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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.

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.

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 novelPichia-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.

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.

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.

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.

The aim of this study was to evaluate the bioaccumulation of titanium dioxide nanoparticles (TiO2NPs) in edible mussels bred in polluted artificial seawater. An in vivo study was conducted by exposing mussels to different concentrations of TiO2NPs (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 TiO2NP 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 TiO2NPs after 3 days’ depuration. These results show the potential for consumer exposure to TiO2NPs when contaminated mussels are consumed without a proper depuration process.

Analytical methods able to differentiate fresh from frozen/thawed fish products are needed for suppliers and official authorities to prevent food frauds. In this study we applied a metabolomics approach to identify markers of freezing/thawing in skinless European sea bass (Dicentrarchus labrax) fish fillets. To this end, one hundred and twenty fish samples (sixty fresh and sixty frozen/thawed) were analysed in positive and negative ion modes by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). A combination of a supervised classification method by partial least square-discriminant analysis (PLS-DA), and Recursive Feature Elimination (RFE) allowed us to discover the most informative metabolites useful to codify fresh and defrosted European sea bass. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) metabolites were identified as the most relevant predictors. These were used to build a model capable of classifying European sea bass samples according to their fresh or frozen/thawed condition. The model showed accuracy, sensitivity and specificity of 100 %, when tested to an independent set of European sea bass undergoing the same experimental conditions. EPA and DHA were found to be altered even in a set of twenty fresh and frozen/thawed Atlantic salmon (Salmo salar), opening the way for the development of a targeted method aimed at providing robust species-independent tests to be applied in the seafood chain by producers and competent authorities.

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.

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 astacicauses crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction ofA. astaciinto Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carryA. astaciin their cuticle as a benign infection.Aphanomyces astacirapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis ofA. astacipure cultures characterized five genotype groups (A, B, C, D and E). CurrentA. astacigenotyping 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 developA. astacigenotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts ofA. astacipure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence ofA. astacigenotype groups A and B in UK waters.

Mycobacteriosis is a chronic and often lethal disease in fish, affecting a wide range of cultured and wild species worldwide. Several species of the genus Mycobacterium have been isolated from fish, with M. marinum being the most significant due to its wide host spectrum, economic impact in aquaculture, and zoonotic potential. The damage caused by mycobacteriosis to intensive fish farming and the ornamental trade has been extensively reported, and there is currently no effective treatment other than depopulation and facility disinfection. In this study, we describe a case of cutaneous granulomatous mycobacteriosis in gilthead seabream (Sparus aurata) reared in two onshore recirculating aquaculture systems (RAS) in Israel. External clinical signs of disease (cutaneous hyperplastic nodules) in the two RAS were similar in their appearance but their prevalence differed: in RAS-A, the prevalence was relatively low (1%), whereas in RAS-B, prevalence varied with time (up to 33%). Histopathological findings in both RAS showed the presence of granulomatous lesions limited to the hypodermis in all specimens, and less frequently in the coelomic parenchymas. Most of the cutaneous nodules consisted of multiple discrete to coalescent mature granulomas and pre-granulomatous lesions. In all analyzed specimens, acid-fast bacilli, consistent to the genus Mycobacterium, were positive by Ziehl–Neelsen staining. Bacteria were isolated on selective media (solid and liquid), and identified as M. marinum by molecular and mass spectrometry (MALDI-TOF) analyses. The spectra of M. marinum isolates in this study were compared to M. marinum isolates from clinical cases in humans. Mycobacterium marinum isolated from both RAS showed similarity to those in four human cases, diagnosed by the National Mycobacterium Reference Centre of Israel, which underline the zoonotic potential of these isolates.

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.

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.

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.

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.

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 476–2834 μg/kg and 346–2826 μg/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.

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/m3 and high density, HD: 30 kg/m3), 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.

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. © Inter-Research 2013 · www.int-res.com.

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. Resume Contexte La maladie d'Hikui est une atteinte deformante bien connue de la carpe koi (Cyprinus carpio) affectant primairement les poissons a pigmentation rouge. Elle cause des patchs orange clair a jaune, multifocaux a coalescents, partant des zones cutanees rouges. Certains cas repondent a la chirurgie ou au traitement topique mais les recidives sont frequentes. Objectifs Decrire la presentation clinique et pathologique de la maladie d'Hikui et de ses causes. Sujets Neufs carpes koi atteintes appartenant a un proprietaire prive. Methodes Huit poissons ont subi une chirurgie ou une biopsie; un a ete euthanasie. Les tissus ont ete soumis pour histologie, immunohistochimie et microscopie electronique a transmission. Resultats Cinq poissons ont montre les lesions typiques de la maladie d'Hikui, tandis que quatre poissons ont montre une presentation atypique caracterisee par des nodules ou des plaques cutanees rouges sombres, œdemateuses focales ou multifocales. L'histologie a montre des cellules neoplasiques encapsulees, infiltrantes et denses composees de cellules simples arrangees en amas, lignes et faisceaux frequemment centres sur des capillaires. L'immunohistochimie pour l'actine des cellules musculaires lisses ont montre des cellules neoplasiques dans tous les cas. L'ultrastructure a montre des cellules neoplasiques avec processus cytoplasmique allonges entourant les capillaires, une membrane basale fine et des vesicules plasmalemmales occasionnelles. Conclusions et importance clinique Toutes les donnees supportent un processus neoplasique produisant des tumeurs des parois vasculaires. L'immunoreactivite a l'actine des cellules musculaires lisses et les criteres ultrastructuraux etaient evocateurs d'une origine perycytaire (hemangiopericytome). Ceci est la premiere description de la maladie d'Hikui qui aboutit a une conclusion diagnostique. La nature neoplasique de cette atteinte suggere l'utilite potentielle d'une approche chirurgicale pour la gestion clinique des cas les moins severes. Resumen Introduccion La enfermedad de Hikui es una enfermedad bien conocida deformante de los peces koi (Ciprinus carpio) que afecta primariamente a los peces con pigmentacion roja. Produce una descolocacion anaranjada a amarillenta multifocal confluente en placas elevadas empezando en las zonas de color rojo. Algunos casos responden a cirugia o tratamiento tropico, pero es frecuente observar recidiva. Objetivos describir la presentacion clinica y patologica de la enfermedad de Hikui y su causa Animales nueve carpas koi de propietarios privados afectadas por la enfermedad Metodos ocho de los peces sufrieron cirugia o biopsia; uno fue eutanasiado. Los tejidos fueron remitidos para estudio histopatologico, inmunohistoquimica y microscopia electronica de transmision. Resultados cinco peces mostraron signos tipicos de la enfermedad de Hikui y 4 peces mostraron una presentacion atipica caracterizada por nodulos o placas focales o multifocales, edematosas, de color rojo oscuro. La histopatologia demostro la presencia de neoplasias no encapsuladas, infiltrativas y densamente celulares de celulas fusiformes organizadas en haces, filas y espirales frecuentemente centradas alrededor de capilares. La inmunohistoquimica para actina de musculo liso marco las celulas neoplasicas en todos los casos. El analisis ultraestructural mostro celulas neoplasicas con procesos citoplasmicos delicados alrededor los capilares y una fina membrana basal y ocasionalmente vesiculas en el plasmalema Conclusion e importancia clinica toda la informacion indica que la enfermedad de Hikui es un proceso neoplasico de celulas perivasculares. La reactividad para actina de musculo liso y las caracteristicas ultraestructurales son indicativas de un origen en pericitos (hemangiopericitoma). Este es la primera publicacion tratando la enfermedad de Hikui que llega a un diagnostico conclusivo. La naturaleza neoplasica del proceso sugiere el uso potencial de la cirugia en el manejo clinico en los casos menos severos. Zusammenfassung Hintergrund Die Hikui Erkrankung ist eine gut bekannte verunstaltende Erkrankung des Koi Karpfen (Cyprinus carpio), die in erster Linie Fische mit Rotfarbung betrifft. Sie verursacht hell orange bis goldgelb verfarbte, multifokale bis koaleszierende erhabene Flecken, die von den roten Hautgebieten ausgehen. Einige Falle werden durch chirurgische Intervention oder durch topische Behandlung besser, aber ein Wiederauftreten kommt haufig vor. Ziele Eine Beschreibung der klinischen und pathologischen Prasentation der Hikui Erkrankung und ihre Ursache. Tiere Neun betroffene Koi Karpfen, die privaten Hobbyhaltern gehorten. Methoden Acht Fische wurden chirurgisch behandelt oder es wurden Biopsien genommen; ein Fisch wurde euthanasiert. Die Gewebeproben wurden zur histologischen und zur immunhistochemischen Untersuchung sowie zur Untersuchung mittels Transmissionselektronenmikroskop eingesendet. Ergebnisse Funf Fische zeigten typische Veranderungen der Hikui Erkrankung, wahrend vier Fische eine atypische Prasentation aufwiesen, die durch fokale oder multifokale, odomatose, dunkelrote kutane Plaques oder Knoten charakterisiert war. Histologisch zeigte sich das Bild von nicht eingekapselten, infiltrierenden und dichten zellularen Neoplasmen, die aus Spindelzellen zusammengesetzt waren, die in Reihen und Wirbeln angeordnet waren, die sich haufig um die Kapillaren drangten. Immunhistochemisch wurde mittels markiertem Aktin der glatten Muskulatur in allen Fallen neoplastische Zellen nachgewiesen. Die Ultrastruktur zeigte neoplastische Zellen mit zarten zytoplasmatischen Fortsatzen in der Umgebung der Kapillaren, eine dunne Basalmembran und gelegentliche Membranblaschen. Schlussfolgerungen und klinische Bedeutung Alle Untersuchungsergebnisse bekraftigten das Vorkommen eines neoplastischen Prozesses, durch den perivaskulare Wandtumoren entstanden. Die Immunreaktion auf Aktin der glatten Muskulatur und die ultrastrukturellen Merkmale waren ein Hinweis auf perizytaren Ursprung (Hamangiperizytom). Es handelt sich hierbei um den ersten Bericht, welcher sich mit der Hikui Erkrankung befasst, und eine eindeutige Schlussfolgerung zulasst. Die Tatsache, dass es sich hierbei um einen neoplastischen Prozess handelt, weist auf die potentielle Sinnhaftigkeit eines chirurgischen Eingriffs im Zuge des klinischen Managements in weniger schweren Fallen hin. 要約 背景 緋喰い症は、主に赤い色素をもつ魚に罹患する、鯉 (Cyprinus carpio)の外見を損なうよく知られた疾患である。皮膚の赤い部位から始まる、淡いオレンジ色から黄金色の多病巣性から癒合性の隆起性局面を生じる。外科手術や外用療法に反応する症例もあるが、一般的には再発する。 目的 緋喰い症の臨床および病理学的な所見とその原因を解説することである。 供与動物 個人愛好家が所有する9匹の罹患した鯉 方法 8匹の鯉は外科手術あるいは生検を行い、1匹は安楽死された。組織は組織学、免疫組織化学、および透過電子顕微鏡法のために提出された。 結果 5匹の鯉は緋喰い症の典型的な病変を示していた一方、4匹は限局性あるいは多病巣性で浮腫性の暗赤色の皮膚局面あるいは結節を特徴とする非典型的な外貌を示していた。組織学では被膜に覆われていない、浸潤性および束状、一列に並び、しばしば毛細血管が中央に存在する螺旋状に配列する紡錘形細胞により構成される細胞が密に詰まった腫瘍を特徴としていた。平滑筋アクチンに対する免疫組織化学ではすべての症例で腫瘍細胞が標識されていた。超微細構造法では毛細血管を取り囲むわずかな細胞質を伴った腫瘍細胞、薄い基底膜、および時折みられる細胞質内小胞が認められた。 結論および臨床的な重要性 すべてのデータは血管周囲壁腫瘍を形成している腫瘍過程であることを支持していた。平滑筋アクチンに対する免疫反応性および超微細構造的特徴は周皮細胞由来(血管周皮腫)であることを示していた。この報告は確定診断が得られた緋喰い症の最初の報告である。この疾患の腫瘍の性質は、重症度の低い症例の臨床的な管理における、外科的なアプローチの潜在的な有用性を示唆している。 摘要 背景 众所周知,Hikui病是一种影响锦鲤美观的疾病,主要感染鱼的红色皮肤区域。该病从红色区域开始,皮肤变浅橙色或金黄色,引起多病灶或多斑块融合。有些病例需要手术或局部治疗,但经常复发。 目的 描述Hikui病临床症状和病理学表现以及病因。 动物 9条属于私人发烧友的Hikui患病锦鲤。 方法 8条鱼进行外科手术或活组织取样;其中一条进行安乐死。将取样组织进行组织学、免疫组化和透射电子显微镜检查。 结果 五条鱼表现Hikui病的特征病变,四条鱼表现为单独病变或多灶性病变、水肿、深红色皮肤斑块或结节等非典型特征病变。组织学表现为无明显界限的、浸润性、密集的肿瘤细胞,由成簇、列或常见螺旋状围绕在毛细血管周围的纺锤形细胞组成。所有病例免疫组化用平滑肌肌动蛋白标记肿瘤细胞。超微结构显示肿瘤细胞细长的细胞质包围着毛细血管、薄基底膜,偶尔围绕质膜小泡。 总结和临床意义 所有的数据显示,该病为血管壁肿瘤。平滑肌肌动蛋白的免疫反应和超微结构特征。提示为外膜细胞源性(血管外皮细胞瘤)。这是第一篇有关Hikui病确诊方面的报道。对于临床少数需要手术治疗的严重病例,肿瘤的性质对于手术通路的选择具有提示意义。

Cherax destructor is a crayfish endemic to south-eastern Australia and one of the last alien crayfish to be introduced in Italy. In the Laghi di Ninfa Natural Reserve (Latium region, Central Italy), the species was probably introduced in 1999, but only reported for the first time in 2008. Nearby this area, the most widespread alien crayfish is the red swamp crayfish Procambarus clarkii. In the Natural Reserve, between 2008 and 2013 and during spring and summer, crayfish sampling was carried out with baited traps to assess the distribution of C. destructor and its possible relationship with P. clarkii. Cherax destructor was first recorded in 2008; few P. clarkii were detected in the cultivation ponds where C. destructor was present in 2012 and 2013. Moreover, crayfish plague analyses evidenced a positive result in two out of the 12 sampled P. clarkii. Cherax destructor is now completely absent from the Natural Reserve, while P. clarkii has spread in the area and was probably responsible for this eradication since C. destructor is vulnerable to crayfish plague which was also detected in the area. An ecosystem restoration project in the area favoured the spread of. P. clarkii; the implications of this intervention are discussed.

Vibrio harveyi è una specie patogena emergente per l’acquacoltura marina a livello globale, nelle specie ittiche e nei gamberi peneidi. In Dicentrarchus labrax, V. harveyi è stato isolato in episodi di mortalità durante le fasi di ingrasso (40-160 gr). 41 ceppi identificati biochimicamente come V. harveyi, appartenenti alla ceppoteca dell’Università di Bologna e del CSI (IZSVe), isolati in branzino a partire dal 2008 da 12 impianti di allevamento italiani (gabbie a mare e vasche a terra), sono stati sottoposti ad analisi comparative fenotipiche, genetiche e del profilo proteico. La capacità discriminante dei vari metodi è stata valutata raffrontando questi ceppi di campo con ceppi di referenza di V. harveyi e di Vibrionaceae filogeneticamente associate al clade Harveyi quali V. rotiferianus, V. owensii, V. campbelli, V. alginolyticus, V. parahaemolyticus, V. natriegens, V. mytili. Sono state inoltre comparate specie causa di patologia in branzino (Photobacterium damselae ssp. piscicida, P. damselae ssp. damselae, V. anguillarum, V. ordalii, V. scophthalmi) e specie isolate in ambiente marino con caratteristiche fenotipiche simili (V. vulnificus, V. gigantis, V. xuii). I ceppi rivitalizzati sono stati valutati a 25°C con prove biochimiche in macrometodo, prove in micrometodo con inoculo al 2% NaCl (API 20E, bioMérieux) e crescita su terreni differenziali e selettivi (TCBS, CHROMAgar™ Vibrio, MacConkey). L’analisi molecolare è stata condotta mediante il sequenziamento e l’analisi filogenetica del gene uridina monofosfato chinasi (PyrH) e l’amplificazione mediante PCR-end point specie-specifica (gene toxR). La metodica in spettrometria di massa MALDI-TOF ha analizzato mediante il MALDI Biotyper Microflex LT con software Maldi Biotyper 3.0 (BrukerDaltonics), il contenuto proteico ribosomiale di colonie di 24 h. Gli spettri MSP ottenuti dai ceppi in esame sono stati comparati con il database BrukerDaltonics implementato con ceppi Vibrio spp. di referenza standardizzati (93 ceppi) in quanto privo di queste specie. I risultati dei dati fenotipici (28 parametri) elaborati statisticamente applicando il coefficiente di Gower hanno permesso una clusterizzazione efficace (40 ceppi su 41 analizzati) dei ceppi di campo con i due V. harveyi di referenza. Sono risultate discriminanti le prove in micrometodo di arginina deidrolasi (ADH), lisina decarbossilasi (LDC), ornitina decarbossilasi (ODC), e del citrato (CIT) associate alla colorazione di CHROMAgar™ Vibrio a 48 h e TCBS. L’analisi molecolare delle sequenze del gene PyrH ha permesso una clusterizzazione coerente dei ceppi di campo e di referenza con le sequenze in letteratura. L’analisi del gene toxR speciespecifico ha prodotto un amplificato di 380 bp unicamente nei ceppi di V. harveyi di campo e di referenza L’esito MALDI-TOF ha rispecchiato l’assegnazione di specie ottenuta dal genotipo. 40 ceppi di campo sono stati assegnati con I e II best match a V. harveyi (score > 2,3); un solo ceppo di campo ha presentato best match (score 2,25) con V. diabolicus e II match (score 2,24) con V. harveyi. Le analisi sin qui condotte hanno evidenziato un’ottima concordanza nell’identificazione di ceppi di V. harveyi. Le prove fenotipiche sono state complessivamente in grado di discriminare V. harveyi dai congeneri, tuttavia dai dati in bibliografia si riscontra una certa variabilità nei risultati biochimici per le prove ritenute discriminanti. L’analisi del gene toxR risulta più rapida del sequenziamento del gene PyrH. L’identificazione mediante MALDI-TOF appare economica, rapida ed efficiente grazie all’implementazione preliminare del database BrukerDaltonics.

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.

An episode of mortality in farmed redclaw crayfish, Cherax quadricarinatus, reared in Sicily, is described. Histological and PCR methods were used to identify the oomycetes, Aphanomyces astaci, as the causative agent of the disease. Mortality up to 30%, and haemocytic melanising response against hyphae in affected specimens, indicates a certain resistance of C. quadricarinatus towards A. astaci. Procambarus clarkii reared in the same facility is hypothesized to be the carrier of the pathogen. Evidence of mortality in redclaw crayfish held at higher temperatures than any previously investigated episodes is discussed in this paper.

MedAID (Mediterranean Aquaculture Integrated Development) is a European H2020 Research and Innovation Project that started in 2017. The main objective was to increase the overall competitiveness and sustainability of the whole value chain of the Mediterranean marine aquaculture sector by improving its technical and business performance and by shifting to a sustainable market-oriented approach with a more positive social and consumer perception. In the field of disease control and welfare MedAID aims to provide essential components for a better health and welfare management system for the Mediterranean marine aquaculture industry overall and at company level. One of the gaps addressed by MedAID was the lack of reference methodologies for disease diagnostics for European seabass and Gilthead seabream, the main species produced in Mediterranean marine fish farming. The present Diagnostic Manual was conceived in the form of up-to-date guidelines providing current methodologies for a harmonised approach to the health challenges due to viral and bacterial pathogens in the farming of seabass and seabream., https://om.ciheam.org/option.php?IDOM=1037