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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.

Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.

Listeria monocytogenes is a ubiquitous pathogen found both in the environment and food. It can cause listeriosis in a wide range of animals as well as in humans. Investigations on presence, spread and virulence are still limited to terrestrial and human environments. Embracing the One Health Approach, investigating the presence and spread of L. monocytogenes in marine ecosystems and among wildlife, would provide us with useful information for human health. This study investigated the presence of L. monocytogenes and Listeria spp. in two species of sea turtles common in the Mediterranean Sea (Caretta caretta and Chelonia mydas). A total of one hundred and sixty-four carcasses of sea turtles (C. caretta n = 161 and C. mydas n = 3) stranded along the Abruzzo, Molise, Campania, and Calabria coasts, were collected. Brain and fecal samples were taken, enriched, and cultured for the detection of Listeria spp. From the specimens collected, strains of L. monocytogenes (brain n = 1, brain and feces n = 1, multiorgan n = 1 and feces n = 1), L. innocua (feces n = 1 and brain n = 1), and L. ivanovii (brain n = 1) were isolated. Typical colonies were isolated for Whole Genome Sequencing (WGS). Virulence genes, disinfectants/metal resistance, and antimicrobial resistance were also investigated. L. monocytogenes, L. innocua, and L. ivanovii were detected in C. caretta, whilst only L. monocytogenes and L. innocua in C. mydas. Notable among the results is the lack of significant differences in gene distribution between human and sea turtle strains. Furthermore, potentially pathogenic strains of L. monocytogenes were found in sea turtles.

The levels of polycyclic aromatic hydrocarbons, PAHs, were determined in the liver and salt gland of 19 loggerhead turtles (Caretta caretta Linnaeus, Cheloniidae) stranded along the coasts of the south Tyrrhenian Sea, Italy, from 2019 to 2021. The 16 EPA’s priority PAHs were determined by gas chromatography coupled with mass spectrometry (GC-MS). The average values of ΣPAHs in liver, 139 ± 55.0, were exceptionally high and up to one hundred times those of literature and appeared even more worrying in salt gland, 320 ± 97.6 ng/g, w.w. Naphthalene was the predominant contributor to PAHs richness and accounted for 90.0% and 93.7% of ΣPAHs in the two matrices, highlighting the petrogenic source of exposure. An overall higher bioaccumulation of NAP, more than two-fold, was detected in salt gland and especially in female and adults. Data of PAHs richness highlighted a potential risk of neoplastic disease development and that anthropogenic activities may seriously impair healthy state conditions of C. caretta. populations in south Tyrrhenian Sea.

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea.

Caretta Caretta are endangered sea turtles leaving in the Mediterranean Sea. We carried out a study on 195 sea turtles stranded along the coast of Southern Italy, evaluating anthropogenic and opportunistic factors affecting animal health status and probably threatening the species. More than 60% of the animals showed body injuries, mainly caused by marine traffic and fishing. Ingestion of marine litter of various origins was observed in the digestive tract of 38.5% of the turtles investigated. Chelonid herpesvirus 5 was detected (for the first time in this basin) in 10.8% of the turtles, which were however free from fibropapillomatosis. Analyses on trace elements showed a time-dependent reduction in the mean concentrations of Cd and Hg, a decrease in the levels of Pb, and a time-dependent increase of As. Particularly, a site-dependent accumulation of As was detected in turtles from the coasts of Calabria. There is a significant correlation between the levels of As in the kidney of Campania turtles and their straight-line carapace length, indicating a size-dependent accumulation of this metal. Overall, besides dangerous debris items, high levels of trace elements may weaken the immune system of Caretta caretta, the most common turtle of the Mediterranean Sea, making it more likely vulnerable to viruses.

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.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Marine pollution is one of the major threats affecting loggerhead turtles, which due to their long life span, highly migratory behavior, and carnivorous diet, may be exposed to elevated levels of toxic elements throughout their life. The transfer of chemicals from mothers to their offspring is of particular conservation concern because it may affect embryonic development and hatching success. In this study, the concentrations of 16 toxic and potentially toxic trace elements, 6 indicator polychlorinated biphenyls (PCBs), and organochlorine pesticide residues (OCPs) were determined in 138 eggs from 46 loggerhead turtle nests laid during the 2021 nesting season in Campania, Italy, western Mediterranean Sea. The possible impact of pollutant levels on hatching success and early embryonic death was also investigated. Trace element analysis was performed using an ICP-MS, except for mercury, which was determined using a Direct Mercury Analyzer® (DMA). PCBs and OCPs were analyzed with high-resolution gas chromatography coupled with high-resolution mass spectrometry (HRGC-HRMS) and gas chromatography tandem quadrupole mass spectrometry GC-MS /MS, respectively. The concentrations of essential elements in the eggs were higher than those of non-essential elements. In addition, the highly chlorinated PCBs (153, 138, and 180) contributed the most to the total PCBs, while OCPs were not detected. No correlations were found between contaminant concentrations and reproductive parameters (hatching success and no obvious embryos). The results obtained suggest that the levels of contaminants found in the eggs do not affect the reproductive success of the species in the study area.

Information on demographic and life-history traits of endangered vertebrate species, such as sea turtles, is crucial for planning management and conservation actions. We applied skeletochronology of phalanges to estimate the age of loggerhead turtles, Caretta caretta, found dead stranded along the beaches of Campania (western Mediterranean) from 2013 to 2017. To obtain maturity data, we examined gonads from a subsample of 7 males and 11 females. Overall, curved carapace length (CCL) ranged from 5.6 to 90.8 cm, but for most turtles (89%) it was 50–79.9 cm. Predominance of stranded females (62%) was recorded. Ten out of the eighteen histologically examined gonads allowed estimating maturity. Based on the lines of arrested growth counting, the estimated age of the examined specimens ranged from 0 (hatchling) to 26 years. The modal age was 14 years for males and 17 years for females. The smallest male with spermatogenetic activity had a CCL of 65 cm and was estimated to be 16 years old. The smallest female with follicular development stage, characterising the transition towards adulthood, had a CCL of 69.5 cm and was estimated to be 20 years old. Anthropogenic factors were responsible for 36% of the mortality of individuals, followed by parasitic/infective pathologies (20%).

Abstract Background The Spirorchiidae is a family of blood flukes parasitizing turtles. Spirorchiids may cause a wide range of inflammatory reactions in the vascular system of their host being frequently implicated with stranding and death of sea turtles worldwide. Recent studies revealed the presence of two spirorchiid species in the Mediterranean basin. Our study presents comparative epidemiological data of spirorchiid infections in loggerhead turtles (Caretta caretta) stranded during an eight-year period from Adriatic and Tyrrhenian Seas, and the first report of Neospirorchis Neogen-11 in a green turtle (Chelonia mydas). Methods We screened a total of 319 carcasses of loggerhead turtles stranded from January 2011 to December 2018 along the Tyrrhenian coast (n = 111) and the north-western Adriatic coast (n = 208) of Italy using traditional (copromicroscopy and histopathology) and molecular assays. Three green turtles from the Tyrrhenian coast were also included in the study. Results A total of 56 (17.5%) loggerhead turtles and one green turtle (33.3%) were found to be infected with spirorchiid flukes. Amplification, sequencing of the ITS2 region of the ribosomal RNA gene cluster and BLAST analysis confirmed the presence of Hapalotrema mistroides and Neospirorchis Neogen-11 in 51 (16.0%) and 24 (7.5%) loggerhead turtles, respectively, and Neospirorchis Neogen-11 in an infected green turtle. Differences in prevalence of infection between the two sampling areas were found. Conclusions The risk of spirorchiid infection in the Tyrrhenian Sea is lower than in the Adriatic Sea and in general the risk of infection in the Mediterranean is lower than in other geographical locations. Differences in the prevalence of infection between the two sampling areas were related to the differences of regional habitats supporting different abundance of spirorchiid intermediate hosts. A systematic monitoring to evaluate the progress of the infection is recommended, as well as studies on the occurrence and distribution of spirorchiid species from other Mediterranean areas.

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.

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.

The interplay between actions of Lie groups and monotone quantum metric tensors on the space of faithful quantum states of a finite-level system observed in recent works is here further developed.

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.

Wild animals are potential vectors of antibiotic-resistant bacteria in the environment. The present study aimed to investigate the occurrence of antimicrobial resistance among Salmonella serovars isolated from wildlife and the environment in Italy. A total of 164 Salmonella isolates were analyzed, and six different subspecies and 64 serovars were detected. High proportions of Salmonella isolates proved resistant to streptomycin (34.1%), followed by trimethoprim-sulfamethoxazole (23.2%), tetracycline (17.7%), ciprofloxacin (14.63%) and ampicillin (11.59%). By source, the lowest level of resistance was observed in Salmonella serovars isolated from a water environment, while antimicrobial resistance was frequent in strains collected from shellfish, reptiles and birds. Multidrug-resistant strains were recovered from seafood (n = 11), mammals (n = 3) and water (n = 1). Three S. Typhimurium monophasic variant strains showed asimultaneous resistance to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole, which represents a recognized alert resistance profile for this serovar. These data indicate the environmental dissemination of resistant strains due to anthropogenic activities, which, in southern Italy, probably have a higher impact on marine ecosystems than on terrestrial ones. Moreover, as most of the animals considered in the present study are usually consumed by humans, the presence of resistant bacteria in them is a matter of great concern.

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts.

Nel presente lavoro viene riportato il risultato del rilevamento degli spiaggiamenti e dei rinvenimenti in mare di Cetacei avvenuti negli anni 2006 - 2011 lungo le coste della Campania. I dati sono stati raccolti in base ad una collaborazione scientifica tra l’Università degli Studi di Napoli Federico II, l’Istituto Zooprofilattico Sperimentale del Mezzogiorno, il Centro Studi Cetacei e il Centro Studi Ecosistemi Marini, collaborazione, che nel 2008 è stata ratificata da un protocollo di intesa finalizzato agli interventi sui Cetacei spiaggiati lungo le coste della Campania. Nel 2009 la Giunta Regionale della Campania, su richiesta del Settore Veterinario dell’Assessorato alla Sanità, ha decretato la “Costituzione di un Comitato di Coordinamento per l’attuazione del protocollo di intesa per gli interventi in caso di Cetacei e tartarughe spiaggiate” formato dai rappresentanti dei citati enti, delle AA.SS.LL. costiere, della Direzione Marittima di Napoli e dell’A.R.P.A.C. al fine di approvare le linee guida relative alla procedura operativa da adottarsi in caso di intervento per il recupero di carcasse di Cetacei spiaggiati. In totale sono stati raccolti i dati riguardanti 73 esemplari di almeno 6 specie, così suddivisi: 1 Balaenoptera acutorostrata; 4 Balaenoptera physalus; 2 Grampus griseus; 1 Delphinus delphis; 9 Tursiops truncatus; 40 Stenella coeruleoalba; 10 Delfinidi indeterminati e 6 Cetacei indeterminati. Si riporta anche il primo caso di mal rossino diagnosticato in un esemplare di Stenella striata mai segnalato prima per le coste dell’Italia continentale.