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We studied the bioaccumulation of metals and PAHs, the pathological conditions, regressive phenomena and pathogens in wild Mytilus galloprovincialis taken along the North Pier facing the former second Italian largest steelworks of Bagnoli. There was no Cd and Pb bioaccumulation with respect to the EU role 221/2002. Metal shell index decreased as follows: Pb > Ni > Zn > Cu and correlates with the pollution state. The level of BaP was up to thirtysixfold higher the EU rule 835/2011. The sum of 4 hydrocarbons, PAH4, were up to seventeen-fold the rule. PAH levels increased toward the coast. Prevalence values of tissue necrosis and inflammatory lesions were between 50 and 100%. In May animals showed lesion like granulocytomas and inflammatory capsules. Signs of atresia, necrotic oocytes and diffused cases of hermaphroditism were detected. An appropriate localization of farming to avoid contamination from sediment turbulence and risks for consumer health is needed.

— Clinical laboratory investigations as well as light, electron microscopic and X-ray microanalytical studies on skin biopsy specimens and hair from 10 yorkshire terriers suffering from Colour Dilution Alopecia (CDA) were carried out. Biochemical data detected a marked melanin reduction in hair from affected areas. Histologically, melanin clumps were mainly evident in the epidermal basal layer cells as well as inside the bulb and hair shaft. Ultrastructurally, basal keratinocytes were overloaded by fully melanized melanosomes disrupting cytoplasmic structures. Intracytoplasmic and free melanosomes were also evident in all cutaneous layers. Scanning electron microscopy showed holes and bulges scattered in the hair shaft. X-Ray microanalysis demonstrated silicate particles in the more superficial cuticular holes and calcium in the deeper fractured layers. Based on these observations a new hypothesis on the pathogenetic mechanism(s) of alopecia is proposed. Resume— Diverses investigations, microscopie optique, microscopie electronique, etudes microanalytiques aux rayons X ont ete realisees sur des biopsies cutanees et des poils provenant de dix yorkshire Terriers presentant une Alopecie des Robes Diluees. Les examens biochemiques montrent une diminution nette de la melanine dans les poils provenant des regions malades. Des aggregate de melanine sont observes dans les cellules de l'assise basale epidermique ainsi qu'a l'interieur des bulbes pileux et des tiges pilaires. Sur le plan ultrastructural, les keratinocytes basaux montrent une surcharge en melanosomes detruisant ainsi les structures cytoplasmiques. Des melanosomes libres et intra-cytoplasmiques sont egalement observes dans toutes les couches epidermiques. La microscopie electronique a balayage montrent des trous et des bosses disperses dans la gaine folliculaine. Des analyses en rayons X demontrent la presence de particules de silicate dans les trous les plus superficiels de la cuticule et du calcium dans les couches plus profondes. A partir de ces observations, une nouvelle hypothese sur la pathogenie de dette alopecie est proposee. [Roperto F., Cerundolo, R., Restucci, B., Vincensi, M. R., De Caprariis, D., De Vico, G., Maiolino, P. Colour dilution alopecia (CDA) in ten yorkshire Terriers (Alopecie des robes diluees (ARD) chez dix yorkshire Terriers). Resumen— Se llevaron a cabo analisis clinicos asi como estudios microanaliticos con rayos X y microscopia electronica sobre biopsias cutaneas tomadas a diez yorkshire Terriers que padecian de Alopecia de Color Diluido (CDA). Los estudios bioquimicos detectaron una fuerte reduccion en melanina en el pelo de las zonas afectadas. A nivel histologico, los agregados de melanina se localizaban principalmente en la capa basal de la epidermis asi como en el bulbo piloso y en en pelo. A nivel ultraestructural, los queratinocitos basales estaban sobrecargados de melanosomas totalmente melanizados, alterando su estructura citoplasmatica. Tambien se identificaron melanosomas en forma libre o a nivel intracitoplasmatico en todas las capas cutaneas. La microscopia electronica de escaner mostro cavidades y prominencias diseminadas a lo largo del pelo. Mediante microanalisis por rayos X se detectaron particulas de silicato en las cavidades cuticulares mas superficiales y calcio en las capas mes profundas. Teniendo en cuenta estos hallazgos, se propone una nueva hipotesis sobre el/los mecanismo/s patogenico/s de la alopecia. [Roperto F., Cerundolo, R., Restucci, B., Vincensi, M. R., De Caprariis, D., De Vico, G., Maiolino, P. Colour dilution alopecia (CDA) in ten yorkshire Terriers (Alopecia de color diluido (CDA) en diez yorkshire Terriers).

Introduction: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particu- lar for its artemisinin content, an active ingredient at the base of most antimalarial treatments, used every year by over 300 million people. In the last years, owing to low artemisinin content, research of new ways to increase the yield of the plant matrix has led to the use of the total extract taking advantage from the synergic and stabilising effects of the other components. Objective: In this work we evaluated and compared the content of artemisinin and scopoletin in extracts of A. annua collected in the Campania Region (southern Italy), by two different extraction processes. Methodology: Artemisia annua plants were extracted by traditional maceration (TM) in hydroalcoholic solution as a mother tincture prepared according to the Euro- pean Pharmacopeia and by pressurised cyclic solid–liquid (PCSL) extraction, a new generation method using the Naviglio extractor. Results: The results showed that the PCSL extraction technique is more effective than traditional methods in extracting both phytochemicals, up to 15 times more, reducing the extraction times, without using solvents or having risks for the opera- tors, the environment and the users of the extracts. Conclusion: The Naviglio extractor provides extracts with an artemisinin and scopoletin content eight times higher than the daily therapeutic dose, which should be evaluated for its stability over time and biological properties for possible direct use for therapeutic purposes.

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such &ldquo, lower risk&rdquo, areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

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.

Due to high rates of proliferation and DNA synthesis, neoplastic cells have higher requirements of iron than normal cells. For that reason, neoplastic cells have remodelled iron metabolism pathways, over‐expressing genes encoding for iron uptake proteins, among which Transferrin Receptor‐1 (TFR‐1). Accumulating evidence has proven that overexpression of TFR‐1 and high Iron concentration, are both widespread condition of cancer cells, both essential to tumour onset and progression. We studied TFR‐1 and PCNA immunohistochemical expression in fifteen (15) Canine osteoblastic osteosarcomas (COS). After immunohistochemical staining, counting of TFR‐1 positive cells by two independent observers showed that 85%–95% of neoplastic cells were strongly labelled at cytoplasmic level by anti‐TFR‐1 antibody in all examined COS. Furthermore, 70%–80% of neoplastic cells were positively labelled at the nuclear level by PCNA. Surprisingly, about 100% of intratumour vascular endothelial cells were also positive, whereas extratumour vascular endothelial cells were negative. The latter is an interesting finding, as TFR‐1 is usually not expressed in normal vasculature, with the exception of normal brain vascular endothelium, where it allows transport of transferrin, and thus iron, into tissues, suggesting a similar function here to support cancer growth. The early results presented highlight the relevance of TFR‐1 expression in canine OS, suggesting therapies involving both TFR‐1 and Iron metabolisms in dogs with osteosarcoma should be developed., Neoplastic cells have higher requirements of iron than normal cells. For that reason, neoplastic cells over‐express genes encoding for iron uptake proteins, including Transferrin Receptor‐1 (TFR‐1). We studied TFR‐1 immunohistochemical expression in ten Canine osteoblastic osteosarcomas (OS). In all examined OS, 85–95% of neoplastic cells showed a strong cytoplasmic immunostaining with anti‐TFR‐1 antibody. Surprisingly, about 100% of intratumor vascular endothelial cells were also positive, whilst extra‐tumor vascular endothelial cells were negative. The early results presented highlight the relevance of TFR‐1 expression in canine OS, suggesting therapies involving both TFR‐1 and Iron metabolisms in dogs with osteosarcoma should be developed.

Disease is an increasing threat for marine bivalves worldwide. Recently, a mass mortality event (MME) impacting the bivalve Pinna nobilis was detected across a wide geographical area of the Spanish Mediterranean Sea and linked to a haplosporidian parasite. In 2017–2018, mass mortality events affecting the pen shell Pinna nobilis were recorded in two different regions of Italy, Campania and Sicily, in the Tyrrhenian Sea (Mediterranean Sea). Histopathological and molecular examinations of specimens showed the presence of Haplosporidium sp. in only one specimen in one area. Conversely, in all of the surveyed moribund animals, strong inflammatory lesions at the level of connective tissue surrounding the digestive system and gonads and linked to the presence of intracellular Zhiel-Neelsen-positive bacteria were observed. Molecular analysis of all of the diseased specimens (13) confirmed the presence of a Mycobacterium. Blast analysis of the sequences from all of the areas revealed that they were grouped together with the human mycobacterium M. sherrisii close to the group including M. shigaense, M. lentiflavum and M. simiae. Based on pathological and molecular findings, it is proposed that a mycobacterial disease is associated with the mortality episodes of Pinna nobilis, indicating that, at this time, Haplosporidium sp. is not responsible for these events in Campanian and Sicilian waters.

In the recent years, heliciculture has rapidly developed in Italy both in terms of number of companies (about 8000) and average extension of snail farms (5000–10,000 m 2 ). Studies aimed to evaluate the whole environmental performance of heliciculture are still not available in literature. This paper presents a Life Cycle Assessment (LCA) performed for Helix aspersa maxima snail production in the South of Italy. The system boundary ( cradle to farm gate ) of the production chain considered the following stages: indoor breeding, outdoor fattening, cleaning out and packaging. The outdoor fattening displayed the largest part of total burdens (about 90%) for all impact categories due to the relevant contribution of its processes (enclosures set-up, fodder cultivation, supplementary feeding and irrigation). The process that highly affected all impact categories was the supplementary feeding used in fattening pens which reached the highest value for Terrestrial acidification (84%) and the lowest one for Ozone depletion (41%). Nevertheless, the impact of enclosures set-up and fodder cultivation appeared marked in particular for Ozone depletion (46%) and Marine Eutrophication (30%), respectively. Results showed the key factors (supplementary feeding, HDPE mesh) to be considered and discussed to proper design a semi-intensive snail management in order to enhance benefits and constrain limitations. Moreover, the snail farming highlighted a lower environmental load, for almost all impact categories, compared to literature data on conventional meat (beef, pig and chicken).

Objectives The objective of this study was to assess local vascular architecture in atrophic-erosive oral lichen planus (OLP). Materials and Methods We investigated the capillary structure of the oral mucosa in 31 patients with OLP and 32 healthy controls. Capillaries images were captured in vivo through a capillaroscope. We applied fractal analysis to quantify the microvasculature morphometric changes in the oral mucosa of atrophic-erosive OLP patients in terms of their fractal dimension (D). Results The oral vascular networks of atrophic-erosive OLP lesions had a significantly higher D, both in buccal mucosae (D = 1.167, P = .019) and in tongue (D = 1.196, P = .038), compared with the control population (1.123 for both locations, respectively). Conclusions The present study confirms previous literature data on a close relationship between abnormal vascular architecture and atrophic-erosive OLP. Fractal analysis provided a quantitative descriptor of the complexity of the vascular patterns, which increases in the OLP samples. These data may provide new information on the OLP pathogenesis, as well as serve as morphologic quantifiers for monitoring treatment strategies.

This article proposes a systematic evaluation of methods pertaining to the pathology discipline (histology, histochemistry, ultrastructure, in situ techniques, image analysis including morphom-etry and fractal dimension analysis) employed to study liver and intestine status during a series of nutritional trials performed on flatfish species (common sole, Solea solea; Senegal sole, Solea senegalensis and turbot, Scophthalmus maximus) of commercial interest. Histology is the first step able to detect the normal architecture of the tissues or the possible occurring changes related to inflammation, degeneration and cell death. The evaluation of liver should include a scoring system to obtain semi-quantitative numerical data that are statistically analysable; histochemisty is a valid tool for identifying the type of cell content, together with ultrastructure that also permits the detection of even mild signs of cell damage. For the evaluation of the intestine, in situ techniques are useful together with fractal dimension analysis to determine the mucosal tro-phism, while ultrastructure can demonstrate cell injury. These considerations were resumed in a methodological flowchart as a valid tool to assess the fish health status when nutritional trials are planned.

The bivalve Mytilus galloprovincialis has a broad geographic distribution, represent an important species for the ecology of coastal waters, also constituting a major aquaculture species. In the present work, molecular and tissue biomarkers were examined in mussel populations (M. galloprovincialis) located in four different areas of the coastal water of the Campania Region. During an annual life cycle, we analyzed the expression patterns of several genes commonly used to estimate cellular stress response and damage, namely p53, p63, HSP70, MT-10, and MT-20, related tissue lesions (pathogens, inflammations, digestive tubules damage), oxidative stress indicators (H 2 O 2 , SOD specific activity) and associated environmental data. The computed Principal Component Analysis showed that the areas were discernible based on the environmental data and biomarker results. About animal health status, mussels from Gulf of Pozzuoli and Naples's harbor did show a thinnest epithelial cell of digestive tubules compared to mussels sampled from other sampling sites; moreover, high prevalence of cases of intersex in three of the examinated areas were observed. The presence of a potential zoonotic pathogen (Nocardia crassostreae) was identified, appearing as an important possible emerging disease. We also reported the OIE notifiable protozoa Marteilia refringens in three areas out of four. The likely impact of both observed pathogens on the mussel health and shellfish aquaculture needs to be urgently addressed. Results are discussed considering animal histopathological health parameters and biological effects.

The Mediterranean land snail Theba pisana (Mollusca: Helicidae) is an introduced agricultural pest in manycountries around the world, including Australia, Israel, USA and South Africa. In addition, this snail is an intermediate host of parasites of importance in both human and veterinary medicine. In this study, a natural population of T. pisanasnails on the Domitian coast of Italy was surveyed following a mass mortality event. By light microscopy, 30% of the collected individuals showed in the calcium cells of the digestive gland thepresence of hypertrophied nuclei containing eosinophilic to weakly basophilic inclusion bodies. Ultrastructural examination by transmission electron microscopy (TEM) showed nuclear inclusions constituted by a reticulatedstroma into which unenveloped, roundish virus-like particles (38 ± 4 nm in diameter) were present. To the bestof our knowledge this could be thefirst evidence for a virus-like agent infecting the gastropodT. pisana, whichmay open new biocontrol perspectives of the this pest worldwide (14) (PDF) Early evidence for a virus-like agent infecting the pest snail Theba pisana (Gastropoda: Pulmonata) in Southern Italy. Available from: https://www.researchgate.net/publication/316866417_Early_evidence_for_a_virus-like_agent_infecting_the_pest_snail_Theba_pisana_Gastropoda_Pulmonata_in_Southern_Italy [accessed Mar 18 2020].

The use of aquatic invertebrates in biomedical research and as environmental sentinels has dramatically grown in recent decades, with an increased need in understanding of comparative pathology. The Unionids freshwater mussels are a group of worldwide distributed bivalves residing small ditches and ponds, lakes, canals and rivers, often used as animal test in eco-toxicological studies. Once one of the most abundant bivalve molluscs in ancient rivers around the world, now many of them are declining in many countries and consequently are nearly extinct in many areas. The causes of this decline are not fully understood but alteration and degradation of the freshwater habitat seemed to play a central role. To date, link causality to the observed losses during episode of mussel die-offs has been more difficult to establish, and disease and pathogen presence have been scarcely considered. In this article we provide a brief overview of unionids freshwater mussel conservation status, also describing reported diseases and pathogens and illustrating a few relatively well-documented studies.

Heliciculture for food production has huge potential and new opportunities for rural development and young entrepreneurs in Italy. No studies have yet been performed on the environmental performance of snail rearing which also might be a beneficial tool for producers. The aim of the present paper is to evaluate the impact of snail meat by a cradle-to-farm gate life cycle assessment centred on the carbon footprint (CF). The study considered greenhouse gas (GHG) emissions linked to cultivation stages (indoor breeding, outdoor fattening, cleaning out and packaging) of Helix aspersa maxima meat production in a semi-intensive rearing system in Southern Italy. The shell potential for CO2 sequestration was also taken into account. Snail CF amounted to 0.7 kg CO2 eq per kg fresh edible meat, with the highest share (about 60%) from the supplementary feeding production. Due to the combined effect of relevant amount input and restrained lifetime of HDPE mesh applied in the open field, the impact of breeding enclosures appeared considerable (about 29%). Greenhouse gas emissions linked to fodder cultivation and to the cleaning-out phase appeared restrained (nearly 4% and 5%, respectively), whilst the share of reproduction system, irrigation and packaging was negligible ( The environmental load of supplementary feeding resulted to mainly ascribable (about 74%) to maize and field bean grain cultivation (for feed mixture). It was followed by grain transport (about 17%) and processing (about 4%) to feed mill and further transport of manufactured feed components (maize–field bean–limestone) to the snail farm (about 5%). The CF score might be reduced by 18%, including potential long term CO2 sequestration in shells. As compared to other conventional macro-livestock meat sources, snail meat showed reduced GHG emissions.

Comparative pathology as a scientific discipline studies animal diseases in relation to their aetiology, pathogenesis and prognosis. Among the main aspects of this discipline, regressive changes, host defense responses with pathological implications and progressive changes, represent the majority of the possible responses of cells and tissues to pathogens and exposure to chemicals. One of the most persistent issues in the field of invertebrate pathology is the variability in terminology and definition, which has led to confusion in scientific communication. The aim of this paper is to provide an overview of the pathological basis of bivalve disease (defensive, regressive and progressive phenomena) and contribute to the standardised terminology for bivalve molluscan disease in the context of comparative pathology.

Haemic neoplasia (HN) in bivalves has been reported in association with mass mortality events in various species of molluscs. The aim of this work was to quantify the nuclear morphometry and DNA content of neoplastic cells of mussels Mytilus galloprovincialis affected by HN using nuclear densitometry in Feulgen-stained preparations. The results were also compared with a population of normal mussel haemocytes. We captured 256 images of 3 different neoplasia stages and 120 images of normal haemocytes; thus, a total of 120,166 nuclei were analysed. We extracted 21 morphological parameters from normal and neoplastic nuclei. Eighteen of these parameters were different (P