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.

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.

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.

Due to marine mammals’ demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

The presence of cetaceans along the Campania coast has always been documented. Between 2016 and 2022, out of 65 cetaceans stranded along the Campania coast, 46 were studied for bacteriological, virological, parasitological, and histopathological investigations. The results highlighted that for 59% (n = 27) of the specimens, the cause of death was of natural origin, while for only 2% (n = 1) of animals, the origin of death was ascribed to anthropic causes. Unfortunately, for 39% (n = 18) of the cetaceans, it was impossible to determine the cause of death. All the cetaceans that died of natural causes showed viral, bacterial, and parasitic infections. The primary pathogens detected were Cetacean Morbillivirus (CeMV, 65.2%, n = 30/46), Toxoplasma gondii (10.9%, n = 5/46), and Brucella ceti (8.7%, n = 4/46). The animals showed typical lesions of the isolated pathogens, such as systemic infection, meningoencephalitis, and pneumonia. Moreover, even with a lower frequency, other relevant pathogens like Photobacterium damselae, Salmonella enteritidis, Erysipelothrix rhusiopathiae, and Chlamydia abortus were isolated. These data were useful to understand the spread and circulation of these pathogens, some zoonotic, in the coastal marine waters of the Campania region.

Data collected by C. Re. Di. Ma over a 3-year period (2020–2022) were considered to assess anthropic pressure on cetaceans living in the Ligurian sea. Out of a total of 37 stranded cetaceans, a complete post mortem examination was performed on 23 cases. Of these, 14 were further selected considering at least one of these conditions: (i) confirmed, probable, or suspected interaction with fishing activities through the application of a standardized diagnostic framework (7/14; 50%), (ii) toxicological stress through the evaluation of OCs hazardous levels (14/14; 100%), and (iii) terrestrial pathogen-associated disease (systemic infection and/or associated lesions) (7/14; 50%). For 9 animals out of a total of 14 selected, the cause of death was classified as natural (6/14; 42,8%), anthropic (3/14; 21,4%), or not determined (5/14; 35,7%) based on gross and histological pathology and ancillary testing. These findings extend our knowledge of the anthropic pressure to which cetaceans stranded along the Ligurian coastline are subjected from a multidisciplinary point of view.