Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet’s ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1–5000 μm) and nanoplastics (<1 μm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells’ medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity.

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.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Between 2019 and 2021, 21 bottlenose dolphins were found stranded dead along the Ligurian Pelagos Sanctuary coast (Italy). For 11 animals, out of a total of 14 recovered, the cause of death was classified as natural, anthropic, or not determined based on gross and histological pathology and ancillary testing. Hexachlorobenzene (HCB), dichlorodiphenyltrichloroethanes (DDTs), and polychlorinated biphenyls (PCBs) were examined in their blubber, and results were discussed according to their toxicological properties. All specimens showed the following trend: PCBs > DDTs >> HCB, and the levels of cancerogenic, mutagenic, and teratogenic organochlorine compounds (T-OCs) were >50% of total OCs. Immunosuppressant organochlorine compound (IS-OC) levels in 10 out of 11 animals were above 50% of the total OCs. PCB levels always exceeded the threshold of 17 mg/kg lipid weight for PCB-induced adverse health effects. The results suggest that bottlenose dolphins living in the Pelagos Sanctuary undergo a high level of exposure to pathogens and OCs, betraying the designation of the Cetacean Sanctuary and, consequently, of a region created for their conservation. Immune dysfunction and infectious disease susceptibility appear to be highly connected with high levels of OC xenobiotics. These data are useful to understand health and mortality trends in cetacean populations, as well as for developing policies for cetacean conservation and management in this important protected area of the Mediterranean.

Toxoplasma gondii constitutes a major zoonotic agent but also has been frequently identified as an important cause of clinical disease (e.g., abortion, pneumonia, encephalitis) in wildlife; specifically, T. gondii has been associated with neurological disease in cetaceans. This study investigated the genetic diversity of T. gondii strains involved in infections in dolphins found stranded in the Mediterranean coastlines of Italy. Tissue samples from 16 dolphins (Stenella coeruleoalba and Tursiops truncatus species) positive for T. gondii-DNA presence by PCR were examined by histology and subjected to further genetic characterization of strains detected by PCR-RFLP and multilocus PCR-sequencing assays. According to fully genotyped samples, the genotypes ToxoDB#3 (67%) and #2 (22%) were detected, the latter being reported for the first time in cetaceans, along with a mixed infection (11%). Subtyping by PCR-seq procedures provided evidence of common point mutations in strains from southwestern Europe. Despite evidence of T. gondii as a cause of neurological disease in dolphins, sources of infections are difficult to identify since they are long-living animals and some species have vast migration areas with multiple chances of infection. Finally, the genetic diversity of T. gondii found in the dolphins studied in the Mediterranean coastlines of Italy reflects the main genotypes circulating inland in the European continent.