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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.

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 in-field 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.

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.

Cichlids include hundreds of species with a high economic value for aquaculture. These fish are subjected to intensive trade and farming that expose them to the risk of infectious diseases. This work focuses on ornamental cichlids held in an aquarium commercial facility presenting emaciation, in order to evaluate the presence of lesions in fish skin and organs. The fish were sampled during routine management activities and subjected to pathological and molecular investigations. The presence of lymphocystis disease virus, typically associated with cutaneous nodular disease, was ruled out. Histologically, they presented granulomas in the spleen, sometimes extending to the other visceral organs. Bacterial heat-shock protein 65 PCR products were detected in tissues associated, in the majority of cases, with granulomas; molecular investigation identified Mycobacterium spp. in two cases and Cutibacterium acnes in seven cases. Immunoreactivity to anti-Mycobacterium and anti-C. acnes antibodies was detected within granulomas. The presence of C. acnes within granuloma is elucidated for the first time in fish; however, similarly to what is found in humans, this bacterium could be harmless in normal conditions, whereas other contributing factors would be required to trigger a granulomatogenous response. Further confirmation by bacterial culture, as well as using large-scale studies in more controlled situations, is needed.

Viral nervous necrosis (VNN) is the most important viral disease affecting farmed fish in the Mediterranean. VNN can affect multiple fish species in all production phases (broodstock, hatchery, nursery and ongrowing) and sizes, but it is especially severe in larvae and juvenile stages, where can it cause up to 100% mortalities. European sea bass has been and is still the most affected species, and VNN in gilthead sea bream has become an emerging problem in recent years affecting larvae and juveniles and associated to the presence of new nervous necrosis virus (NNV) reassortants. The relevance of this disease as one of the main biological hazards for Mediterranean finfish farming has been particularly addressed in two recent H2020 projects: PerformFISH and MedAID. The presence of the virus in the environment and in the farming systems poses a serious menace for the development of the Mediterranean finfish aquaculture. Several risks associated to the VNN development in farms have been identified in the different phases of the farming system. The main risks concerning VNN affecting gilthead seabream and European seabass have been identified as restocking from wild fish in broodstock facilities, the origin of eggs and juveniles, quality water supply and live food in hatcheries and nurseries, and infected juveniles and location of farms in endemic areas for on-growing sites. Due to the potential severe impact, a holistic integrated management approach is the best strategy to control VNN in marine fish farms. This approach should include continuous surveillance and early and accurate diagnosis, essential for an early intervention when an outbreak occurs, the implementation of biosecurity and disinfection procedures in the production sites and systematic vaccination with effective vaccines. Outbreak management practices, clinical aspects, diagnostic techniques, and disinfections methods are reviewed in detail in this paper. Additionally, new strategies are becoming more relevant, such as the use of genetic resistant lines and boosting the fish immune system though nutrition.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Swim bladder disorders and consequent buoyancy problems are encountered in ornamental fish, including koi carp. Nevertheless, beyond clinical and pharmacological management, 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–pathological 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.

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 aim of the present study was to evaluate the presence of Salmonella spp., verotoxigenic E. coli (VTEC), Vibrio spp., and Norovirus GI-GII in bivalve molluscs, cockles, and European grooved carpet shells (Cerastoderma spp. and Ruditapes decussatus) collected from a class B growing natural bed in Sardinia (Italy). All of the samples were analysed for Salmonella spp. detection according to European Commission Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp. were performed according to previously published methods. Presumptive identification of Vibrio spp. isolates was performed by means of conventional biochemical tests. E. coli VTEC was isolated following a direct multiplex polymerase chain reaction (PCR) screening test. Norovirus GI and GII were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). No Salmonella spp. were detected. The prevalence of Vibrio spp. was 90%, and the average contamination levels were 3.19 ± 1.07 and 2.84 ± 0.31 Log10 cfu/g in cockles and European grooved carpet shells, respectively. The prevalence of E. coli VTEC was 6.6%. All of the isolates showed a complete pathogenicity profile. The presence of Norovirus was highlighted in 25% of European grooved carpet shells samples. Results showed the typical microbiological profile of bivalve molluscs collected from backwaters and confirmed the capability of shellfish to accumulate E. coli VTEC, pathogenic vibrios, and Norovirus. The presence of such pathogens in shellfish is of major concern for the safety of consumers.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by bone marrow expansion and the proliferation of one or more myeloid cell lineages, predominantly driven by the expression of the constitutively active fusion product tyrosine kinase BCR:ABL1. Rarely, CML patients directly develop a blast crisis (BC), mostly of myeloid origin. CML at blast crisis with a T-cell phenotype at diagnosis, without any prior history of CML, is extremely rare. Herein, we describe one rare CML case, in a young man showing an unusual and early T-lymphoid blastic crisis at diagnosis, as the first onset of a previously unknown CML. The multidisciplinary collaboration between laboratorians and clinicians for the diagnosis and management of this atypical case was crucial in outlining both a targeted pharmacological treatment and a successful hematopoietic stem cell transplantation.

Ewing sarcoma (EWS) is a paediatric aggressive malignant tumour of bones and soft tissues. Multidisciplinary chemotherapies, surgical resection, and radiation represent the only strategies counteracting the disease, however spreading and relapse of disease still remain a clinical issue. Circulating tumour cells (CTCs) are an important feature of EWS but the prognostic significance has not been, yet, clarified. CTCs have been found both in patients with localized disease and in those who recur or metastasize. The identification of markers that can detect recurrences and metastasis remains an important challenge for research. Unfortunately, even most of patients with localized cancer relapsed and the reason has not yet been fully understood. In this clinical study on EWS patients, we evaluated the expression of CD99 antigen and beta-3 adrenergic receptor (β3-AR) on CTCs and bioptic derived cells by flow cytometry. The preliminary data revealed a higher β3-AR expression on cells derived from metastatic or relapsed patients, suggesting a role for the β3-AR as a possible predictive maker of disease recurrence in both patients with metastatic and localized disease.