Background/Objectives: European Brown Hare Syndrome (EBHS) is an acute and highly contagious viral disease of hares that causes considerable economic losses on wild and captive-reared hares. No preventive treatments are currently available to defeat the disease. Immunoprophylactic and biosafety measures could be applied to prevent EBHS only in captive-reared hares, where vaccination is proposed as an effective strategy. Due to the lack of a cellular substrate for virus growth, commercially available vaccines are autovaccines produced from inactivated liver suspensions of hares dead for EBHS. Therefore, using a recombinant vaccine based on VP60 major capsid protein seems a viable alternative to overcome such a problem. Methods: the 6xHis C-terminal tagged VP60 protein of EBHSV was expressed and produced in baculovirus, purified by affinity chromatography and the self-assembled recombinant (rEVP60-His₆) protein. To establish the protective properties of rEVP60-His₆-based VLPs, hares were immunised with 50 and 100 µg of VLPs and parenterally challenged with EBHSV. Results: all hares vaccinated with 100 µg of VLPs survived after the experimental infection, demonstrating the excellent protective ability of this prototype VLPs-based vaccine. Conclusions: self-assembled EBHSV rEVP60-His₆ protein was successfully produced following a rapid, simple, low-cost protocol. Although the protective efficacy of such VLPs were experimentally demonstrated, some key aspects remain to be clarified, including the duration of protection, the entity of the antibody response, and the ability to stimulate cell-mediated response. Last, an additional aspect to be evaluated is whether the use of an adjuvant can determine whether its presence improves the performance of the recombinant VLPs vaccine.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in the post-transcriptional regulation of gene expression. Since miRNAs modulate host immune responses, they represent promising molecular biomarkers of paratuberculosis (PTB), particularly during early or subclinical stages, when conventional diagnostic tests may lack sensitivity. In this context, faecal miRNAs could provide valuable insights into intestinal immune responses and mucosal damage associated with Mycobacterium avium subsp. paratuberculosis (MAP) infection. Although miRNAs have been extensively investigated in serum, blood, and tissues, their detection and characterization in bovine faeces remain poorly explored. The aim of this study was to evaluate the expression profiles of selected candidate faecal miRNAs in Marchigiana beef cattle naturally exposed to MAP and to assess their association with different infection phenotypes. Thirty-four cows were classified into three phenotypic groups: healthy exposed, MAP-infected, and PTB-affected based on longitudinal diagnostic records including interferon-γ assay, serological testing (ELISA), and faecal qPCR. Five candidate miRNAs were selected from previous studies and quantified in faecal samples by RT-qPCR. Four of the five selected miRNAs were consistently detected across samples. Bta-miR-92a was significantly downregulated in both MAP-infected and PTB-affected animals compared with healthy cattle, suggesting early modulation during MAP infection. Bta-miR-223 was significantly upregulated in PTB-affected animals compared with both healthy and MAP-infected groups, consistent with its established role in the regulation of intestinal inflammation. The ortholog of hsa-miR-501-5p was significantly upregulated in MAP-infected cattle, potentially reflecting early host–pathogen interactions at the intestinal mucosal level, while bta-miR-24-3p showed no significant differences among groups. Overall, these findings support the feasibility of faecal miRNA analysis as a complementary non-invasive molecular approach to support traditional diagnostic tests for PTB, especially during the early and subclinical stages of MAP infection.