Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Cetacean Morbillivirus and Toxoplasma gondii Co-infection in Mediterranean Monk Seal Pup, Italy.

A Mediterranean monk seal (Monachus monachus) pup from the southern Adriatic coast of Italy showed cetacean morbillivirus (CeMV) and disseminated Toxoplasma gondii co-infection, which probably resulted from CeMV-induced immunosuppression. These findings are of concern for the conservation of this critically endangered species.

Dolphin Morbillivirus in Eurasian Otters, Italy.

We report biomolecular evidence of dolphin morbillivirus in 4 wild Eurasian otters (Lutra lutra) from southern Italy; 2 animals showed simultaneous immunohistochemical reactivity against morbilliviral antigen. These cases add further concern and support to the progressively expanding host range of dolphin morbillivirus in the western Mediterranean Sea.

Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia.

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min-1 as the limit for the heart rate for an extended dive of over 3 min. Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain.

Dolphin Morbillivirus Associated with a Mass Stranding of Sperm Whales, Italy.

In September 2014, seven sperm whales were stranded along Italy's Adriatic coastline. Postmortem investigations on 3 female adult whales and 1 male fetus carried by the largest female revealed molecular and immunohistochemical evidence of dolphin morbillivirus infection. A possible role of the virus in the stranding event was considered.

Mediterranean Fin Whales (Balaenoptera physalus) Threatened by Dolphin MorbilliVirus.

During 2011-2013, dolphin morbillivirus was molecularly identified in 4 stranded fin whales from the Mediterranean Sea. Nucleoprotein, phosphoprotein, and hemagglutinin gene sequences of the identified strain were highly homologous with those of a morbillivirus that caused a 2006-2007 epidemic in the Mediterranean. Dolphin morbillivirus represents a serious threat for fin whales.

Correlation of renal histopathology with renal echogenicity in dogs and cats: an ex-vivo quantitative study.

BACKGROUND: Increased cortical or cortical and medullary echogenicity is one of the most common signs of chronic or acute kidney disease in dogs and cats. Subjective evaluation of the echogenicity is reported to be unreliable. Patient and technical-related factors affect in-vivo quantitative evaluation of the echogenicity of parenchymal organs. The aim of the present study is to investigate the relationship between histopathology and ex-vivo renal cortical echogenicity in dogs and cats devoid of any patient and technical-related biases. RESULTS: Kidney samples were collected from 68 dog and 32 cat cadavers donated by the owners to the Veterinary Teaching Hospital of the University of Padua and standardized ultrasonographic images of each sample were collected. The echogenicity of the renal cortex was quantitatively assessed by means of mean gray value (MGV), and then histopathological analysis was performed. Statistical analysis to evaluate the influence of histological lesions on MGV was performed. The differentiation efficiency of MGV to detect pathological changes in the kidneys was calculated for dogs and cats. Statistical analysis revealed that only glomerulosclerosis was an independent determinant of echogenicity in dogs whereas interstitial nephritis, interstitial necrosis and fibrosis were independent determinants of echogenicity in cats. The global influence of histological lesions on renal echogenicity was higher in cats (23%) than in dogs (12%). CONCLUSIONS: Different histopathological lesions influence the echogenicity of the kidneys in dogs and cats. Moreover, MGV is a poor test for distinguishing between normal and pathological kidneys in the dog with a sensitivity of 58.3% and specificity of 59.8%. Instead, it seems to perform globally better in the cat, resulting in a fair test, with a sensitivity of 80.6% and a specificity of 56%.

Report on the brain of the monk seal (Monachus monachus, Hermann, 1779).

The Mediterranean monk seal (Monachus monachus, Hermann, 1779) is an endangered species of pinniped endemic to few areas of the Mediterranean Sea. Extensive hunting and poaching over the last two centuries have rendered it a rare sight, scattered mainly in the Aegean Sea and the western coast of North Africa. In a rare event, a female monk seal calf stranded and died in southern Italy (Brindisi, Puglia). During due necropsy, the brain was extracted and fixed. The present report is the first of a monk seal brain. The features reported are remarkably typical of a true seal brain, with some specific characteristics. The brain cortical circonvolutions, main fissures and the external parts are described, and an EQ was calculated. Overall, this carnivore adapted to aquatic life shares some aspects of its neuroanatomy and physiology with other seemingly distant aquatic mammals.

Bottlenose dolphin (Tursiops truncatus) immortalized fibroblasts on novel 3D in vitro collagen-free scaffolds.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species.

First Detection of Gammacoronavirus in a Striped Dolphin (Stenella coeruleoalba) from the Adriatic Sea.

This case report presents the first molecular identification of a gammacoronavirus in a free-ranging striped dolphin (Stenella coeruleoalba) that was found stranded along the Croatian coastline in 2022. The dolphin exhibited a concurrent infection with cetacean morbillivirus. The gammacoronavirus strain was amplified and sequenced from heart tissue imprinted on an FTA®card, revealing a notable genetic distance (approximately 8%) from previously characterized cetacean gammacoronaviruses. This finding highlights the importance of including gammacoronaviruses in routine diagnostics for stranded dolphins to gather epidemiological data on their prevalence and potential role in causing disease in cetaceans. This study sets the premises for a further understanding of the diversity and distribution of gammacoronaviruses in marine mammals and highlights the necessity for ongoing surveillance of emerging infectious diseases in wild populations.

Are we even close? Five years marine litter ingestion monitoring in loggerhead turtles along Italian coast reveals how far we are from the Good Environmental Status.

The loggerhead sea turtle Caretta caretta has been chosen as bioindicator to monitor the amount of litter ingested by marine animals within the European Marine Strategy Framework Directive and the Barcelona Regional Sea Convention. European Member States and Contracting Parties are committed to achieve the Good Environmental Status (GES), which is reached when the quantity of ingested litter does not adversely affect the health of the species concerned. Although the monitoring strategy has been outlined for more than a decade, to date no threshold values have been adopted to verify GES achievement. After five years of extensive monitoring along the Italian coasts, this study evaluates the suitability of five different GES scenarios and proposes a new threshold value (i.e., "there should be less than 33% of sea turtles having more than 0.05 g of ingested plastic in the GI") for its implementation in the European seas and the Mediterranean basin.

Pathogen Prevalence in Cetaceans Stranded along the Italian Coastline between 2015 and 2020.

The monitoring of stranded marine mammals represents a strategic method to assess their health, conservation status, and ecological role in the marine ecosystem. Networks worldwide track stranding events for the passive monitoring of mortality patterns, emerging and reemerging pathogens, climate change, and environmental degradation from a One Health perspective. This study summarizes pathogen prevalence data from the Italian Stranding Network (ISN) derived from post-mortem investigations on cetaceans found dead stranded along the Italian coastline between 2015 and 2020. The decomposition of the carcasses and logistics limited the post-mortem examination to 585 individuals, out of 1236 single-stranding reports. The most relevant pathogens identified were Cetacean Morbillivirus, Herpesvirus, Brucella spp., and Toxoplasma gondii, whose roles as environmental stressors are well known, despite their real impact still needing to be investigated in depth. Statistical analysis showed that age and sex seem to be positively related to the presence of pathogens. This study represents the first step in harmonizing post-mortem investigations, which is crucial for evidence-based conservation efforts. Implementing diagnostic and forensic frameworks could offer an indirect insight into the systematic monitoring of diseases to improve the identification of regional and temporal hotspots in which to target specific mitigation, management, and conservation strategies.

Network analysis to reveal the most commonly detected compounds in predator-prey pairs in freshwater and marine mammals and fish in Europe.

Marine and freshwater mammalian predators and fish samples, retrieved from environmental specimen banks (ESBs), natural history museum (NHMs) and other scientific collections, were analysed by LIFE APEX partners for a wide range of legacy and emerging contaminants (2545 in total). Network analysis was used to visualize the chemical occurrence data and reveal the predominant chemical mixtures for the freshwater and marine environments. For this purpose, a web tool was created to explore these chemical mixtures in predator-prey pairs. Predominant chemicals, defined as the most prevalent substances detected in prey-predator pairs were identified through this innovative approach. The analysis established the most frequently co-occurring substances in chemical mixtures from AP&P in the marine and freshwater environments. Freshwater and marine environments shared 23 chemicals among their top 25 predominant chemicals. Legacy chemical, including perfluorooctanesulfonic acid (PFOS), brominated diphenyl ethers (BDEs), polychlorinated biphenyls (PCBs), hexachlorobenzene and mercury were dominant chemicals in both environments. Furthermore, N-acetylaminoantipyrine was a predominant pharmaceutical in both environments. The LIFE APEX chemical mixture application (https://norman-data.eu/LIFE_APEX_Mixtures) was proven to be useful to establish most prevalent compounds in terms of number of detected counts in prey-predator pairs. Nonetheless, further research is needed to establish food chain associations of the predominant chemicals.

Dolphin morbillivirus infection in a captive harbor seal (Phoca vitulina).

During the second morbillivirus epidemic (2007 to 2011) in cetaceans along the Italian coastline, dolphin morbillivirus (DMV) was detected by molecular analyses in a captive harbor seal (Phoca vitulina), with pathological findings consistent with morbillivirus infection. This report confirms interspecies DMV transmission from cetaceans to pinnipeds.

Environmental and pathological factors affecting the hatching success of the two northernmost loggerhead sea turtle (Caretta caretta) nests.

In recent years, the report of loggerhead sea turtle (Caretta caretta) Mediterranean nesting range has expanded together with new records of nests becoming northward on the Italian coastline of the Tyrrhenian and Adriatic seas. These areas are characterized by intensive human activities, such as tourism, fishery, and marine traffic, all possibly involved in the influence of the use of coastal habitat by marine species. These anthropic threats, in addition to the natural ones and the changing environmental characteristics of the beach, may influence the growth of microorganisms causing hatching failures. Among microorganisms, fungal infection by the genus Fusarium (Link, 1809) is considered one of the main causes of globally declining sea turtle populations. In summer 2021, the two northernmost worldwide loggerhead sea turtle nests were monitored along the Northern Adriatic coastline (Veneto, Italy). These first records may potentially candidate this area as suitable for a large part of the loggerhead turtle's life cycle and it could represent a minor sea turtle nesting area that, according to Prato and colleagues, remained unnoticed due to the lack of specific monitoring. Sea Turtle Egg Fusariosis (STEF) was deemed to have deeply compromised the hatching success of the northmost one. Climate change and anthropogenic impacts have been scored as one of the highest hazards to sea turtle health and could have played a role in the STEF development. Environmental changes, human activities, and emerging pathogens deserve the highest attention in terms of health research, and conservation management.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles.

Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-ß. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.

Comparison between Sampling Techniques for Virological Molecular Analyses: Dolphin Morbillivirus and Herpesvirus Detection from FTA® Card and Frozen Tissue.

Stranded animals offer valuable information on marine mammal physiology and pathology; however, the decomposition state of the carcasses and lack of a rigorous cold chain for sample preservation can sometimes discourage diagnostic analyses based on nucleic acid detection. The present paper aims at evaluating the reliability of FTA® card tissue imprints as an alternative matrix to frozen tissues for virological analyses based on biomolecular methods. Given the contribution of Cetacean morbillivirus (CeMV) to strandings and the increase of herpesvirus detection in cetaceans, these two pathogens were selected as representative of RNA and DNA viruses. Dolphin morbillivirus (DMV) and herpesvirus presence was investigated in parallel on tissue imprints on FTA® cards and frozen tissues collected during necropsy of dolphins stranded in Italy. Samples were analysed by nested RT-PCR for DMV and nested-PCR for herpesvirus. Only one animal was positive for herpesvirus, hampering further considerations on this virus. DMV was detected in all animals, both in FTA® card imprints and tissue samples, with differences possibly related to the decomposition condition category of the carcasses. Tissue sampling on FTA® cards seems a promising alternative to frozen tissues for biomolecular analyses, especially when ensuring adequate storage and shipment conditions for frozen tissues is difficult.

Age-related changes in the primary auditory cortex of newborn, adults and aging bottlenose dolphins (Tursiops truncatus) are located in the upper cortical layers.

Introduction: The auditory system of dolphins and whales allows them to dive in dark waters, hunt for prey well below the limit of solar light absorption, and to communicate with their conspecific. These complex behaviors require specific and sufficient functional circuitry in the neocortex, and vicarious learning capacities. Dolphins are also precocious animals that can hold their breath and swim within minutes after birth. However, diving and hunting behaviors are likely not innate and need to be learned. Our hypothesis is that the organization of the auditory cortex of dolphins grows and mature not only in the early phases of life, but also in adults and aging individuals. These changes may be subtle and involve sub-populations of cells specificall linked to some circuits. Methods: In the primary auditory cortex of 11 bottlenose dolphins belonging to three age groups (calves, adults, and old animals), neuronal cell shapes were analyzed separately and by cortical layer using custom computer vision and multivariate statistical analysis, to determine potential minute morphological differences across these age groups. Results: The results show definite changes in interneurons, characterized by round and ellipsoid shapes predominantly located in upper cortical layers. Notably, neonates interneurons exhibited a pattern of being closer together and smaller, developing into a more dispersed and diverse set of shapes in adulthood. Discussion: This trend persisted in older animals, suggesting a continuous development of connections throughout the life of these marine animals. Our findings further support the proposition that thalamic input reach upper layers in cetaceans, at least within a cortical area critical for their survival. Moreover, our results indicate the likelihood of changes in cell populations occurring in adult animals, prompting the need for characterization.

Loggerhead Sea Turtle as Possible Source of Transmission for Zoonotic Listeriosis in the Marine Environment.

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1-0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl-Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment.

Assessment of Antimicrobial Effects on Broiler Gut Barrier Through Histopathology and Immunohistochemistry of Tight-Junction Proteins.

In recent years, antimicrobial (AM) use in poultry farming has been attracting attention worldwide mainly due to AM resistance spreading. The role of AM prophylaxis in the modulation of gut microbiota, as well as of gut health, is still not clearly understood. Therefore, this study aimed to investigate the role of different prophylaxis protocols in the modulation of the gut barrier in broilers by applying a histopathological approach. Intestinal tissue samples were collected from a total of 240 male broilers (Ross 306), reared and treated with different AM protocols. Haematoxylin and Eosin (HE) staining and a multiple scoring system were used to evaluate the presence of lesions in ileum, cecum and colon of treated broilers. Moreover, immunohistochemistry (IHC) was performed to assess the expression of claudin-3 and ZO-1 proteins in intestinal tissues. The application of a semi-quantitative scoring system was used in IHC stained samples. HE results revealed that intestinal tissues were mainly characterized by epithelial detachment and fusion of the intestinal villi, but also by the presence of lymphocytic infiltrate in the mucosa and submucosa of AM-treated broilers. However, the IHC approach for the evaluation of claudin-3 and ZO-1 proteins showed that their expression was not affected by the different AM treatments. Nevertheless, the presence of intestinal lesions highlighted by histopathology suggests that AM treatments could harm the gut health of broilers, inducing an inflammatory response and consequent epithelial lesions. In order to clarify the role of AM treatments in the modulation of gut barrier in broilers, further studies are needed.