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Wild ruminants often harbor pathogens transmissible to other animals and humans, but their epidemiological role is not always defined for all microorganisms. In this survey, spleens, kidneys, and hearts sampled from 162 fallow deer (Dama dama) were subjected to molecular analyses to detect bacterial (Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Brucella spp., Chlamydia abortus, Coxiella burnetii, Francisella tularensis, Leptospira spp.) and protozoan (piroplasms, Neospora caninum, Toxoplasma gondii) pathogens. Five (3.08%) spleens were positive for A. phagocytophilum, and twelve (7.40%) spleens were positive for Theileria cervi. The remaining pathogens investigated were not detected, and no coinfections were found. The analyzed animals do not seem to have a relevant role in the spreading of these pathogens; however, monitoring is pivotal to understand the epidemiological scenarios and take appropriate preventive measures in areas frequently visited by people.

We propose a design-based strategy to exploit pellet group counts performed within plots of a prefixed size using the clearance count technique with the purposes of analyzing habitat selection, mapping the pellet group presence throughout the study area, and estimating the abundance of deer populations. As is customary in design-based inference, the strategy is free of model assumptions, and the precision and statistical consistency of the proposed estimators are determined by the probabilistic sampling scheme adopted to locate plots. The unique necessary assumptions are the absence of migratory movements during the survey period, the accurate recording of the number of pellet groups deposited within sample plots between the 2 visits, and a precise approximation of the daily defecation rate of the population. In addition to these assumptions, which can be attained by a suitable design of the survey, the statistical soundness of the strategy rests on the use of tessellation stratified sampling, a stratified sampling scheme that ensures an even distribution of plots throughout the study area. The scheme also allows for the estimation of the standard errors and the construction of confidence intervals without involving any other assumptions. We applied this strategy in summer 2019 in a protected area of a Mediterranean coastal region to estimate the density of a fallow deer (Dama dama) population. We estimated the corresponding standard error considering the uncertainty entailed by the estimation of the daily defecation rate, with the purpose of performing reliable monitoring. The proposed strategy provided precise estimates of deer abundance and is readily implementable in the field, standardized, and easily repeatable over time, thus allowing reliable monitoring and comparisons across time and space, which are fundamental attributes for management of deer populations.

After facing a great decline all over Europe during the past centuries, starting from the second half of the XX century the roe deer (Capreolus capreolus) was reintroduced and strongly managed throughout its range, as other ungulate species. Overhunting and habitat change were the main factors threatening roe deer populations in Italy, where small remnant populations of putatively native roe deer survived in a few localities of eastern Alps and central-southern Italy. We investigated the genetic variation of a roe deer population inhabiting the northern Apennines in the province of Massa-Carrara (Tuscany, Italy), analysing both mitochondrial DNA control region and a total of 11 autosomal microsatellite loci, to identify possible sources and recolonisation patterns, as well as the local prevalence of native Capreolus capreolus italicus gene pool. Analyses revealed an admixed nature of roe deer in this area, merging both native and non-native lineages, with a dominance of italicus haplotypes in the matriline and a majority of non-native genetic component in the autosomal markers. The high similarity with roe deer from neighbouring areas suggests a natural population origin by immigration. Two scenarios may explain the observed pattern of genetic variation: a colonisation by a limited number of immigrants from a single admixed source (either north or south-east), or a two-step recolonisation, firstly from the south, where the italicus ancestry was prevalent, and then from the north, mostly by individuals carrying C. c. capreolus genes. This study shows the genetic consequences of translocations even in populations not directly targeted by human interventions and highlight how investigating genetic variation might be essential in species management.

Wild animals are recognized as significant reservoirs for various zoonotic pathogens, including antibiotic-resistant bacteria. This study aimed to investigate the presence of Staphylococcus spp. strains in fallow deer (Dama dama) inhabiting a natural preserve in Central Italy and to examine the phenotypic and genotypic antimicrobial resistance and the presence of some virulence genes among the isolates. During July and December 2022, nasal swabs were collected from 175 fallow deer, which were then analyzed through bacteriological cultures. In total, 176 Staphylococcus spp. strains were isolated and subsequently identified using MALDI-TOF mass spectrometry. S. aureus was the most abundant species with 66 (37.5%) strains, followed by S. hyicus, 34 (19.31%) strains, S. sciuri, 32 (18.18%) strains, S. chromogenes, 27 (15.34%) strains, S. xylosus, 11 (6.25%) strains, S. warneri, 5 (2.84%) strains, and S. devriesei, 1 (0.56%) strain. Antimicrobial susceptibility was assessed for each isolate via the agar disk diffusion method, testing a panel of 13 molecules belonging to 9 antimicrobial classes. The highest resistance rates were detected for penicillin (29.55%), rifampicin (22.73%), and amikacin (20.45%). Notably, intermediate susceptibility was observed for erythromycin (61.93%), enrofloxacin (28.41%), and ceftiofur (21.02%). Conversely, the strains exhibited particularly high susceptibility to amoxicillin/clavulanic acid (99.43%), cefoxitin (97.73%), and vancomycin (96.02%). Based on the results, 32 (18.18%) isolates were classified as multidrug-resistant (MDR). Two strains of S. chromogenes and one strain of S. xylosus, both resistant to penicillin, tested positive for the blaZ gene. No methicillin-resistant strains were found, and none of the isolates harbored genes associated with enterotoxin and toxic shock syndrome toxin production. This study highlights the potential role of wildlife, particularly fallow deer, as reservoirs of antibiotic-resistant Staphylococcus spp. strains. Such findings underscore the importance of monitoring wildlife for antimicrobial resistance, which could have implications for public health and veterinary medicine.

The aim of the study was to assess which kill site characteristics were selected by a lone wolf living in a protected Mediterranean coastal area near the city of Pisa, Italy, where both wild and domestic ungulates were available as potential prey. Between 2017 and 2019, we monitored the wolf’s predatory behaviour through a combination of camera trapping and active search for kill sites and prey carcasses. The main prey found was the fallow deer (n = 82); only two wild boars and no domestic ungulates were found preyed upon. The features and habitat of kill sites were modelled to test for selection by the wolf. The habitat type of kill site was composed of meadows and pastures (89.3%), woods (7.3%), degraded coastal areas (1.9%), roads and rivers (1.1%), and marshes (0.5%). We calculated their distance from landscape features and ran a binomial generalised linear model to test the influence of such landscape variables. The distance of kill sites from landscape elements was significantly different from random control sites, and a positive selection for fences was found. In fact, the wolf pushed fallow deer towards a fence to constrain them and prevent them from escaping. We also analysed the body condition of predated fallow deer as a percentage of fat content in the bone marrow of the hind legs. Our results revealed the selection of the lone wolf for deer in good body condition. This is a possible outcome of the habitat selection shown by fallow deer in the study area, where fenced open pastures are the richest in trophic resources; therefore, our findings suggest a high efficacy for the lone wolf hunting strategy, but also the adoption of a high risk feeding strategy by deer. This study suggests that a lone predator can take advantage of human infrastructures to maximise its predatory effectiveness.

Carcass utilization by scavengers is crucial for ecosystem dynamics, influencing energy transfer and nutrient cycling. Through the provision of unconsumed carcasses, the return of wolves ( Canis lupus ) to European ecosystems can trigger complex cascading effects on scavenger communities. Here, we investigate the scavenging dynamics of mammalian species in a prey-rich Mediterranean coastal area close to a large city where wolves have recently returned. Using camera traps, we monitored the scavenging activity on fallow deer ( Dama dama ) carcasses preyed upon by wolves over a five-year period following the predator’s return. We developed models to assess how competition between red foxes ( Vulpes vulpes ), wild boars ( Sus scrofa ), and wolves evolved over time, focusing on their carcass use, direct interactions, and spatiotemporal associations. Wolves exhibited consumption of only minor portions of prey and a negligible reuse of carcasses, while foxes and wild boars engaged in indirect competition for access to this emerging trophic niche. We found a notable temporal change in scavenging activity over the monitoring period, with wild boars progressively emerging as the primary scavenger and providing a substantial removal of carcass biomass. Our findings suggest that wild boars can assume a prominent scavenging role in environments where carcass availability is both abundant and predictable. This study underscores the importance of understanding scavenger temporal dynamics in the context of apex predator recolonization and highlights the need for further research to explore the long-term impacts of these interactions on community structure and ecosystem services as wolf populations continue to expand across Europe. Significance statement Carcass utilization and competition among scavengers play critical roles in shaping ecosystem dynamics and services. While previous studies have often viewed interspecific competition among scavengers as a static process, our research delves into the temporal progression of scavenging on unconsumed carcasses, killed in large number and only partially consumed by wolves upon their return to a prey-rich, human-dominated landscape. The simple scavenger community inhabiting the area proved remarkably efficient in removing the carcasses, primarily due to the increasing success of wild boars over red foxes and even wolves themselves in exploiting this emerging niche. As wolf populations continue to recover and expand across Europe, insights from this study are of utmost importance for predicting future scenarios of food chain dynamics and for devising strategies to mitigate potential biological hazards associated with carcass release in human-dominated landscapes.

In order to assess the spreading of tick-borne bacteria and protozoa in a protected area largely frequented by people and in which numerous domestic and wild animals live, molecular analyses were carried out in ticks collected from fallow deer (Dama dama) to detect Anaplasma phagocytophilum, Borrelia sp., Coxiella burnetii, Francisella tularensis, Hepatozoon sp., and piroplasms. A total of 148 tick pools, for a total of 475 ticks collected from fallow deer and identified as female adult Ixodes ricinus, were submitted to DNA extraction and different PCR assays. One hundred and two (68.92%) pools were positive for one or more pathogens: three (2.02%) for C. burnetii, 21 (14.19%) for Borrelia sp., 35 (23.64%) for piroplasms, and 87 (58.78%) for A. phagocytophilum. All tick pools were negative for F. tularensis and Hepatozoon sp. Sixty-seven (45.27%) pools were positive for only one investigated pathogen, whereas in 35 (23.64%) pools DNA of two or more pathogens were found. Sequencing analyses identified 28 piroplasm amplicons as Theileria sp. OT3 and seven amplicons as possible Theileria cervi. Sequencing of the 21 Borrelia amplicons identified six samples as B. miyamotoi and eight as B. lusitaniae, whereas seven amplicons had 100% homology with a Borrelia sp. found in France and 99.37% with a B. theileri strain detected in Zambia. Monitoring tick-borne pathogens in ticks is pivotal to assess the spread of these microorganisms, the evolution of their epidemiology, and the risk of infections for animals and humans.

Graphene and its various derivatives, known as graphene-based nanomaterials (GBNs), hold tremendous potential across many fields due to their exceptional properties. As with any novel material, concerns about their safety have emerged alongside their widespread production and use. Several studies have shown that GBNs can have diverse effects on various cell lines and organisms under different exposure conditions. This study intends to evaluate the potential toxicity of few-layer graphene (FLG) in human bronchial BEAS-2B. Cells were exposed to different concentrations of FLG for 24 h, and the cyto-genotoxic, oxidative, and inflammatory effects were evaluated. We found an increase in cytotoxicity in terms of cell death, cell apoptosis, and membrane damage at the highest concentration. We also detected a slight increase in direct DNA damage and the percentage of comets. Oxidative DNA damage was observed at the highest concentration. FLG exposure did not induce notable cytokine release. Overall, this study suggests that exposure to FLG can induce cytotoxicity, apoptosis, and DNA damage in BEAS-2B, particularly at high concentrations. These findings contribute to a better understanding of the potential health effects of FLG and highlight the importance of considering dose-dependent effects when assessing the safety of GBNs.

This study is aimed at evaluating whether the occupational exposure to TiO2 during the industrial production process is able to induce genotoxic, oxidative, and inflammatory effects on blood, biomonitoring the same workers that showed micronucleus induction in the exfoliated buccal cells, as previous published. The final aim was to find sensitive and suitable biomarkers to evaluate potential early toxicity of occupational exposure to TiO2. On the same 40 workers involved in the manufacture of TiO2 pigment, 5 office workers, and 18 controls previously studied, we used formamidopyrimidine glycosylase- (Fpg-) comet assay on lymphocytes to evaluate genotoxic/oxidative effects and detected cytokine (IL-6, IL-8, and TNFα) release by ELISA to evaluate proinflammation. Moreover, we studied the possible influence of single nucleotide polymorphisms of XRCC1 and hOGG1 DNA repair genes and of GST metabolism-related genes (GSTT1 and GSTM1) on the evaluated effects. We did not find statistically significant differences in the mean values of the analysed Fpg-comet assay parameters; only the percentage of DNA damaged cells appearing in the test as comets (% comets) resulted higher in the exposed workers compared to controls. Also, the data analysed taking into account the specific task (bagging, industrial cleaning, mobile operations, maintaining, and production) showed differences only for % comets which resulted higher in industrial cleaners compared to controls. We found variations of IL-6 and IL-8 levels in the exposed workers with concentrations that were lower for IL-6 and higher for IL-8 compared to the control group. XRCC1, hOGG1, and GSTT1 polymorphisms did not influence neither comet parameters nor cytokine release. These findings demonstrate that TiO2 production process is able to induce slight proinflammatory effects in terms of IL-8 increased release but not significant genotoxic/oxidative effects on lymphocytes, which do not seem to be a target of TiO2, prevalently inhalable particles, generated in the studied production site.

The human muscle isoform of lactate dehydrogenase (hLDH5) is one of the key enzymes of the glycolytic process. It is overexpressed in metastatic cancer cells and is linked to the vitality of tumors in hypoxic conditions. With the aim of identifying new hLDH5 inhibitors, a fully automated docking-based virtual screening platform was developed by considering different protein conformations and the consensus docking strategy. In order to verify the reliability of the reported platform, a small database of about 10,000 compounds was filtered by using this method, and the top-ranked compounds were tested for their hLDH5 inhibition activity. Enzymatic assays revealed that, among the ten selected compounds, two proved to efficiently inhibit enzyme activity with IC50 values in the micromolar range. These results demonstrate the validity of the methodologies we followed, encouraging the application of larger virtual screening studies and further refinements of the platform. Furthermore, the two active compounds herein described may be considered as interesting leads for the development of new and more efficient LDH inhibitors.