Neurofilament light chain (NfL) is a neuron specific cytoskeletal protein released into cerebrospinal fluid (CSF) and blood following axonal injury, serving as a sensitive biomarker of neuroaxonal damage. Ultrasensitive immunoassays now permit reliable detection of NfL at very low concentrations, with Simoa™ and Ella™ being the most widely used platforms. Direct comparisons between these technologies remain limited, particularly in non human matrices. This study evaluated the analytical performance and agreement between Simoa™ and Ella™ platforms in CSF and serum samples from healthy cattle and bovine presenting neurological symptoms. In CSF, Passing–Bablok (PB) regression analysis indicated good agreement with no systematic or proportional bias. In serum, Simoa™ produced significantly lower serum NfL concentrations compared to Ella™, and PB regression revealed a proportional bias between methods. Overall, Ella™ showed a significantly lower coefficient of variation than Simoa™, indicating higher repeatability. In conclusion, both assays showed strong agreement in quantifying NfL in bovine CSF, whereas serum measurements might require platform-specific interpretation, especially when NfL is used as a minimally invasive biomarker of axonal injury in cattle.

Monitoring stranded cetaceans represents a strategic method to assess their health, conservation status, and ecological role in the marine ecosystem. Efficient stranding networks and standardized protocols are essential to monitor this phenomenon and investigate its causes. This study assesses the evidence of fishery interaction on stranded cetacean carcasses found along the Italian coastline from 1986 to 2023. Evidence assessment and post-mortem investigation methods evolved over three macro-periods, from non-standardized reporting (1986-2014) to an integrated national stranding network (2015-2019), and finally to the creation of a new standardized, evidence-based diagnostic framework under the EU-funded LIFE DELFI project (LIFE18 NAT/IT/000942) (2020-2023). A total of 5355 cases were selected for this analysis. A literature review and evidence of interaction on stranded carcasses supported the categorization of findings, ranging from case history to pathological observations, allowing the assessment of temporal variation, demographic parameters, geographical distribution, and fishing gear identification. Evidence of fishery interaction was found in 12.89% of the cases (690/5355), with an annual average of 18.15 affected animals, and fishery interaction was identified as the likely cause of death in 10.32% of the cases. The most frequently reported species were Stenella coeruleoalba and Tursiops truncatus, showing significant differences in fishery interactions, particularly in relation to sex, age class, and geographical distribution. Adult male Tursiops truncatus exhibited a higher susceptibility to gillnet interaction in the Adriatic Sea. The results of this study emphasize the importance of standardized post-mortem investigations and long-term monitoring to identify risk hotspots, implement species- and region-specific mitigation strategies, and establish threshold values for cetacean conservation.

Neurofilament light chain (Nf-L) is a biomarker for axonal damage in human neurology but is understudied in cattle. With this study we wanted to determine Nf-L stability at two different storage temperatures and Nf-L levels in healthy cattle and the relationship with age, evaluate whether Nf-L holds diagnostic potential for neurological disorders, and whether an association exists between Nf-L in serum and in cerebrospinal fluid (CSF). To do this, we measured Nf-L levels in CSF and serum samples from 49 healthy and 75 sick cattle. Storage at −80 °C or −20 °C had no impact on Nf-L concentration. Physiological median Nf-L levels were 6.3 pg/mL (serum) and 414 pg/mL (CSF) in calves and 5.5 pg/mL (serum) and 828 pg/mL (CSF) in adult cattle. There was no association between Nf-L levels in CSF and calf age (r2 0.07, p = 0.13), while a weak association was found for Nf-L in serum (r2 0.26, p = 0.01), and a significant association in adult cattle (CSF, r2 0.69, p = 0.0001; serum, r2 0.68, p = 0.0003). CSF Nf-L levels were higher in samples from animals with degenerative (median Nf-L 49971 pg/mL) and infectious central nervous system (CNS) disorders (median Nf-L, age < 2 months 8863 pg/mL; age 2–12 months 17474 pg/mL; age 1–6 years 3546 pg/mL), CNS anomalies and metabolic/toxic disorders. There was a significant association between CSF Nf-L and serum Nf-L in cattle with neurological disorders (r2 0.2, p = 0.009). Taken together, these findings suggest the potential of Nf-L as a diagnostic tool in cattle neurology.

CWD surveillance and diagnosis are important issues in Europe since its detection in Norway, as some of its strains, like that of classical scrapie, are contagious. In addition, there are concerns as several matters about CWD are not yet known. Although diagnostic methods for the active surveillance in bovine and small ruminants have been able to detect the European CWD strains, a retrospective study on Italian wild red deer (Cervus elaphus) samples was performed to compare the results obtained from rapid screening tests, authorized according to EU Regulation 999/2001, and the RT-QuIC, a highly sensitive method in the detection of prion disease infection. A total of one hundred brainstems and medial retropharyngeal lymph nodes were selected out of those received from the CWD Italian surveillance system. Confirmed CWD-positive and -negative samples were included in the study as controls. All of the samples were first tested with the HerdChek BSE–Scrapie Antigen Test and then using the RT-QuIC. The rapid test was negative in all brainstem and lymph node samples. RT-QuIC analyses showed only one red deer brainstem sample positive for seeding activity, while all lymph nodes were negative, including the one from this case. This positive brainstem sample was then re-extracted and retested using two different recombinant prion protein substrates (Ha90-231; BV23-231) and their different batches from the first analyses. Seeding activity was consistently confirmed across both substrates and extractions, with positive signals detected down to dilutions of 10−4 using rPrP Ha90-231 and as low as 10−6 with rPrP BV23-231. The additional diagnostic investigations performed on this red deer using the alternative rapid test (TeSeE SAP Combi), Western blot, and immunohistochemistry showed negative results both in the brainstem and lymph nodes. This study showed that overall, the results obtained with the HerdChek BSE–Scrapie Antigen Test and RT-QuIC agree except in one case. Our findings highlight the potential of the RT-QuIC method to detect very low levels of PrPSc-associated seeding activity that may escape detection using classical methods. While seeding activity does not always equate to infectivity, only a bioassay will confirm the real disease status of this Italian case. These findings support the integration of RT-QuIC as a powerful complementary tool within existing surveillance frameworks to strengthen early detection and diagnostic accuracy.

Marine litter represents a growing threat to marine biodiversity, particularly to cetaceans, yet its impacts on these sentinel species remain insufficiently quantified. This study provides the first comprehensive, transboundary assessment of litter ingestion in stranded cetaceans along the Italian coastline and across the wider Adriatic basin, including Croatia and Slovenia, between 2009 and 2023. Through harmonized post-mortem examinations, and focusing on the period of consistent data collection and analysis (2009-2023), this study documented plastic litter ingestion in 2.9% of necropsied cetaceans in Italy and 3.7% in the broader Adriatic subregion, with sperm whales (Physeter macrocephalus) showing the highest frequency (50% FO) and susceptibility. In 11 cases, ingestion was associated with health deterioration and mortality. The most commonly ingested items were plastic sheets and fragments. The Italian Adriatic subregion emerged as a hotspot for plastic interactions, reflecting regional hydrodynamics and anthropogenic pressures. Applying criteria from regional and international frameworks, the results showed that 60% of P. macrocephalus had ingested more than 1 kg of plastic, with 40% exhibiting harmful effects. These data provide baseline values that can serve as reference points for proposing thresholds to achieve Good Environmental Status under the Marine Strategy Framework Directive. Despite these results supporting the use of sperm whales as suitable sentinel species for monitoring macroplastic pollution, the available data are limited to Italy and influenced by distributional patterns and unusual mortality events. A combined approach, where T. truncatus is monitored for its broad spatial representativeness and P. macrocephalus for its ecological susceptibility, may be a useful strategy to guide further research and inform management measures in the future. These findings underscore the need for standardized monitoring protocols, enhanced cross-border data sharing, and policy measures to mitigate plastic impacts. This work provides crucial baseline knowledge for conservation planning and reinforces the role of cetaceans as indicators of ecosystem health in the Mediterranean.

Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an endogenous control and a conserved 92 bp region shared by WNV and USUV genomes. Degenerate primers for ACTB ensure RNA extraction quality and PCR performance while enabling simultaneous detection of both viruses. A total of 1002 mosquito pools collected in Piedmont, Italy, during the 2024 vector season under the National Surveillance Plan for Arboviruses (PNA), were tested. The assay showed 100% accuracy—ACTB mRNA was detected in all pools, and six pools tested positive for WNV or USUV (three each). Diagnostic specificity was confirmed on 40 horse and bovine serum samples. Sanger sequencing confirmed ACTB identity across multiple mosquito species. The assay also demonstrated reproducibility across different operators and thermocyclers. The limit of detection (LOD) evaluation showed that the assay is capable of detecting viral RNA at very low concentrations, confirming its high analytical sensitivity. The duplex RT-PCR here developed is a reliable, sensitive, and specific tool for arbovirus surveillance, combining pathogen detection with internal quality control of RNA extraction and amplification, thus improving early warning and rapid response to mosquito-borne disease threats.

Neurobrucellosis is a shared condition of cetaceans and humans. However, the pathogenesis and immune response in cetacean neurobrucellosis has not been extensively studied. In this multicentric investigation, 21 striped dolphin ( Stenella coeruleoalba) neurobrucellosis ( Brucella ceti) cases diagnosed over a 10-year period (2012-2022) were retrospectively evaluated. For each case, morphological changes were assessed by evaluating 21 histological parameters. Furthermore, the immunohistochemical expression of Brucella antigen, glial fibrillary acid protein (GFAP), and a selection of inflammatory cell (IBA-1, CD3, and CD20) and cytokine (tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], interleukin [IL]-1β, IL-2, and IL-6) markers were investigated. Inflammation of the leptomeninges, ependyma, and/or choroid plexus was lymphohistiocytic, containing macrophages/microglia (IBA-1+), T-cells (CD3+), and B-cells (CD20+) in equal proportion. B-cells occasionally formed tertiary follicles. GFAP expression showed astrocytosis in most cases. Expression of TNF-α, IFN-γ, and IL-2 indicated an intense proinflammatory response, stimulating both macrophages and T-cells. Our results showed that the inflammation and neuroinflammation in neurobrucellosis of striped dolphins mimic human neurobrucellosis and in vitro and in vivo studies in laboratory animals. Cetacean disease surveillance can be exploited to expand the knowledge of the pathogenesis and immunology of infectious diseases, particularly brucellosis, under a One Health approach.

Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications.

Despite attacks of bottlenose dolphins towards conspecifics and other species of dolphins being reported worldwide, inside the Mediterranean Sea these behaviours are still considered sporadic and have been, to date, only recorded in Spain and France, mostly directed to striped dolphins. In this work, we reported the necropsy outcomes of a dead striped dolphin that suggest a fatal interaction with bottlenose dolphins inside the Italian side of the Pelagos Sanctuary, Western Ligurian Sea. Results from the post-mortem examination showed multiple, multifocal external rake marks spaced 1-1.2 cm and several internal injuries, consisting of subcutaneous and muscular haematomas and haemorrhages, multiple vertebral and rib fractures, haemothorax and lung laceration. The inter-tooth distance and all the internal findings were consistent with a fatal traumatic interaction with bottlenose dolphins as reported in similar cases in other parts of the world where the same results were found. No other significant gross and microscopic findings and concurrent significant pathogens were detected. This case highlights the existence of negative interspecific interactions in an area where they have never been reported before and adds new important information to the literature for understanding their occurrence, expanding the geographical range of observation in the western Mediterranean Sea.

Cetacean brains are uniquely adapted to diving, but can be affected by diseases and exposure to toxins, triggering neurodegenerative processes that may cause stranding. Some species exhibit a significant post-reproductive lifespan (PRLS), increasing the likelihood of observing cumulative and age-related pathology. Immunohistochemistry against amyloid-β and hyperphosphorylated tau proteins is increasingly implemented to assess Alzheimer’s Disease-like neuropathology in cetaceans, but comparisons between geographically distinct populations, animals of different age groups, sex, and with concomitant pathologies are lacking. We tested 43 cetaceans’ (30 Tursiops truncatus; 13 Stenella coeruleoalba) parietal cortex, our most consistently archived cerebral tissue, in immunohistochemical analyses with amyloid-β oligomer 42 (Aβ-42) and hyperphosphorylated tau (pTau AT180 and AT8) antibodies. Aβ-42 antibody cross-reacted with plaques in three aged bottlenose and two aged striped dolphins, but was more often detected within neurons, glia, and blood vessels of all the dolphins. Histoscore comparisons between dolphins of different ages, sexes, and pathologies revealed significant correlations between older age, viral infections, and plaque presence. Protozoan cysts cross-reacted with Aβ-42 antibody. pTau signal was observed as single foci in neurons and neuropil in two young and two aged bottlenose dolphins. To our knowledge, this study is the first of its kind for the Mediterranean region and will help establish baseline understanding of physiological and pathological expression of proteins associated with human neurodegenerative disease in cetacean brains.

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.

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.

BackgroundKnowledge of the distribution and abundance of disease-causing mosquito vectors is fundamental for assessing the risk of disease circulation and introduction. Aedes caspius (Pallas, 1771) and Aedes vexans (Meigen, 1830) have been implicated, to different extents, in the circulation of several arthropod-borne viruses (arboviruses). These two mosquitoes are vectors of Tahyna virus in Europe and are considered potential vectors of Rift Valley fever virus, a virus not present but at risk of introduction on the continent.MethodsIn this work, we analysed abundance data collected during West Nile virus (WNV) surveillance in northern Italy (Po Plain) via 292 CO2-baited traps to evaluate the distribution and density of these two non-target mosquitoes. We modelled the distribution and abundance of these two mosquito species in the surveyed area using two distinct spatial analysis approaches (geostatistical and machine learning), which yielded congruent results.ResultsBoth species are more abundant close to the Po River than elsewhere, but Ae. caspius is present in the eastern and western parts of the plain, linked with the occurrence of rice fields and wetlands, while Ae. vexans is observed in the middle area of the plain.ConclusionsPresence and abundance data at the municipality level were obtained and made available through this work. This work demonstrates the importance of maintaining and improving entomological surveillance programs with an adequate sampling effort.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

Aedes japonicus is an invasive Asian mosquito species, and to date it is widespread in many European countries. In Italy, it was first recorded in 2015 at the Austrian border and it then spread throughout the Northeast of the country. In 2019, it was also identified in Piedmont region, near the Swiss border. In the framework of the Italian program for prevention, surveillance, and response to Arboviruses, from June to November 2021, biweekly entomological surveillance was performed in the Liguria region (Northwest Italy). The collected mosquitoes were morphologically and genetically identified and molecularly analysed for the detection of West Nile and Usutu viruses. Six female mosquitoes, trapped on the 6th of July 2021 using a gravid trap in Albenga (Savona province), were morphologically identified as Ae. japonicus and the identification was genetically confirmed. The pool tested was negative for the presence of West Nile and Usutu viruses. The detection of Ae. japonicus was performed in a coastal area characterized by the presence of many floriculture activities. Considering the distance from the established Ae. japonicus mosquito populations in Italy and other European countries, this could represent an independent introduction in this country.

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.