Baseline pathological data of the wedge clam Donax trunculus from the Tyrrhenian Sea (Mediterranean Basin).

In recent years, a collapse in Donax trunculus fishing yields has occurred in the Tyrrhenian Sea (Mediterranean Basin). There is little information available on the impact disease may have had on D. trunculus populations. For the first time, a pathological survey was performed on the natural beds of the bivalve on the Campania and Lazio coasts, western Italy. Detected pathogens and related diseases were analysed, and their prevalence and mean intensity values were calculated. Viral particles, Chlamydia-like organisms, ciliates, coccidians, microcells and trematodes were observed. An unknown ciliate was linked to severe inflammatory and necrotic lesions in the digestive gland. Metacercariae of the trematode Postmonorchis sp. were also strongly represented in almost all samples, reaching high levels of infection; however, none of the pathogens described required the World Organisation for Animal Health to be notified. Initial results indicated that further surveys related to environmental data are necessary in order to assess the relevance of these early observations in managing the declining D. trunculus population in the Tyrrhenian Sea.

Comparative pathology in bivalves: Aetiological agents and disease processes.

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.

Quantitative histopathology of the Mediterranean mussel (Mytilus galloprovincialis L.) exposed to the harmful dinoflagellate Ostreopsis cf. ovata.

Ostreopsis cf. ovata is a benthic dinoflagellate that produces palytoxin-like compounds that adversely affect both marine vertebrates and invertebrates and are reported to be responsible for human intoxication in aerosol form. In this work, a histopathological analysis accompanied by quantitative evaluation of tissue injury in mussels (Mytilus galloprovincialis) exposed to O. cf. ovata cells under natural and experimental conditions, provided baseline data on the health status of the mussels in terms of defensive and regressive pathological changes. We recorded a total of 15 health parameters in the digestive system, muscle, kidney and gills in mussels exposed to O. cf. ovata both in the laboratory and at sea. Animals exposed to different concentrations of O. cf. ovata cells (300, 500 and 1000cellsml(-)(1)) for 48h showed activation of the inflammatory response, which increased with the cell concentration, mainly characterized by haemocyte aggregates actively enclosing the algae, while mussel mortality was also recorded in some cases. Moreover the use of image analysis for the evaluation of digestive tubule damage revealed a pronounced increase in the lumen in terms of its area, perimeter and circularity, with a shift in a high percentage of tubules from an adsorbing profile to an atrophic profile. Animals collected from the natural environment during a summer bloom of O. cf. ovata in the Gulf of Naples (Italy) showed comparable lesions in terms of types and severity. This is the first quantitative study assessing damage to the digestive epithelia in terms of lumen modifications in mussels exposed to O. cf. ovata. The presented methodology provides a new technique for automating the evaluation of epithelial tubule modifications. Our results highlight the importance of monitoring the presence of O. cf. ovata in this area, taking into account the effects on the residing marine species.

Gorgonian disease outbreak in the Gulf of Naples: pathology reveals cyanobacterial infection linked to elevated sea temperatures.

In recent years, mass mortality events of benthic invertebrates in the Mediterranean Sea have been documented to coincide with the increased seawater temperatures associated with global climate change. Following a disease outbreak in gorgonians during the summer seasons of 2008 and 2009 in the Gulf of Naples (Tyrrhenian Sea), we conducted gross and microscopic analyses of healthy and diseased specimens of Eunicella cavolinii and E. singularis using both light and electron microscopy (SEM). Macroscopically, diseased colonies exhibited evident tissue thinning, and dead colonies showed a complete loss of polyps and coenenchyme, exposing their skeletons to settlement by fouling organisms. Histopathology revealed chronic inflammatory lesions at the polyp and axial level, characterized by amoebocyte infiltration of tissue accompanied by new apposition of melanin/gorgonin sheets. We interpreted this response as a defense against different kinds of pathogens-identified as mainly a heterogeneous consortium of filamentous cyanobacteria-and which gradually led to enlargement and hardening of the coral axis, which resembled a wood-like structure at the final stage of the disease. These processes elicited the formation of multiple inflammatory nodules and capsules, some of which were macroscopically visible. A parallel 16S rRNA and ITS analysis of the diseased tissue identified Synechococcus, Arthrospira and other uncultured cyanobacteria grouped within the Oscillatoriales. These results suggest that a cyanobacterial consortium is involved in the pathogenesis of the inflammatory disease leading to the mortality of Gorgoniaceae in the area. Finally, there were anomalously high temperatures (up to 25°C) between 10 and 20 m depth during the sampling period, particularly in June 2009. This supports the hypothesis that the coral skeleton may serve as a reservoir for the pathogens in cooler seasons, with warmer conditions leading to pathogen reactivation and recurring mortality events.

Postmonorchis sp. inq. (Digenea: Monorchiidae) metacercariae infecting natural beds of wedge clam Donax trunculus in Italy.

The wedge clam Donax trunculus Linnaeus, 1758 is one of the most common bivalve molluscs inhabiting the sandy shores of the Mediterranean Sea and is considered an important commercial resource. In this study, we report the first molecular, morphological and histopathological descriptions of metacercariae from a trematode belonging to the genus Postmonorchis (Digenea: Monorchiidae) that infects D. trunculus in natural beds of the Italian Tyrrhenian coast (Campania, Lazio and Tuscany). Morphological analysis of the parasite revealed a combination of features that exist in the 3 previously identified species of Postmonorchis, viz. P. donacis, P. variabilis and P. orthopristis, with the addition of new, distinctive morphological characteristics. The pathogen exhibited a predilection for the gill; however, it was also present in the labial palp and mantle in addition to the gut, kidney epithelium and foot. The inflammatory response was characterised by either a focal or diffuse haemocyte infiltration followed by the formation of multiple, large multi-layered capsules associated with tissue destruction. The prevalence of the pathogen ranged from 75 to 100%, while the infection intensity fluctuated among the study areas. Further studies regarding the life cycle of this parasite and the identification of other larval and adult stages and their respective hosts may confirm the identification of a new species of Postmonorchis that infects wedge clams in Mediterranean waters. The study of the parasite is completed by molecular analysis of the ITS1 and ITS2 rDNA sequences.

A mycobacterial disease is associated with the silent mass mortality of the pen shell Pinna nobilis along the Tyrrhenian coastline of Italy.

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.

Histological, histochemical and morphometric changes of splenic melanomacrophage centers (SMMCs) in Sparicotyle-infected cultured sea breams (Sparus aurata).

Diseases caused by parasites are much more frequently described in cultured fish, which suffer from artificial conditions and numerous stress factors. This study investigates the histological, histochemical and morphometric modifications of splenic melanomacrophage centers (SMMCs) infected by Sparicotyle chrisophrii (Monogenea, ectoparasite of the gills) in sea breams (Sparus aurata), cultured in floating cages in the Gulf of Gaeta (Italy). Infected fish swam near the water surface, showing severe signs of anemia. Several spleens were collected from both healthy and dead fish (70-100 gr. body weight). A spleen histopathology was evaluated by using traditional stainings, such as Haematoxylin and Eosin (HE), Periodic Acid-Schiff reaction (PAS), Perl's reaction for haemosiderin and Schmorl's reaction for lipofuscins. Furthermore, SMMCs morphometry was performed on PAS-stained sections to study 7 morphometric parameters [Mean SMMCs profile area (MPA), Mean SMMCs maximum diameter (Media), Mean SMMCs minimum diameter (media), Mean SMMCs diameter (Dia), Mean SMMCs Perimeter (P), Mean SMMCs Form Factor (FF) and Mean SMMCs number per square millimeter of spleen tissue (MN)]. A light microscope of HE stained sections of spleen revealed a dramatic increase in the size and number of SMMCs in parasitized animals. Morphometric data illustrated statistically significant differences (p < 0.01) of all studied parameters between healthy and diseased fish. This study emphasizes the importance of using histopathological investigations to unravel the complex biological host/parasite interaction, which results in systemic lesions affecting reared marine species.

Nuclear morphometry in relation to lymph node status in canine mammary carcinomas.

The assessment of nuclear area and nuclear shape by morphometric analysis, has been investigated in 40 canine mammary carcinomas in relation to their metastatic behaviour to regional lymph-nodes. The tumours were reviewed by two experienced pathologists blinded regarding their lymph-node status, and were classified according to the histogenetically based criteria suggested by Benjamin et al. (1999). Twenty of these tumours showed lymph-node metastases (node-positive), and the other twenty were node-negative. Node-positive tumours included 6 simple adenocarcinomas, 10 ductular carcinomas, 2 anaplastic carcinomas and 2 carcinomas in mixed tumours; node-negative tumours included 18 adenocarcinomas %96, 10 simple adenocarcinomas, 8 complex adenocarcinomas %96, and 2 carcinomas in mixed tumours. Node-positive tumours showed MNA and mean SDA values significantly higher (p<0.001) than node-negative carcinomas. Data of this study, seems to confirm the importance of an histogenetically based classification in canine mammary tumours, also suggesting that morphometry may increase our prognostic performances allowing a reproducible method for detecting individual tumours with higher metastatic potential.

A novel workflow for computer guided implant surgery matching digital dental casts and CBCT scan.

Nowadays computer-guided "flap-less" surgery for implant placement using stereolithographic tem-plates is gaining popularity among clinicians and patients. The advantages of this surgical protocol are its minimally invasive nature, accuracy of implant placement, predictability, less post-surgical discomfort and reduced time required for definitive rehabilitation. Aim of this work is to describe a new protocol (Smart Fusion by Nobel Biocare), thanks to which is now possible to do a mini-invasive static guided implant surgery, in partially edentulous patients with at least 6 remaining teeth, without the use of a radiographic guide. This is possible thanks to a procedure named surface mapping based on the matching between numerous points on the surface of patient's dental casts and the corresponding anatomical surface points in the CBCT data. The full protocol is examined focusing the attention on the clinical and laboratory procedures. CONCLUSIONS: Also with some critical points and needing an adequate learning curve, this protocol allows to select the ideal implant position in depth, inclination and mesio-distal distance between natural teeth and or other implants enabling a very safe and predictable rehabilitation compared with conventional surgery. It represents a good tool for the best compromise between anatomy, function and aesthetic, able to guarantee better results in all clinical situations.

Smart fusion vs. double scan: a comparison between two data-matching protocols for a computer guided implant planning.

AIM: To make a comparison between two different approaches of data matching during the preparation of a computer guided implant planning. MATERIAL AND METHODS: Electronic and manual literature searches were performed to collect information about the double-scan protocol and the smart fusion protocol. The two systems were then tested to identify or confirm their advantages and disadvantages. RESULTS: The double scan protocol is a reliable and well-known process to obtain a fusion between the anatomical data (CBCT) and the prosthetic data (radiographic template). It is possible to use this protocol both in dentate and edentulous patients. The newly developed smart fusion technique offers the possibility to superimpose the anatomical data (CBCT) onto the prosthetic data (cast + wax-up scan) without the production of a radiographic template. This system is still being tested by the clinicians even though dental manufacturers already consider it as the best solution for the dentate patients selected for a static computer guided implant surgery. CONCLUSIONS: Smart fusion protocol offers a reasonable time and cost reduction even though its application is limited to dentate patients. A noticeable drawback of the workflow is the matching step: often a manual intervention is necessary to obtain a correct alignment of the CBCT data with the lab scan of the cast. This issue is partially due to the use of non-volume stable materials during the preparation of the cast. Future improvements could be made by combining CBCT data with direct optical scans of patient dental arches in order to create the so-called 'virtual patient'.

Fractal organization of feline oocyte cytoplasm.

The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display self-similar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400x with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

Nucleomorphometric analysis of canine cutaneous mast cell tumours.

Thirty-five canine cutaneous mast cell tumours (CCMCTs) were analysed by computerized nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), mean nuclear perimeter (MNP) and mean nuclear form factor (FF) were calculated. Significant differences in respect of MNA and MNP occurred between tumours of grades I and III and between those of grades II and III (P<0.01) but not between tumours of grades I and II (P>0.01). No significant differences in respect of FF were observed between tumours of different grades. The results obtained indicate that nuclear morphometric analysis may assist in the grading of CMCTs.

Transcrestal guided sinus lift without grafting materials: a 36 months clinical prospective study.

PURPOSE: This study describes the ability to perform a technique for bone regeneration in maxillary posterior deficit (TGSL) without the use of bone grafting materials using a highly minimally invasive protocol. MATERIALS AND METHODS: Sixty six implants have been inserted in the sinus floor of a total of 39 patients through the transcrestal guided sinus lift technique (TGSL). All patients have been followed for at least three years in function. The drilling protocol was adapted on the basis of bone density of each implant site to achieve a torque between 45 and 55 Ncm. Healing titanium abutments tightened to 35 Ncm have been used. A CAD/CAM metal ceramics final prosthetic restoration has been generated a six months after the tissues healing and the provisional functionalization of the occlusion. Survival rate of implants and prostheses, biological and biomechanical complications, changes in marginal bone levels, and total height of alveolar crest bone before and after surgery have been evaluate and measured by the results obtained in this prospective study. It was also measured the periodontal parameters as well as levels of perception of pain by the patient during the entire recovery period. RESULTS: The result of the data of follow-up was 41.96 (24 to 36) months. Cumulative implant survival was 98.53% at 3 years. There were no biological and mechanical complications and there were no prosthetic failures during the whole period of follow-up. The Marginal Bone Loss (MBL) average during the first year of operation was from 0.33 to 0.36 mm, while the 3-year follow-up, the MBL average was 0.51 to 0.29 mm. The average of residual bone height of alveolar ridge before treatment was 6.7 to 1.6 mm (range 5.1 to 9.2 mm), while the average bone height was gained 6,4 - 1.6 mm (range 3.2 to 8.1 mm). All patients reported lower pain levels and found to have normal periodontal parameters. CONCLUSIONS: This study suggests that the use of guided surgery to perform transcrestal maxillary sinus lift to increase the sub-antral crestal height is a minimally invasive technique of success for the short and medium-term of follow-up, thus avoiding the extended treatment time and reducing the morbidity associated with the lifting of the floor of the maxillary sinus with traditional technique using bone grafting materials. Furthermore, this protocol without the use of graft materials does not vary the final outcome that have demonstrated the presence of newly formed bone around implants offering always predictable results, and giving a further reduction in the costs of the procedure rehabilitation.

Morphometric study of nucleolar organiser regions (AgNOR) in HPV-associated precancerous lesions and microinvasive carcinoma of the oral cavity.

A morphometric study of silver-stained nucleolar organiser regions (NOR) was performed on histological sections from routinely paraffin-embedded blocks of 26 oral biopsy specimens (21 cases of leukoplakia consisting of 13 low, 4 moderate, 4 severe degree of dysplasia, and 5 cases of microinvasive carcinomas). In situ hybridisation (ISH) for HPV-DNA was performed on serial sections of the same samples. The following parameters were studied: V NOR (single AgNOR volume per nucleus), TV NOR (total AgNOR volume per nucleus), and R.I. (AgNOR's roundness index). The results highlight that not all the morphometric features of AgNORs allow the discrimination between lesions with dysplasia of low, moderate and severe degree and microinvasive carcinoma. TV NOR appeared useful, while the other morphometric parameters appeared statistically not significant i differentiating between the different lesions. These findings suggest that high values of TV NOR in oral dysplasia could represent a risk marker, identifying a subgroup of lesions with a worse prognosis, constituting then a possible indication for rigorous clinical management and/or for complex treatment of these HPV-associated preneoplastic lesions.

Tissue damage and haematophagia due to praniza larvae (Isopoda: Gnathiidae) in some aquarium seawater teleosts.

Over a 5 yr period, severe infestations due to Gnathia vorax larvae affecting different teleosts have been registered at the Civic Aquarium of Messina, Italy. Here, the damage caused by these parasites is evaluated from a macroscopical and histopathological point of view. We also attempt to evaluate the amount of blood sucked up by a single parasite and make some observations about the haematophagia. The histopathological changes confirm mucosal electrolytic failure as the main problem, even though the mean value of blood sucked up by a single parasite, about 1.9 mg, suggests that haematophagia may play an important role in the pathogenesis of such infestation. In conclusion, it was found that the G. vorax larvae caused 2-fold damage that was responsible for the death of the fish. Little by little, the parasitic problem was successfully removed by washing the tank, using fresh water rather than costly and dangerous chemotherapeutans.

Prognostic value of nuclear morphometry in feline mammary carcinomas.

This study of 30 cats with mammary carcinoma was designed to investigate the relationship between (1) six nuclear morphometric parameters (mean nuclear profile area [MNA], standard deviation of MNA [SDa], coefficient of variation of MNA [CVa], nuclear form factor (p2/4 pi area) [FF], standard deviation of FF [SDf], and coefficient of variation of FF [CVf]) assessed by image analysis, and (2) survival for > 1 year or < 1 year after surgical removal of the tumour. Only the SDf and the CVf appeared to be related to survival. Cats that died within 1 year had an SDf or CVf (or both) higher than the corresponding mean values (SDft and CVft) for all 30 cats; but only four of 16 cases (25%) with a SDf lower than SDft and five of 17 cases (29.4%) with a CVf lower than CVft died within 1 year. The authors conclude that SDf and CVf represent reliable prognostic parameters in feline mammary carcinomas.

Expression of beta 1 integrin in normal, dysplastic and neoplastic canine mammary gland.

The expression of beta 1 integrin was evaluated immunohistochemically in a series of normal, dysplastic and neoplastic canine mammary glands, and in lymph node metastases. The tissues were formalin-fixed and paraffin wax-embedded. In malignant neoplasms, beta 1 integrin was decreased and redistributed along the entire cell membrane. In lymph nodes, strong immunohistochemical staining was seen intercellularly in clusters of metastatic cells within subcapsular sinuses and at the periphery of intranodal metastases. These results suggest that the expression of integrin molecules may be related to malignancy and to the metastatic potential of neoplastic cells.

Number and size of silver-stained nucleoli (Ag-NOR clusters) in canine seminomas: correlation with histological features and tumour behaviour.

Twenty spontaneous canine seminomas were examined. A statistically significant difference (P < 0.01) was detected in both the mean silver-stained nucleolar area/cell and the mean silver-stained nucleolar number/cell between (1) two intraductal seminomas with no signs of invasion, and (2) three intraductal ones with signs of invasion and 15 diffuse-type seminomas (two of which metastasized 3 and 12 months after surgical treatment). Furthermore, the metastasizing seminomas showed a mean silver-stained nucleolar area/cell which was significantly greater than that observed in all the other seminomas examined (P < 0.01). It is concluded that quantitative studies of nucleolar changes are useful in evaluating the biological behaviour of spontaneous canine seminomas.

Silver-stained nucleolar (Ag-NOR) cluster size in feline mammary carcinomas: lack of correlation with histological appearance, mitotic activity, tumour stage, and degree of nuclear atypia.

The relationship between Ag-NOR cluster size, mitotic index, tumour stage, degree of nuclear atypia and histological appearance was evaluated in a series of feline mammary carcinomas. The mean Ag-NOR cluster area per cell in these tumours ranged from 1.86 to 6.25 microns 2 and was not related to any other parameter studied. This suggests that Ag-NOR cluster size measurement is of poor diagnostic and prognostic value for feline mammary carcinomas.

Expression of vascular endothelial growth factor in basal cell tumours and in squamous cell carcinomas of canine skin.

The expression of vascular endothelial growth factor (VEGF) was evaluated immunohistochemically in 20 basal cell tumours (BCTs) and 15 squamous cell carcinomas (SCCs) of canine skin. VEGF was identified in all the SCCs and was particularly striking in those occurring on the toe. On the other hand, VEGF was absent in the great majority of BCTs, occurring only in those of the solid type. The results suggest that presence of VEGF is a useful additional criterion for evaluating malignancy and growth potential in tumours of these types.