Alessandra Martucciello

Introduction: Milk is more than a traditional nutrient source; it serves as a crucial carrier for milk-derived extracellular vesicles (mEVs), food-derived nanoparticles with diverse effects connecting animal health and human nutrition to ecological stability. The primary goal of this study was to characterize the phenotype and functional impact of mEVs from water buffalo ( Bubalus bubalis ) across the lactation curve. The researchers aimed to determine how the transition from colostrum (Colo EVs) to early milk (EM-EVs) and mature milk (MM-EVs) alters the vesicles’ physical properties and their ability to modulate the immune response in macrophages. Material and methods: The study utilized Flow Cytometry (FC) as a core tool, first validating specific antibody clones for use in the buffalo species. The functional impact was tested on both a bovine cell line (BoMac) and primary water buffalo macrophages, focusing on cellular uptake, mitochondrial oxidative stress (ROS) and phagocytic activity. Results and conclusion: MM-EVs play the most important role in boosting phagocytosis in bubaline macrophages, accompanied by the highest increase in CD14 surface density, whereas Colo-EVs demonstrated the highest degree of internalization and the strongest antioxidant activity. In conclusion, the study demonstrates that mEVs provide a biological bridge during the calf’s development. While Colo-EVs focus on antioxidant protection and systemic signaling, MM-EVs actively enhance the innate immune defense. This provides a mechanistic explanation for why continued consumption of maternal milk beyond the colostral phase is vital for the immunological resilience and survival of buffalo calves. Furthermore, BoMac cells and bubaline macrophages exhibit distinct cytokine profiles, as IL-1α displays an increasing trend that did not reach statistical significance in MM-EV-treated BoMac cells, whereas a non-significant increasing trend is observed in macrophages treated by both Colo EVs and MM-EVs. Ultimately, the conserved nature of tetraspanins and miRNA profiles underscores the broad cross-species translational potential of buffalo mEVs, extending to human applications.

Animal welfare and ethical responsibility are central considerations in animal research. In Italy, European Directive 2010/63/EU was implemented through Legislative Decree No. 26/2014, establishing Animal Welfare Bodies (AWBs) to ensure ethical project evaluation and promote the Replacement, Reduction, and Refinement (3Rs) principles. Despite their regulatory importance, long-term analyses of AWB activities remain limited, particularly within the Italian context. This study presents a ten-year retrospective analysis of the AWB of the Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM) from 2016 to 2025. The study collected data on project evaluations, species involved, opinions issued, retrospective assessments, personnel competence management, and welfare monitoring procedures. Over the study period, the AWB evaluated 33 projects, issuing 26 positive opinions, 6 positive opinions with reservations, and 1 negative opinion, and conducted 11 retrospective assessments. The data showed marked fluctuations in authorised animal numbers, including a substantial reduction in mouse authorisations between successive five-year regulatory testing cycles. These trends occurred alongside the implementation of alternative methods and changes in regulatory testing strategies. The findings describe the activities and longitudinal outcomes of a single institutional AWB and are consistent with a potential contribution to 3Rs implementation, while the single-institution observational design does not allow causal attribution of these changes to AWB activities alone.

Milk is a primary source of vital nutrients and bioactive components fundamental to the growth and development of both newborn animals and humans. Produced by economically significant livestock species (including cattle, buffaloes, goats, sheep and camels), milk is a complex matrix rich in caseins, vitamins, fats, and proteins. Beyond its classical nutritional profile, milk serves as a pivotal vehicle for milk-derived extracellular vesicles (mEVs). These specialized food-derived EVs (fEVs) exert pleiotropic effects that resonate with the One Health paradigm, linking animal well-being and human nutrition to broader ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stability, also rendering them potential therapeutic tools as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their molecular cargo. This review provides a comprehensive comparative analysis of mEVs across a diverse taxonomic range, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, highlighting their distinct functional signatures. Indeed, a critical issue in mEV research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing flow cytometry (FC) approaches are discussed. Key insights include the use of conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of bead-based assays to overcome resolution limits) and imaging flow cytometry (IFC). In both technical approaches, the application of different EV generic fluorescent markers and the strategic selection of tetraspanins (i.e., CD9, CD63, CD81), is mandatory: we emphasize that selecting the correct antibody clones and accounting for inter-species cross-reactivity are essential steps for ensuring the accuracy and reproducibility of mEV research across mammalian species.

Milk is a primary source of vital nutrients and bioactive components fundamental for the growth and development of both newborn animals and humans. Produced by economi-cally significant livestock species (including cattle, buffaloes, goats, sheep and camels) milk is a complex matrix rich in caseins, vitamins, fats and proteins. In addition to its nu-tritional profile, milk serves as a vehicle for milk-derived extracellular vesicles (mEVs), a specialized class of food-derived EVs (fEVs) that exert pleiotropic effects aligned with the One Health concept, relating animal health, human nutrition, and ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stabil-ity, rendering them also potential therapeutic tools, as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their mo-lecular cargo. This review provides a comparative analysis of mEVs across diverse spe-cies, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, with a focus on their unique functional profiles. Indeed, a critical issue in mEVs research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing Flow Cytom-etry (FC) approaches are discussed. Key insights include the use of Conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of Bead-Based Assays to overcome resolution limits) and of Imaging Flow Cytometry (IFC). In both technical approaches, the application of different EVs generic fluorescent markers and the strategic selection of tetraspanins (i.e. CD9, CD63, CD81), is mandatory, empha-sizing the importance of selecting appropriate antibody clones or considering cross-reactivity when targeting these antigens across different mammalian species.

Background: Bovine alphaherpesvirus 1 (BoAHV-1) is a major respiratory and reproductive pathogen in cattle worldwide. Both innate and adaptive immune responses contribute to protection against this virus; however, virus-host interactions remain partly undefined. In this study, the impact of BoAHV-1 infection on calves’ immune responses was investigated in detail. Methods: Six calves were intranasally infected with wild-type BoAHV-1, and blood samples were collected longitudinally. Leukocyte subset dynamics were assessed by complete haematological assay and flow cytometry, while multiplex ELISA was used to quantify serum levels of ten cytokines. For each parameter, post-infection values (days 2, 4, 8, 10, and 14) were compared with pre-infection baseline values (day 0). Results: Infection induced an initial phase of immunosuppression, reflected by decreased circulating αβ and γδ-T cells. However, infected animals rapidly developed a protective immune response, characterised by increased circulating classical and intermediate monocytes and elevated levels of the related chemokine MIP-1β. Early post-infection, rises in serum IFN-γ and IL-10 were also detected. Conclusions: Our data suggest that monocyte recruitment and increased serum levels of IFN-γ and IL-10 are positively associated with the ability to overcome infection. A better understanding of the immunopathogenic mechanisms underlying BoAHV-1 infection will support the development of more effective vaccines against this virus.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as “Infectious Bovine Rhinotracheitis” (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 10^5.00 TCID₅₀/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

Bisphenol A (BPA) and some of its analogues are well known as endocrine-disrupting chemicals (EDCs), i.e., compounds that can affect the physiological hormonal pathways in both animals and humans, causing adverse health effects. The intake of these substances through diet represents a public concern, compounded by the scarce data in the literature about contamination levels in food. In the framework of a research project, funded by the Italian Ministry of Health, we determined the contamination levels of BPA and 19 EDCs in the production chain of buffalo milk, analysing feed, drinking water, buffalo milk, and blood sera. Overall, 201 feed, 9 feed additive, 62 drinking water, 46 milk, and 190 blood serum samples were collected from 10 buffalo farms in the Campania region, Southern Italy, between 2019 and 2020, and analysed. Moreover, 15 retail bovine milk samples packaged both in Tetra Pak and in PET were analysed to further evaluate consumers’ exposure to EDCs. The results of our work showed no contamination by EDCs in drinking water samples, whereas in 43% of all of the other samples from the farms at least one bisphenol was detected. The most abundant bisphenol detected was BPA (32% of the samples from the farms and 80% of the retail milk samples), thus proving that this compound is still widely used for plastic production.

Paratuberculosis is a chronic enteric disease affecting virtually all ruminants, but only anecdotal information is currently available about the occurrence of this disease in water buffaloes (Bubalus bubalis). We carried out a survey study aimed at determining the prevalence of paratuberculosis in 2 provinces in the region of Campania, Italy, where about half of all Italian buffaloes are reared. From May 2017 to December 2018, we collected 201,175 individual serum samples from 995 buffalo herds. The sera were collected from animals over 24 mo old and were tested using a commercial ELISA test. The herd-level apparent prevalence result was 54.7%, and the animal-level apparent prevalence was 1.8%. The herd-level true prevalence was estimated using a Bayesian approach, demonstrating a high herd-level prevalence of paratuberculosis in water buffaloes from the Campania area. These findings suggest that the urgent adoption of paratuberculosis herd-control programs for water buffaloes in this area would be beneficial.