Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Pathology, epidemiology, and phylogeny of mussel egg disease due to the microsporidian Steinhausia mytilovum (Field, 1924) in the Mediterranean mussel (Mytilus galloprovincialis).

Microsporidia are well known fungal pathogens of aquatic animals. However, the taxonomy of microsporidia is generally poorly resolved, which has consequently constrained our understanding of their pathology and epidemiology in marine animals. To date, microsporidia have been reported in both bivalves and gastropods, and microsporidia from mollusks have been classified in different genera. Despite ongoing work to better describe these genera, including detailed microscopic and ultrastructural images, so far we lack information on microsporidian phylogeny and pathogenicity of species within these genera. Here we investigate the microsporidian parasite Steinhausia mytilovum associated with the mussel, Mytilus galloprovincialis, in natural beds and farms along coast of southern Italy. A survey of M. galloprovincialis was conducted in 13 mussel farms and one natural bed between 2009 and 2020. We found the presence of S. mytilovum in 10 of the investigated farms, with a prevalence ranging between 14 and 100% of female mussels, depending on the population and season in which they were sampled. The parasite developed in the oocytes within a sporophorous vesicle (SV) where it produced 1-3 spores per cell, both in the cytoplasm and in the nucleus. Stenhausia mytilovum elicited an infiltrative (24.8%) or a strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective tissue, in some cases accompanied by gonadal atresia (24.8%), leading to loss of gonadal architecture. In 7% of cases no reaction was observed. Ultrastructural observations revealed a mitochondrial re-organization to interact with all the phases of parasite development; the mitochondria were arranged outside the parasitophorous vesicle (PV) or directly interacting with the spore inside vesicle. There are five taxonomic clades of microsporidians as identified by SSU ribosomal gene sequence data. Maximum likelihood analysis assigned S. mytilovum within the Clade IV, defined as the Class Terresporidia, with closest genetic relationship (83.6% identity) to an undetermined invertebrate ovarian microsporidian. The constant presence, prevalence, and severity of S. mytilovum in coastline populations of M. galloprovincialis populations in southern Italy may indirectly reflect immunocompetence at both individual and population levels.

A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression.

Introduction: The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. Methods: We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. Results and discussion: In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104-1.60 × 105 ml-1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus-a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.

Cytotoxicity of an Innovative Pressurised Cyclic Solid-Liquid (PCSL) Extract from Artemisia annua.

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid-liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression.

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 "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Expression of transferrin receptor-1 (TFR-1) in canine osteosarcomas.

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.

Nodular-like growth and axial thickening in gorgonians are a defensive response to endolithic cyanobacteria, involving amyloid deposition.

An accurate approach to coral disease study is critical for understanding the global decline of coral populations. Such an approach should involve the proper use of medical concepts and terminology to avoid confusion and promote clarity in the coral disease literature. Inflammatory and neoplastic disorders have been frequently confused in corals. They are both reported as 'growth anomalies' because of their possible gross similarity, but in fact they are very different types of lesions and pathologic phenomena. In this work, we assessed the distribution and prevalence of growth anomalies, externally visible as nodular-like lesions, in the soft corals Eunicella cavolinii and E. singularis in 2008-2009 in 3 different areas along the Campanian coastline of Italy. Histopathology revealed them as chronic inflammatory lesions, resembling chronic inflammatory lesions of vertebrates, encapsulating an unidentified pathogen. Congo red and Masson Fontana histochemistry highlighted an amoebocyte infiltration with the presence of new apposition of melanin coupled with amyloid sheets intended as part of the defensive response, as reported in other invertebrates. A parallel molecular analysis of 16S rRNA of the lesions suggested that the causative agent is an endolithic cyanobacterium belonging to the order Nostocales. This is the first study assessing the presence of amyloid fibrils in corals.

Optimisation of artemisinin and scopoletin extraction from Artemisia annua with a new modern pressurised cyclic solid-liquid (PCSL) extraction technique.

INTRODUCTION: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particular 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 European 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 operators, 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.

Assessment of the Health Status of Mussels Mytilus galloprovincialis Along the Campania Coastal Areas: A Multidisciplinary Approach.

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 (H2O2, 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.

Urtica dioica (Stinging Nettle): A Neglected Plant With Emerging Growth Promoter/Immunostimulant Properties for Farmed Fish.

Urtica dioica (stinging nettle), is a perennial plant belonging to the family of Urticaceae, genus Urtica. Despite the use of nettle in folk veterinary medicine is well documented, U. dioica is today an underestimated and frequently neglected plant, considered by the contemporary agriculture as a weed to be eliminated. This mini review focus on very recent studies on dietary administration of U. dioica, both as a single herb or in combination with other herbs, to enhance growth and stimulate farmed fish immunity, thus enabling the fish to be more resistant against bacterial infections. Such an emerging feature, together with cost-effectiveness, adequate availability, and easy processing of nettle, could make this herb an excellent, inexpensive and widely used dietary supplement on intensive fish farms.

Early evidence for a virus-like agent infecting the pest snail Theba pisana (Gastropoda: Pulmonata) in Southern Italy.

The Mediterranean land snail Theba pisana (Mollusca: Helicidae) is an introduced agricultural pest in many countries 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. pisana snails 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 the presence of hypertrophied nuclei containing eosinophilic to weakly basophilic inclusion bodies. Ultrastructural examination by transmission electron microscopy (TEM) showed nuclear inclusions constituted by a reticulated stroma into which unenveloped, roundish virus-like particles (38±4nm in diameter) were present. To the best of our knowledge this could be the first evidence for a virus-like agent infecting the gastropod T. pisana, which may open new biocontrol perspectives of the this pest worldwide.

Nuclear morphometry and ploidy of normal and neoplastic haemocytes in mussels.

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<0.05). Among those (expressed in pixel units-inter-pixel distance of 0.45 micrometres-as: normal vs. neoplastic) nuclear area (117.1±94.1 vs. 423.1±226.9), perimeter (44.9±14.0 vs. 79.0±21.3) and (IOD) integrated optical density (13.47±34.5 vs. 177.1±150.8) were relevant features to discriminate between normal and neoplastic cells. Those differences allowed identifying two distinctive populations of neoplastic nuclei, occasionally in the same individuals at a given phase of the disease. Moreover, neoplastic haemocytes in less extended lesions showed a ploidy value of 6.2 n along with the presence of a second population of circulating cells with a DNA content of 10.7n. In samples with moderate disease only one peak at 7n was observed. Finally, in more severe conditions, a further ploidy peak of 7.8n was recorded, accompanied by a shallow but broad peak of 31n. This latter extreme value is thought to be due to the presence of giant multinucleated cells where individual nuclei overlap in space and cannot be discerned individually. Computer-based imaging allowed the direct visualization of the cell populations and simultaneous collection of ploidy data as well as morphological features of nuclei.

Disease and Disorders of Freshwater Unionid Mussels: A Brief Overview of Recent Studies.

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.

Fractal analysis of mucosal microvascular patterns in oral lichen planus: a preliminary study.

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.

Proliferation, apoptosis, and fractal dimension analysis for the quantification of intestinal trophism in sole (Solea solea) fed mussel meal diets.

BACKGROUND: The evaluation of intestinal trophism, mainly the mucosal layer, is an important issue in various conditions associated with injury, atrophy, recovery, and healing of the gut. The aim of the present study was to evaluate the kinetics of the proliferation and apoptosis of enterocytes by immunohistochemistry and to assess the complexity of intestinal mucosa by fractal dimension (FD) analysis in Solea solea fed different experimental diets. RESULTS: Histomorphological evaluation of all intestinal segments did not show signs of degeneration or inflammation. Cell proliferation index and FD were significantly reduced with a diet high in mussel meal (MM; p = 0.0034 and p = 0.01063, respectively), while apoptotic index did not show any significant difference for the same comparison (p = 0.3859). Linear regression analysis between apoptotic index (independent variable) and FD (dependent variable) showed a statistically significant inverse relationship (p = 0.002528). Linear regression analysis between cell proliferation index (independent variable) and FD (dependent variable) did not show any significant correlation (p = 0.131582). CONCLUSIONS: The results demonstrated that diets containing increasing levels of mussel meal in substitution of fishmeal did not incite a hyperplastic response of the intestinal mucosa. The mussel meal, which is derived from molluscs, could mimic the characteristics of the sole's natural prey, being readily digestible, even without increasing the absorptive surface of intestinal mucosa. Interestingly, from this study emerged that FD could be used as a numeric indicator complementary to in situ quantification methods to measure intestinal trophism, in conjunction with functional parameters.

Onco-epidemiology of domestic animals and targeted therapeutic attempts: perspectives on human oncology.

The spontaneous tumor biology has been investigated with the support of animalists using animals as a preclinical model allowing translation of results in clinical practice. This review provides an insight into the field of comparative oncology. Evidence shows that companion animal health care is impressively growing in terms of development of new therapies and diagnostic tools, nutrition and disease prevention. However, even if most animal tumors might be a reliable model to study human carcinomas, many open questions, related to the opportunities to select and recruit new models in oncology, along with their legal and ethical implications, remain unanswered.

Nocardiosis in Mediterranean bivalves: first detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy).

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.

Cytomorphology and PCNA expression pattern in bivalves Mytilus galloprovincialis and Cerastoderma edule with haemic neoplasia.

Haemic neoplasia (HN) is a pathologic condition reported in several bivalve species in different geographic areas. In this study we describe the cytomorphological features and the proliferative behaviour, assessed by the proliferating cell nuclear antigen (PCNA), of HN in common cockle Cerastoderma edule and Mediterranean mussel Mytilus galloprovicialis. In mussels the presence of at least 5 types of atypical haemocytes was detected, including A- and B-type cells, previously described in M. edulis and Mytilus sp., with predominance of A-type cells in early phases of the disease and B-type cells in more advanced stages. PCNA immunostaining was positive for 97 to 100% of the neoplastic cells, with both cytoplasmic (A cells) and nuclear patterns (B cells). Conversely, in C. edule there was no distinctive morphological cell sub-population, and staining atypical haemocytes with PCNA (range 93 to 100%) showed nuclear expression in early phases of disease and cytoplasmic expression in more advanced stages. The above findings suggest distinct histo-pathogenetic pathways for HN in mussels and common cockles.

The gonadotropin releasing hormone (GnRH)-like molecule in prosobranch Patella caerulea: potential biomarker of endocrine-disrupting compounds in marine environments.

It has been reported that endocrine disrupter compounds (EDCs) interfere with the endocrine system, mimicking the action of sex steroid hormones in different species of mollusks. Prosobranchs are frequently used as a reliable bioindicator to evaluate EDC exposure. In this article, we evaluate the effects of the xenoestrogen 4-n-nonylphenol (NP) in the prosobranch gastropod Patella caerulea, which exhibits protandrous hermaphroditism as its reproductive strategy. We isolated a partial sequence of a GnRH-like molecule from the gonads of Patella caerulea. The deduced amino acid sequence is highly homologous to that reported for the Lottia gigantea GnRH. Patella caerulea GnRH (pGnRH) mRNA expression is widespread in both male and female germ lines during gametogenesis. We suggest pGnRH as a novel biomarker for the early assessment of presence of EDCs and monitoring short and long-term impacts on Patella caerulea community structure.