affected larvae and postlarvae. Unexpectedly, in 2014-2016 an increasing number of hatcheries in Europe have experienced mass mortalities in sea bream larvae. Two clinical outbreaks were monitored over this time span and findings are reported in this paper. Despite showing no specific clinical signs, the affected fish displayed high mortality and histological lesions typical of VNN. Fish tested positive for betanodavirus by different laboratory techniques. The isolates were all genetically characterized as being reassortant strains RGNNV/SJNNV. A genetic characterization of all sea bream betanodaviruses which had been isolated in the past had revealed that the majority of the strains infecting sea bream are actually RGNNV/SJNNV. Taken together, this information strongly suggests that RGNNV/SJNNV betanodavirus possesses a particular tropism to sea bream, which can pose a new and unexpected threat to the Mediterranean aquaculture.

Background: Hikui disease is a well known disfiguring disease of koi carp (Cyprinus carpio) primarily affecting fish with red pigmentation. It causes light orange to golden yellow, multifocal to coalescing raised patches, starting from the red cutaneous areas. Some cases respond to surgery or topical treatment, but recurrence is common., Objectives: To describe the clinical and pathological presentation of Hikui disease and its cause., Animals: Nine affected koi carp belonging to private hobbyists., Methods: Eight fish underwent surgery or biopsy; one was euthanized. Tissues were submitted for histology, immunohistochemistry and transmission electron microscopy., Results: Five fish showed typical lesions of Hikui disease, whereas four fish showed an atypical presentation characterized by focal or multifocal, oedematous, dark red cutaneous plaques or nodules. Histology showed unencapsulated, infiltrating and densely cellular neoplasms composed of spindle cells arranged in bundles, rows and whorls frequently centred on capillaries. Immunohistochemistry for smooth muscle actin labelled neoplastic cells in all cases. Ultrastructure showed neoplastic cells with slender cytoplasmic processes encircling the capillaries, a thin basal membrane and occasional plasmalemmal vesicles., Conclusions and Clinical Importance: All of the data supported a neoplastic process producing perivascular wall tumours. Immunoreactivity to smooth muscle actin and the ultrastructural features were indicative of a pericyte origin (haemangiopericytoma). This is the first report dealing with Hikui disease that has achieved a conclusive diagnosis. The neoplastic nature of this condition suggests the potential usefulness of a surgical approach in the clinical management of less severe cases., (© 2016 ESVD and ACVD.)

The complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring-Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6-12 month old spat whereas subsequent trials with 2-3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a μdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant μVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The wedge clam Donax trunculus Linnaeus, 1758 is one of the most common bivalve molluscs inhabiting the sandy shores of the Mediterranean Sea and is considered an important commercial resource. In this study, we report the first molecular, morphological and histopathological descriptions of metacercariae from a trematode belonging to the genus Postmonorchis (Digenea: Monorchiidae) that infects D. trunculus in natural beds of the Italian Tyrrhenian coast (Campania, Lazio and Tuscany). Morphological analysis of the parasite revealed a combination of features that exist in the 3 previously identified species of Postmonorchis, viz. P. donacis, P. variabilis and P. orthopristis, with the addition of new, distinctive morphological characteristics. The pathogen exhibited a predilection for the gill; however, it was also present in the labial palp and mantle in addition to the gut, kidney epithelium and foot. The inflammatory response was characterised by either a focal or diffuse haemocyte infiltration followed by the formation of multiple, large multi-layered capsules associated with tissue destruction. The prevalence of the pathogen ranged from 75 to 100%, while the infection intensity fluctuated among the study areas. Further studies regarding the life cycle of this parasite and the identification of other larval and adult stages and their respective hosts may confirm the identification of a new species of Postmonorchis that infects wedge clams in Mediterranean waters. The study of the parasite is completed by molecular analysis of the ITS1 and ITS2 rDNA sequences.