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.

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.

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.

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.

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.

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.

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.

Multimodal Assessment of Bottlenose Dolphin Auditory Nuclei Using 7-Tesla MRI, Immunohistochemistry and Stereology.

The importance of assessing neurochemical processes in the cetacean brain as a tool for monitoring their cognitive health and to indirectly model human neurodegenerative conditions is increasingly evident, although available data are largely semiquantitative. High-resolution MRI for post-mortem brains and stereology allow for quantitative assessments of the cetacean brain. In this study, we scanned two brains of bottlenose dolphins in a 7-Tesla (7T) MR scanner and assessed the connectivity of the inferior colliculi and ventral cochlear nuclei using diffusion tensor imaging (DTI). Serial thick sections were investigated stereologically in one of the dolphins to generate rigorous quantitative estimates of identifiable cell types according to their morphology and expression of molecular markers, yielding reliable cell counts with most coefficients of error <10%. Fibronectin immunoreactivity in the dolphin resembled the pattern in a human chronic traumatic encephalopathy brain, suggesting that neurochemical compensation for insults such as hypoxia may constitute a noxious response in humans, while being physiological in dolphins. These data contribute to a growing body of knowledge on the morphological and neurochemical properties of the dolphin brain and highlight a stereological and neuroimaging workflow that may enable quantitative and translational assessment of pathological processes in the dolphin brain in the future.

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.

The Odontocete Ear Canal-Associated Lymphoid Tissue (ECALT) and Lymph Nodes: Morphological and Pathological Description with Immuno-Phenotypic Characterisation.

A changing marine environment with emerging natural and anthropogenic stressors challenges the marine mammal immune system. The skin and adnexa form a first protective barrier in the immune response, although this is still relatively understudied in cetaceans. The cellular and tissue morphology of the nodular and diffuse lymphoid tissue are not fully charted and the physiological responses are not yet completely understood. The odontocete's external ear canal has a complex relationship with the external environment, with an artificial lumen rendering the inside of the canal a relatively secluded environment. In this work, we studied the odontocete ear canal-associated lymphoid tissue (ECALT) by histo- and immunohistochemistry (HC, IHC) with anti-CD3, anti-CD20, anti-Iba-1, anti-HLA-DR, and anti-vimentin antibodies. The ECALT cellular composition consists mainly of B-lymphocytes with the occasional presence of T-lymphocytes and the dispersed distribution of the macrophages. In cases of activation, the cellular reaction showed a similar pattern with the occasional presence of T-cells, plasma cells, and neutrophils. Nodular lymphoid tissue was generally in line with the description in other odontocetes, although with abundant erythrocytes throughout the entire organ. This study contributes to the understanding of the cellular composition of diffuse and nodular lymphoid tissue in several species of odontocetes, and in association with inflammation of the external ear canal.

Systematic validation and assessment of immunohistochemical markers for central nervous system pathology in cetaceans, with emphasis on auditory pathways.

Cetacean neuropathology is a developing field that aims to assess structural and neurochemical changes involved in neurodegenerative, infectious and traumatic processes, however markers used previously in cetaceans have rarely undergone systematic validation. This is a prerequisite to investigating the potential damage inflicted on the cetacean auditory system by anthropogenic noise. In order to assess apoptotic, neuroinflammatory and structural aberrations on a protein level, the baseline expression of biomarker proteins has to be characterized, implementing a systematic approach to validate the use of anti-human and anti-laboratory animal antibodies in dolphin tissues. This approach was taken to study 12 different antibodies associated with hypoxic-ischemic, inflammatory, plastic and excitatory-inhibitory changes implicated in acoustic trauma within the ventral cochlear nuclei and inferior colliculi of 20 bottlenose dolphins (Tursiops truncatus). Out of the 12 tested antibodies, pro-apoptotic protease factor 1 (Apaf-1), diacylglycerolkinase-ζ (DGK-ζ), B-cell lymphoma related protein 2 (Bcl-2), amyloid-ß peptide (Aß) and neurofilament 200 (NF200) were validated employing Western blot analyses and immunohistochemistry (IHC). The results of the validation process indicate specific patterns of immunoreactivity that are comparable to those reported in other mammals, thus suggesting a key panel of IHC biomarkers of pathological processes in the cetacean brain. As a consequence, the antibodies tested in this study may constitute a valid tool for supporting existing diagnostic methods in neurological diseases. The approach of systematic validation of IHC markers in cetaceans is proposed as a standard practice, in order for results to be transparent, reliable and comparable.

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.

Long-lasting effects of chronic exposure to chemical pollution on the hologenome of the Manila clam.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post-transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas-associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long-lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

Immunohistochemical Markers of Apoptotic and Hypoxic Damage Facilitate Evidence-Based Assessment in Pups with Neurological Disorders.

Seizures in puppies often present a diagnostic challenge in terms of identifying and treating the underlying cause. Dog breeds with mutations of the MDR1-gene are known to show adverse reactions to certain drugs, yet metabolic imbalance exacerbated by physiologically immature organs and other contributing pathologies require consideration before arriving at a diagnosis. This study analysed the brains of two male, 5-week-old Australian Shepherd siblings that died after displaying severe neurological symptoms upon administration of MilproVet® to treat severe intestinal helminth infection. Despite the initial symptoms being similar, their case histories varied in terms of the symptom duration, access to supportive therapy and post-mortem interval. Histopathology and immunohistochemistry were used to obtain more information about the phase of the pathological processes in the brain, employing protein markers associated with acute hypoxic damage (ß-amyloid precursor protein/APP) and apoptosis (diacylglycerolkinase-ζ/DGK-ζ, apoptotic protease activating factor 1/Apaf1, and B-cell lymphoma related protein 2/Bcl-2). The results seem to reflect the course of the animals' clinical deterioration, implicating that the hypoxic damage to the brains was incompatible with life, and suggesting the usefulness of the mentioned immunohistochemical markers in clarifying the cause of death in animals with acute neurological deficits.

Evaluation of per- and poly-fluorinated alkyl substances (PFAS) in livers of bottlenose dolphins (Tursiops truncatus) found stranded along the northern Adriatic Sea.

Per-and poly-fluorinated alkyl substances (PFAS) are a group of chemicals used in a wide variety of commercial products and industrial applications. These chemicals are persistent, can accumulate in humans' and animals' tissues and in the environment, representing an increasing concern due to their moderate to highly toxicity. Their global distribution, persistence and toxicity led to an urgent need to investigate bioaccumulation also in marine species. In 2013 PFAS contamination was detected in a vast area in Veneto region, mainly in Adige and Brenta rivers. In order to investigate any relevant presence of these substances in marine vertebrates constantly living in the area, PFAS were measured in hepatic tissue samples of 20 bottlenose dolphins (Tursiops truncatus) stranded along the northern Adriatic Sea coastline between 2008 and 2020. Using high performance liquid chromatography-mass spectrometry, 17 target PFAS (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTrDA, PFTeDA, PFBS, PFHxS, PFOS, PFDS, PFHpS, PFPeS), were quantified in the samples. PFAS profiles were generally composed of the same five dominant PFAS (PFOS > PFUnA > PFDA ≈ PFDoA ≈ PFTrDA). The greatest PFOS concentration found was 629,73 ng/g wet weight, and PFOS accounted until 71% in the PFAS profiles. No significant differences between sexes were found, while calves showing higher mean values than adults, possibly indicating an increasing ability in the elimination of PFAS with age. Finally, a temporal analysis was carried out considering three different periods of time, but no temporal differences in concentrations were found. The results suggest that long-chain PFAS are widespread in bottlenose dolphins along the North Adriatic Sea. Furthermore, they represent a baseline to investigate the impact of PFAS on marine mammals' conservation and health. Filling an important gap in the knowledge of PFAS accumulation in bottlenose dolphins, this study highlights the relevant role of Environmental and Tissue Banks for retrospective analyses on emergent contaminants.

Analysis of the Gastro-Intestinal Tract of Marine Mammals: A Multidisciplinary Approach with a New Multi-Sieves Tool.

Organs and content of the gastro-intestinal tract (GIT) of marine mammals are relevant for a variety of investigations and provide data to researchers from different fields. Currently used protocols applied to the GIT for specific analysis limit the possibility to execute other investigations and important information could be lost. To ensure a proper sample collection and a multidisciplinary investigation of the GIT of marine mammals, a new multi-sieves tool and a specific protocol have been developed. This new device and approach allowed the simultaneous sampling of the GIT and its content for the main investigations concerned. The samples collected during these preliminary trials were suitable to perform all the different research procedures considered in this work. The obtained results show that with a few and easy procedural adjustments, a multidisciplinary sampling and evaluation of the GIT of marine mammals is possible. This will reduce the risk of losing important data aimed at understanding the cause of death of the animal, but also biology and ecology of marine mammals, and other important data for their conservation and habitats management.

Illegal fishing with electrofishing devices in the Po river basin, Emilia Romagna, Italy.

Electric fishing is an illegal hunting method, unfortunately widely used by poachers to paralyze fish and to catch many animals in a short time. In Italy, it is authorized only for scientific and conservative purposes. Between 2014 and 2018, the Ferrara section of the Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna, Italy, received nine cases of potentially illegal electric fishing in Po river and its tributary rivers. Necropsies were performed following standard protocols and samples of different tissues were collected and examined using histochemical and immunohistochemical techniques. Gross lesions frequently observed were circulatory alteration phenomena (i.e. multi-organ hyperemia, hemorrhages and congestion, hemopericardium), also found histologically, in addition to multifocal degenerative and necrotic muscular processes that could be attributed to injuries from electric current, as already reported in literature. Immunohistochemical investigations confirmed degenerative and necrotic lesions with myoglobin depletion and a corresponding fibrinogen accumulation. Myoglobin globules were also detected in the renal parenchyma, as consequent of rhabdomyolysis. The results of this study allowed to correlate electric fishing to gross, histologic and immunohistochemical lesions, which together constitute a pathognomonic picture to be considered a reference standard in this type of illegal controversy.

SARS-CoV-2, a Threat to Marine Mammals? A Study from Italian Seawaters.

Zoonotically transmitted coronaviruses were responsible for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the dramatic Coronavirus Disease-2019 (CoViD-19) pandemic, which affected public health, the economy, and society on a global scale. The impact of the SARS-CoV-2 pandemic permeated into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home to sea mammals may have dramatic consequences on vulnerable species. The efficient transmission of coronaviruses raises questions regarding the contributions of virus-receptor interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing susceptibility to SARS-CoV-2 for different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-2 expression in the marine mammal respiratory tracts. Furthermore, to evaluate if and how Italian wastewater management and coastal exposition to extreme weather events may led to susceptible marine mammal populations being exposed to SARS-CoV-2, geomapping data were carried out and overlapped. The results showed the potential SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters, putting them at risk when swimming and feeding in specific risk areas. Thus, we highlighted the potential hazard of the reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, while also stressing the need for appropriate action in order to prevent further damage to specific vulnerable populations.

Atypical Toxoplasmosis in a Mediterranean Monk Seal (Monachus monachus) Pup.

The Mediterranean monk seal (Monachus monachus) is the rarest species of pinniped in the world. Necropsy of a Mediterranean monk seal pup that stranded alive on the southern Adriatic Italian coast and died a few hours later revealed co-infection by cetacean morbillivirus (CeMV) and Toxoplasma gondii. Pathological lesions included a multifocal, moderate to severe, necrotizing myocarditis and a diffuse, chronic, moderate interstitial pneumonia with bronchial and bronchiolar epithelial hyperplasia. Lesions of atypical necrotizing arteritis were seen in the aorta and major pulmonary arteries in association with the presence T. gondii organisms. Severe haemorrhagic foci and lesions of non-suppurative meningoencephalitis, together with the presence of protozoal cysts, were seen in the brain. Co-infection of CeMV and T. gondii has not been previously reported in monk seals. The vascular lesions found in this animal can be considered atypical because they have not been reported in other terrestrial or marine mammal species. The disseminated toxoplasmosis associated with the unusual vascular and haemorrhagic brain lesions could be related to the immunosuppressive effects of CeMV infection.

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.