Redspotted Grouper Nervous Necrosis Virus and the Reassortant RGNNV/SJNNV In Vitro Susceptibility against a Commercial Peroxy-Acid Biocide under Different Conditions of Use

Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the infield application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure

Advances in Viral Aquatic Animal Disease Knowledge: The Molecular Methods' Contribution

Viruses are pervasive components of aquatic ecosystems, and most of them are harmless to humans and animals; however, several aquatic viruses can infect animals, leading to diseases, especially when fish are confined, such as in aquaculture facilities. Traditional methods used to detect and study viruses have been widely applied to aquatic animals' viruses, leading to the successful isolation, identification and understanding of several of them. However, they have limits, which can be overcome by molecular methods, such as polymerase chain reaction (PCR)-based assays, sequencing and in situ hybridisation. A standard PCR, followed by the sequencing of purified amplicons, is an effective method for both identifying well-known viruses and discovering new ones. In situ hybridisation, in which a labelled probe binds to a nucleic acid sequence in tissue, is able to correlate the presence of viruses to lesions. Novel molecular isothermal methods, such as loop-mediated isothermal amplification (LAMP), were also developed and applied to viral aquatic animal diseases, bringing molecular diagnosis into the field. This review considers the scientific literature dealing with the molecular methods employed hitherto to study the most relevant finfish and shellfish viral pathogens, stressing their advantages and disadvantages. Aquaculture is the fastest-growing food-producing sector, with a global production of 122.6 million tonnes in 2020. Nonetheless, aquatic animal production can be hampered by the occurrence of viral diseases. Furthermore, intensive farming conditions and an increasing number of reared fish species have boosted the number of aquatic animals' pathogens that researchers have to deal with, requiring the quick development of new detection and study methods for novel unknown pathogens. In this respect, the molecular tools have significantly contributed to investigating thoroughly the structural constituents of fish viruses and providing efficient detection methods. For instance, next-generation sequencing has been crucial in reassignment to the correct taxonomic family, the sturgeon nucleo-cytoplasmic large DNA viruses, a group of viruses historically known, but mistakenly considered as iridoviruses. Further methods such as in situ hybridisation allowed objectifying the role played by the pathogen in the determinism of disease, as the cyprinid herpesvirus 2, ostreid herpesvirus 1 and betanodaviruses. Often, a combination of molecular techniques is crucial to understanding the viral role, especially when the virus is detected in a new aquatic animal species. With this paper, the authors would critically revise the scientific literature, dealing with the molecular techniques employed hitherto to study the most relevant finfish and shellfish viral pathogens

Detection of Human and Fish Viruses in Marine Gastropods

Marine gastropods represent a major food source for higher trophic levels and an important source of animal protein for humans. Like bivalve molluscs, gastropods can accumulate several types of contaminants; however, the bioaccumulation of microorganisms, particularly viruses, has been poorly investigated in these animals. This study focused on gastropods (Tritia mutabilis, Bolinus brandaris and Rapana venosa) collected during the fishing season from 2017 to 2021 in the north-western Adriatic Sea, and on clams (Ruditapes philippinarum) harvested in the same geographical area, in order to evaluate the presence of human and fish viruses in their tissues. A virological investigation was carried out on the digestive gland using molecular methods. The presence of hepatitis A virus was detected in one sample, whereas noroviruses were not present in the investigated specimens. Regarding fish viruses, it was possible to detect the presence of nervous necrosis virus (NNV) in 26.5% of the analyzed gastropods; however, the histological examination did not show any pathological changes in the nervous tissue in both NNV-positive and -negative batches. As a whole, the investigated gastropods showed the ability to bioaccumulate viruses; however, lower contamination by human viruses compared to bivalve molluscs was pointed out, posing a minor concern to human health

Development and diagnostic validation of a one-step multiplex RT-PCR assay as a rapid method to detect and identify Nervous Necrosis Virus (NNV) and its variants circulating in the Mediterranean

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method's detection limit varied between 10 2.3 and 10 3.4 TCID ml -1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains

Swim Bladder Disorders in Koi Carp (Cyprinus carpio)

Swim bladder disorders and consequent buoyancy problems are encountered in ornamental fish, including koi carp. Nevertheless, beyond clinical and pharmacologicalmanagement, they are largely underdiagnosed. In this study, nine koi carp showing abdominal swelling and abnormal swimming behavior were investigated. Clinical approach, varying from case to case, included ultrasonographic and X-ray investigations, bacteriological analysis of the collected fluid, antimicrobial susceptibility pattern, and possibly histological analysis. Diagnostic imaging, corroborating gross examination, documented swim bladder deformation/dislocation and serous fluid within the swim bladder chambers of most animals. Bacteria belonging to the Aeromonas hydrophila/caviae group and Shewanella xiamenensis were identified. S. xiamenensis strains showed a sensibility to all tested molecules except for one strain, which was resistant to tetracycline and cyprofloxacin. Antibiotic treatment succeeded in the full recovery of three cases in which S. xiamemensis infection was detected. Chronic aerocystitis was histologically documented where tissue was available. The swim bladder histopathological findings highlighted a chronic process that had compromised the quality of life of the animals. A multidisciplinary clinical\u2013pathological and microbiological approach is highly suggested to recognize swim bladder conditions as early as possible, aiming to drive medical intervention and raising the chances of fish survival

Yeast-extracted nucleotides and nucleic acids as promising feed additives for European sea bass (Dicentrarchus labrax) juveniles

Nowadays functional ingredients have a significant potential for improving current low fish meal (FM) aquafeed formulation in sustaining growth and enhancing animal robustness for Mediterranean aquaculture. Among them, nucleotides (NT) and nucleic acids (NA) drew attention for their application in the last two decades. NT are organic molecules involved in many life-supporting pathways, and are the building blocks of NA, which stand as genetic repositories. NT are naturally present in organic ingredients, and among them FM is known to be one of the highest NT sources. When this NT source is seriously limited, fish might be under the minimum NT requirements, especially in fast growing life stages of carnivorous species. Hence, a trial on European sea bass juveniles was carried out, testing two dietary FM levels (FM10, FM20 as 10% and 20% FM, respectively) supplemented with 500 mg kg-1 yeast-originate NT or NA dose over 80 days. Thereafter, fish were exposed to one week of sub-optimal thermal and dissolved oxygen condition (30°C and 4.0 mg/L O2) to further explore the effect of NT and NA inclusion on immune response and gut microbiome alteration. At the end of the growth period NT increased feed intake at both FM dietary levels. FM20 combined with NA and NT further improved growth performance, enhancing lipid efficiency and increased anti-inflammatory TGF-b. After sub-optimal environmental conditions both NT and NA exerted prebiotic functions on gut microbiome by promoting beneficial lactic acid bacteria such as Weissella and Leuconostoc. At the same time NT in 10% FM diet increased the abundance of Bacillus taxon. In conclusion, the combination of NT/NA included at 500 mg kg-1 was able to promote growth when included in 20% FM level, assuming higher nutritional NT requirement when combined with 10% FM. On the other hand, NT/NA added in 10% FM upregulate proinflammatory IL-1b and favor beneficial gut bacterial taxa

Cutaneous angiomatosis-like presentation in koi carp (Cyprinus carpio koi): Clinical–pathological investigations

The skin represents an indicator of an animal's health status. Causes of cutaneous diseases in fish most often trace back to biological agents. However, fish skin diseases can also arise from a complex interaction of infectious and non-infectious causes, making it more difficult to identify a specific aetiology. In the period between April and September of the years 2019–2022, four koi carp (Cyprinus carpio koi) from two European countries presented with multifocal, irregularly round, few mm to 1 cm, variably raised cutaneous reddened areas. The fish displayed good general condition. Cutaneous samples, investigated by microbiological and molecular methods and microscopy, did not indicate a primary pathogenic agent. Gross and histological findings of the cutaneous biopsies were consistent with a multifocal/reactive process centred on dermal vessels. The histological features were reminiscent of angiomatosis, a benign proliferative condition affecting the dermal vessels of mammals, including human patients. The clinical–pathological presentation and the dermatologic condition that affected the koi carp are discussed and compared with the veterinary and human literature

Viral investigation in the Ravenna's harbor: mussels as bioindicators for the presence of Norovirus, Hepatitis A virus and Nervous Necrosis Virus

Il porto di Ravenna rappresenta uno dei più grandi porti commerciali Italiani. Si tratta di un ambiente urbanizzato e soggetto ad un’intensa circolazione navale e quindi soggetto a inquinamento e introduzione di specie aliene. La biodiversità in questo ambiente è relativamente alta e include i molluschi bivalvi, specie utilizzata per il biomonitoraggio di contaminanti ambientali grazie alla loro ampia distribuzione, vita sessile, attività di filtrazione e facilità di campionamento. Lo scopo di questo studio è quello di utilizzare i mitili come bioindicatori di contaminazione virale nel porto di Ravenna e nel tratto di mare antistante, considerando sia la variabilità spaziale che quella temporale. In particolare, è stata investigata la presenza di virus umani come il Norovirus (NoV) e il virus dell’epatite A (HAV) e del virus della Necrosi Nervosa (NNV) responsabile nel periodo estivo di focolai di mortalità nei pesci allevati. L’indagine ha mostrato la presenza di contaminazioni da virus umani nei mitili raccolti nella parte più interna del porto con una maggiore contaminazione da NoV nei mesi invernali, similmente a quanto osservato in altre aree. La genotipizzazione dei virus trovati ha mostrato la presenza di ceppi multipli di NoV che rappresentano quelli più diffusi a livello mondiale. È stata inoltre rilevata la presenza di HAV IA, genotipo già isolato in infezioni umane associate al consumo di prodotti ittici crudi. Invece, mitili contaminati da NNV sono stati rilevati in tutti i siti campionati, incluse le piattaforme offshore, mostrando un’ampia diffusione di questo virus. La maggior parte delle contaminazioni da NNV è stata evidenziata durante il periodo estivo. La genotipizzazione ha mostrato la somiglianza dei virus evidenziati con ceppi di NNV precedentemente trovati in pesci e in molluschi bivalvi nel mar Adriatico. Questi risultati suggeriscono che i mitili abbiano accumulato l’NNV rilasciato da pesci selvatici infetti che vivono in quest’area

New knowledge, diagnostic and control tools to limit the impact of viral nervous necrosis (VNN) in the Mediterranean aquaculture sector

The role of aquaculture in satisfying the global seafood demand is essential. The expansion of the aquaculture sector and the intensification of its activities have enhanced the circulation of infectious agents. Among these, the nervous necrosis virus (NNV) represents the most widespread in the Mediterranean basin. The NNV is responsible for a severe neuropathological condition named viral nervous necrosis (VNN), impacting hugely on fish farms due to the serious disease-associated losses. Therefore, it is fundamental to develop new strategies to limit the impact of VNN in this area, interconnecting several aspects of disease management, diagnosis and prevention. This PhD thesis project, focusing on aquatic animals’ health, deals with these topics. The first two chapters expand the knowledge on VNN epidemiology and distribution, showing the possibility of interspecies transmission, persistent infections and a potential carrier role for invertebrates. The third study expands the horizon of VNN diagnosis, by developing a quick and affordable multiplex RT-PCR able to detect and simultaneously discriminate between NNV variants, reducing considerably the time and costs of genotyping. The fourth study, with the development of a fluorescent in situ hybridization technique and its application to aquatic vertebrates and invertebrates’ tissues, contributes to expand the knowledge on NNV distribution at cellular level, localizing also the replication site of the virus. Finally, the last study dealing with an in vitro evaluation of the NNV susceptibility to a commercial biocide, stress the importance to implement proper disinfectant procedures in fish farms to prevent virus spread and disease outbreaks

Cutaneous nerve sheath tumours, non‐infectious polycystic kidney disease, renal granulomas, nephrocalcinosis and crystals: Incidental pathological findings in pond‐living goldfish ( Carassius auratus , Linnaeus)

In an adult male goldfish (Carassius auratus, Linnaeus), an unusual combination of diseases was detected. The owner noted a bilateral, asymmetrical distention of the abdomen, multiple cutaneous masses and an altered swimming behaviour over the course of time; diagnostic work-up was not requested and euthanasia elected. Grossly, the cutaneous masses were whitish, discrete, extremely friable. Histologically, the dermis was multifocally expanded by moderately cellular, unencapsulated neoplastic tissue, composed of dermal spindle cells. The celomic cavity opening revealed a gelatinous multicystic mass, corresponding to the kidney, which replicated several cysts compressing the residual parenchyma. Histologically, sparse, late-stage granulomas, basophilic intratubular deposits and crystals were also detected. No mycobacterial DNA was detected in tissue with granulomas. No parasites were detected in the histological sections examined. Diagnoses of cutaneous nerve sheath tumours, polycystic kidney disease, renal late-stage granulomas, nephrocalcinosis and crystals were formulated. The authors had proposed to recapitulate each single condition