Bluetongue (BT) is a WOAH-notifiable economically important disease of ruminants caused by Bluetongue virus (BTV), transmitted by Culicoides spp. biting midges. Over the past two years, Italy has experienced a marked re-emergence of BT, with thousands of outbreaks reported due to the simultaneous circulation of several BTV strains belonging to serotypes 3, 4, and 8. Moreover, in September 2025, BTV-5 was detected in Sardinia, marking its first occurrence in Europe. Following the first identification by Whole Genome Sequencing, the development of a reliable real-time RT-qPCR-based assay capable of typing the novel BTV-5 ITA 2025 strain was essential, as currently available molecular typing methods targeting BTV segment 2, which encodes the outer capsid protein VP2, are unable to detect this newly emerging strain. Therefore, in this study we developed, optimised, and validated a real-time RT-PCR assay for the detection and typing of BTV-5 ITA 2025 in field samples. The assay is characterised by high sensitivity and specificity, as well as good reproducibility, and can be effectively applied for BTV-5 ITA 2025 diagnosis in the current epidemiological context, supporting surveillance and control strategies.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-borne orbiviruses affecting domestic and wild ruminants. Information on their circulation in Namibian wildlife is limited. This study investigated the molecular detection and serotype distribution of BTV and EHDV in wild ruminants from a commercial game farm in the Khomas Region, Namibia, where wildlife and livestock coexist. Between June and September 2019, spleen samples from 62 clinically healthy animals (kudu, oryx, and red hartebeest) were analysed by real-time RT-PCR using pan-BTV and pan-EHDV assays, followed by serotype-specific tests for selected BTV types. Two animals (3.23%) tested positive for EHDV. BTV RNA was detected in 24/62 animals (38.71%), with Ct values ranging from 28.3 to 38.4. BTV-3 and BTV-4 were the most frequently identified serotypes, while one sample was positive for BTV-1; six BTV-positive samples remained untyped. High Ct values and low RNA loads likely limited sequencing success. Although restricted to a single farm and a limited serotype panel, this study provides preliminary molecular evidence of BTV and EHDV circulation in Namibian wild ruminants, highlighting the need for broader epidemiological investigations at the wildlife-livestock interface.

The "Sentinel Hypothesis" posits that animals often manifest the effects of environmental pollution earlier than humans. This systematic review quantitatively validates this hypothesis within the One Health framework, focusing on three Italian National Interest Sites for Remediation (SINs): Taranto, Campania's "Terra dei Fuochi," and Sardinia. Adhering to PRISMA guidelines, we synthesized data from 76 studies (43 human epidemiological/biomonitoring outcomes, 33 animal sentinel outcomes). In Taranto, excess human cancer incidence (e.g., +40% liver cancer in men) correlates with severe dioxin contamination in the mussel Mytilus galloprovincialis (up to 14.88 pg WHO-TEQ/g). In Campania, polychlorinated biphenyls (PCBs) and dioxins detected in livestock products mirrored contamination found in human breast milk and elevated cancer mortality. In Sardinia, while human cancer epidemiology remains debated, lead accumulation in wild boar (Sus scrofa) liver (mean 6.70 mg/kg) aligns with elevated lead levels in children's hair, highlighting risks from both mining legacies and hunting ammunition. The findings confirm that veterinary surveillance data frequently precede human health alerts, documenting a "systematic delay" in public health response. We recommend integrating veterinary and human surveillance systems to operationalize animal sentinels as early warning tools for environmental health.

Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.

Southern Europe, and Italy in particular, has historically been exposed to repeated incursions of orbiviruses, including multiple bluetongue virus (BTV) serotypes and epizootic haemorrhagic disease virus serotype 8. Sardinia, Italy represents a key sentinel location due to its geographic position in the Mediterranean basin. In August 2025, fatal bluetongue cases in sheep were reported in Sardinia during a period of intense BTV circulation in Italy, characterised by widespread outbreaks associated with BTV-3 and BTV-8. These clinical samples tested positive for BTV but could not be serotyped using the available real-time RT-PCR assays. Whole-genome sequencing identified the causative agent as BTV serotype 5 (BTV-5), representing the first detection of this serotype in Europe. The virus was also successfully isolated in cell culture. Segments 2 and 6 showed the highest nucleotide identity with a BTV-5 strain identified in Nigeria in 1982, whereas the rest of the genome seems to be composed by gene segments originating from multiple BTV serotypes circulating in Africa and Europe. At the time of writing, no official reports of BTV-5 circulation were available from Northern Africa; however, during the editorial process, BTV-5 was reported in Tunisia, further supporting the Mediterranean basin as a single interconnected epidemiological ecosystem for orbivirus emergence and spread.

The "One Health" approach is a multi-sectoral and multidisciplinary collaborative strategy, aiming to promote the health of humans, animals and the environment at local, national, regional and global levels. Effective health collaboration across sectors can be achieved through the "One Health" framework by adopting innovative health policies that shift from the concept of current healthcare services for ill people and animals to the prevention of health risks across entire communities: human, animal, plant, and environmental. This requires epidemiological surveillance, the development of risk and threat reporting systems, and the use of both descriptive and analytical epidemiological data to develop and improve overall health outcomes in humans, animals, plants, and the environment. Additionally, economic modelling can help to predict the potential impact of health threats, ensuring that new strategies are both proactive and sustainable, and are linked to health policies implemented from a One Health perspective and based on sound epidemiological studies. This would require securing the required resources, especially the human knowledge and competence necessary to implement these policies effectively. This study reviews the current concept and application of the One Health approach, its integration with the International Health Regulations (IHR) in epidemic and pandemic control, and its strengths and weaknesses. By analysing these factors, we propose a vision for future strategies that effectively incorporate the "One Health" approach into national and international health policies, with the goal of making "One Health" a practical and implementable framework, ensuring the sustainability of global health systems and fostering a healthier world for all.

Varroa destructor is the main parasitic threat to Apis mellifera colonies worldwide and represents a major concern for honey bee health and apicultural sustainability. Oxalic acid (OA) is widely used for the control of varroosis; however, its efficacy is strongly influenced by the presence of capped brood. This pilot field study aimed to evaluate the efficacy and safety of a combined treatment protocol based on Api-Bioxal® administered by trickling and sublimation. The study was carried out in a permanent apiary located in the province of Bergamo (Northern Italy) from October 2024 to February 2025 and involved ten A. mellifera colonies housed in Dadant-Blatt hives. The experimental protocol consisted of one OA trickling treatment followed by two OA sublimations and a control treatment with amitraz. Treatment-induced mite fall was recorded daily for 117 days; brood presence, ambient temperature and pollen influx were monitored throughout the study period, and colony strength was assessed at the beginning and end of the trial using the Liebefeld method. Differences between pre- and post-treatment measurements were analysed using the Wilcoxon signed-rank test. An overall mean of 563.6 Varroa mites per colony was recorded as treatment-induced mite fall. The highest efficacy of OA was observed when treatments were applied in broodless conditions, whereas the presence of capped brood markedly reduced acaricidal efficacy. No abnormal honey bee mortality or adverse effects on colony health were observed. Colony strength increased in 8 out of 10 colonies, with statistically significant differences between pre- and post-treatment assessments (p < 0.012). These results indicate that Varroa destructor populations remain susceptible to oxalic acid when treatments are properly timed in relation to brood presence. A combined protocol of OA trickling and sublimation may enhance varroosis control without negative effects on colony health. Further large-scale studies are required to confirm these preliminary findings and to support evidence-based adjustments to national Varroa control guidelines.

Parasites represent a major constraint in pond-based aquaculture, as their presence can lead to substantial fish mortality and associated economic losses. This study investigated the extent of Argulus infestation in cultured common carp (Cyprinus carpio) reared in a freshwater hatchery in South Sulawesi Province, Indonesia. Two host groups were examined: broodstock (n = 60; 1091.2 ± 58.16 g) and fingerlings (n = 150; 19.6 ± 0.82 g). Identification of Argulus specimens was conducted using morphological examination complemented by molecular analyses. Parasite prevalence and mean intensity were quantified, and Spearman's rank correlation was applied to determine the relationship between fish body weight and infestation severity. Both morphological and molecular evidence confirmed that the Argulus specimens infesting carp in this study were Argulus foliaceus. Fish body weight was significantly correlated with infestation intensity (P < 0.01), with broodstock exhibiting markedly higher prevalence and mean intensity than fingerlings. Preferred attachment sites differed between life stages: the base of the pectoral fins in broodstock and the body surface in fingerlings. These findings indicate that host life stage and body size strongly influence the severity and spatial pattern of A. foliaceus infestations in common carp. The outcomes provide a scientific basis for developing management guidelines to reduce the incidence and spread of Argulus in aquaculture systems.

Infectious bovine rhinotracheitis (IBR), caused by Bovine Herpesvirus-1 (BoHV-1), represents a significant economic burden on the global dairy industry through reduced productivity, reproductive disorders, and abortion. This cross-sectional study estimated the seroprevalence of IBR and identified associated risk factors among cattle in Eastern and Southern Algeria. Between September and December 2023, blood samples were collected from 380 cattle across 45 unvaccinated dairy farms in thirteen provinces. Sera were tested for BoHV-1 antibodies using a competitive enzyme-linked immunosorbent assay (cELISA), and seropositive samples underwent DIVA testing (Differentiating Infected from Vaccinated Animals) to detect glycoprotein E (gE) antibodies. Multivariable logistic regression analysis assessed risk factor associations with seropositivity. The overall seroprevalence was 38.95% (148/380), with a 100% herd-level prevalence. Among seropositive animals, 66.89% (99/148) were gE-positive, indicating natural infection, while 33.11% (49/148) were gE-negative, suggesting prior vaccination or vaccination exposure. Multivariable analysis identified four significant risk factors (p < 0.05): exotic origin (aOR = 5.33), large herd size (aOR = 2.12), age >3 years (aOR = 1.77), and breed, with crossbreeds showing lower susceptibility (aOR = 0.15) compared to Prim'Holstein cattle. These findings demonstrate widespread circulation of IBR in Algerian cattle, predominantly through natural infection rather than vaccination. The implementation of comprehensive control strategies, including official vaccination programmes utilizing DIVA-compatible vaccines and enhanced biosecurity measures, is crucial to mitigate economic losses in Algeria's dairy sector.

Listeria monocytogenes (L. monocytogenes) is a significant foodborne pathogen, posing a threat to public health. This study investigated the prevalence and genomic diversity of L. monocytogenes in 466 wild animals sampled across Central and Southern Italy (2017-2023), including species such as wild boar, red fox, and wolf, to assess their role as reservoirs and potential links to the food chain. Overall, 22.5% of the animals tested positive, and 118 L. monocytogenes strains were isolated, predominantly from wild boar (n=46), red fox (n=20), and Italian wolf (n=15). Whole Genome Sequencing (WGS) analysis revealed high genomic diversity, classifying the strains into 27 Clonal Complexes (CCs) and 31 Sequence Types (STs). Both hypervirulent clones (e.g., CC1, CC6, CC207) and hypovirulent clones (e.g., CC9, CC19), known for their persistence, were identified, with wild boars harboring a majority of the hypervirulent isolates. All strains carried key virulence genes, and accessory virulence factors, particularly LIPI-3, were detected in hypervirulent strains. Persistence factors, such as the Stress Survival Islet 1 (SSI-1) and genes for metal/disinfectant resistance (cadA, qacA), were also detected, particularly in wild boar isolates. Crucially, core-genome MLST (cgMLST) analysis demonstrated direct genomic links between the wildlife isolates and the Italian National Reference Laboratory database. Multiple clusters were identified, connecting strains from wild animals (wild boars, foxes, wolves) with those from meat products, fresh salads, and food processing environments. A persistent CC9 cluster, circulating in the meat chain for seven years, was strongly correlated with wild boar isolates, underscoring the role of wildlife as a reservoir that continuously introduces both high-virulence and highly persistent strains into the food production system. These findings emphasize the necessity of integrating wildlife surveillance into public health strategies to mitigate the risk of zoonotic transmission, particularly through game meat consumption and handling.