Following the Mass Mortality Events (MMEs) of the pen shell P. nobilis in Campania region and Sicily, a survey of moribund P. nobilis specimens was also conducted in other Italian regions (Campania, Tuscany, Sardinia, and Apulia) and Spain (Catalunya). Histopathological and molecular examination of 27 specimens of P. nobils revealed different types of pathogens associated with tissue lesions, morbidity and mortality. Presence of Mycobacterium, Vibrio species, Haplosporidium pinnae and Perkinsus sp. were detected, differently distributed into the areas. The Mycobacterium sp., previously reported in Campania and Sicily samples, was observed in all the analyzed areas and individuals, associated to systemic inflammatory lesions. In Spain, H. pinnae was observed in 36% of cases, always associated to the Mycobacterium sp. Molecular study using hsp65 genes and Internal Transcriber Spacer ITS support that a new species of Mycobacteria is infecting P. nobilis, close to M. triplex and belonging to the group of M. simiae complex with M. sherrisi. Presence of Perkinsus spp. resembling P. mediterraneus was observed in 2 out of 13 Italian individuals whose presence should be addressed as potential risk for shellfish aquaculture of the area. Vibrio spp. were also detected in some case. The preliminary results of this study suggest that Mycobacterium sp., Vibrio spp., H. pinnae and Perkinsus sp. cooperate to disease pathogenesis, being Mycobacterium and Haplosporidium most of the time involved. Vigilant inspection of those areas where MME is now starting, along with continuous systematic surveys, are crucial to define the spatiotemporal progress of mortality and the role of every single pathogen in the disease outcome.

Nanotechnology is rapidly growing with nanoparticles produced and utilized in a wide range of commercial products worldwide. Among the different types of nanomaterials produced, silver nanoparticles (AgNPs) occupy a predominant position and they are used in electronics, clothing, food industry, cosmetics and medical devices. Nanosilver has also showed excellent performance in antibacterial application. Nowadays, the increasing use of AgNPs has put the evidence on their possible toxicity to the human health and the impact on the environment. This paper focus on adverse effects of AgNPs in adult of Danio rerio. Fishes exposed to increasing concentrations (8, 45, and 70 μg/l) silver nanoparticles (AgNPs, 25 nm in average diameter) and after treatment for 30 days, was quickly euthanized in MS-222. We have evaluated bioaccumulation of AgNPs using ICP-MS and analyzed histological changes, biomarkers of oxidative damage and gene expression in the gut, liver and gills tissues of AgNPs-treated zebrafish. The histological analysis showed lesions of secondary lamellae of the gills with different degrees of toxicity such as hyperplasia, lamellar fusion, subepithelial edema, and even in some cases telangiectasia. Huge necrosis of the intestinal villi was found in the gut. No lesion was detected in the liver. The analysis revealed a high expression of metallothioneins 1 (MTs 1) in animals exposed to AgNPs compared to the control group. The ICP-MS analysis shows that the amount of particles absorbed in all treated samples is almost the same. We can affirm that AgNPs toxicity linked more to their size and state of aggregation than to their concentrations. Silver nanoparticles can damage gills and gut because they are able to pass through the mucosal barrier thanks to their small size. The damage is still reversible because it is not documented injury to the basal membrane.

The effect of the nanoparticles on the marine organisms, depends on their size, chemical composition, surface structure, solubility and shape. In order to take advantage from their activity, preserving the surrounding environment from a possible pollution, we are trying to trap the nanoparticles into new nanomaterials. The nanomaterials tested were synthesized proposing a ground-breaking approach by an upside-down vision of the Au/TiO2 nano-system to avoid the release of nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of TiO2. The non-toxicity of the nano-system was established by testing the effect of the material on zebrafish larvae. Danio rerio o zebrafish was considered an excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test (ZFET) is considered an alternative method of animal test. For this reason zebrafish larvae were exposed to different concentrations of nanoparticles of TiO2 and Au and new nanomaterials. As biomarkers of exposure, we evaluated the expression of metallothioneins by immunohistochemistry analysis and western blotting analysis also. The results obtained by toxicity test showed that neither mortality as well as sublethal effects were induced by the different nanomaterials and nanoparticles tested. Only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis highlighted an increase of the metallothioneins synthesis.

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BackgroundCanine overpopulation is a global issue with serious health and welfare implications. Nonsurgical methods of sterilization could yield positive impacts on this problem, but no long-term data on such methods are available. The objective of the current investigation was to determine the effects of intratesticular injections of calcium chloride dihydrate (CaCl2) in saline in dogs over a one year period. Five concentrations (0%, 10%, 20%, 30%, 60%) of CaCl2 in saline were administered via intratesticular injection to groups of 10 dogs each. Total sperm count and motility, blood levels of testosterone, and side effects were examined at 0, 2, 6, and 12 months post-injection (PI). Testicular size and semen volume were examined at 0 and 12 months PI.ResultsTotal sperm count, semen volume and testosterone showed significant dose-dependent decreases upon treatment with 10%-60% CaCl2 compared with either the control group (0% CaCl2) or baseline for each treatment group. Azoospermia was achieved for at least 12 months PI in 60% and 80% of treated dogs after administration of a 10% and 20% CaCl2, respectively. Treatment with 30% or 60% CaCl2 resulted in azoospermia in 100% of dogs, but more side effects were observed, while no side effects were noticed at lower doses. For each treatment group, testosterone levels had decreased an average of 35%-70% at 6 months following treatment. However, testosterone levels rebounded by the 12-month time point in all groups except the highest dosage group (60% CaCl2), which remained at the low end of physiological range throughout the study. Sperm motility dropped to zero or near zero in all dogs treated with CaCl2. Testicular size was significantly smaller at 12 months PI for all groups when compared to baseline.ConclusionsThis first long-term study confirms reports of the efficacy of CaCl2 sterilization. However, at dosages free of adverse events, calcium chloride in saline may not provide permanent sterilization as previously believed. Future work should explore optimized solvents to increase the permanence of the well-tolerated 20% formulation.