Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Background: Lumpy skin disease virus (Poxviridae family—Capripoxvirus genus) is the aetiological agent of LSD, a disease primarily transmitted by hematophagous biting, affecting principally cattle. Currently, only live attenuated vaccines are commercially available, but their use is limited to endemic areas. There is a need for safer vaccines, especially in LSD-free countries. This research aims to develop and test a safe and efficacious inactivated vaccine. Moreover, in this study, we used keyhole limpet hemocyanin (KLH) as a positive marker to distinguish infected from vaccinated animals (DIVA). Methods: Lumpy skin disease virus was propagated on primary lamb testis cells and Madin–Darby bovine kidney cells (PLT and MDBK, respectively), and four inactivated vaccines were produced. The vaccines differed from each other with the addition or not of KLH and in cells used for virus propagation. To evaluate the safety and immunogenicity, the vaccines and two placebos were administered to six groups comprising six male calves each, and antibody response was investigated using both an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test. In addition, the LSD/γ-interferon test and KLH (IgM-IgG) ELISA were performed on the collected samples. Furthermore, the use of KLH allowed us to distinguish vaccinated animals in the ELISA results, without any interference on the strength of the immune response against the LSDV. Finally, the efficacy of one of four vaccines was investigated through a challenge, in which one group of vaccinated animals and one animal control group were infected with a live field strain of LSDV. Results: Four out of the six control animals showed severe clinical signs suggestive of LSD, and, therefore, were euthanized for overcoming the predetermined limit of clinical score. By contrast, the vaccinated animals showed only mild symptoms, suggesting a reduction in severe disease notwithstanding the incapability of the vaccine in reducing the virus shedding. Conclusion: The vaccines produced were safe and able to elicit both a humoral and a cellular immune response, characteristics that, together with the demonstrated efficacy, make our vaccine a good candidate for countering the LSD spread in disease-free countries, thus also facilitating disease containment throughout the application of a DIVA strategy.

Background: Lumpy skin disease (LSD) is caused by a Capripoxvirus. Live attenuated vaccines, which are commercially available, could be not safe because of the side effects. The aim of this study was the evaluation of the safety, immunogenicity, and effects on the qualitative and quantitative parameters of milk. The feasibility of identifying vaccinated animals using our inactivated vaccine in dairy cows was analysed. The vaccine was tested in guinea pigs as an immunogenicity predictive model. Methods: LSD virus was propagated on Madin–Darby Bovine Kidney (MDBK) cells, then inactivated and supplemented with keyhole limpet hemocyanin (KLH) protein, obtaining a positive marker vaccine. This was inoculated in guinea pigs and in dairy cows, and animal sera were analysed using enzyme-linked immunosorbent assay (ELISA) and a serum neutralisation (SN) test. Quantitative and qualitative analyses were performed on milk. Results: The vaccine was previously tested for efficacy in vaccinated calves, showing a pronounced reduction in clinical symptoms after challenge. The safety and immunogenicity obtained in calves were also confirmed in dairy cows in this study. In fact, high values of the SN test (1:20 to 1:80) and ELISA (90 and 240 S/P%) were obtained after vaccination. Moreover, high immunogenicity of the vaccine was also assessed in guinea pigs. In addition, the results of the milk analyses did not show any differences between vaccinated and control groups. The KLH was able to elicit an immune response detectable using an ELISA (3.0 and 3.5 optical density values). Finally, our vaccine could be used to reduce LSD symptoms and identify vaccinated animals.

The protozoan Trypanosoma equiperdum is the causative agent of Dourine, a contagious sexually transmitted disease of equines that can manifest itself in chronic or acute form. Dourine has serious veterinary and economic implications, as there are no vaccines available for prevention and the management of the disease requires the culling of infected animals, together with rigorous sanitary control measures. This study compares the traditional production of T. equiperdum antigen grown in vivo, with an in vitro cultivation methodology developed to eliminate the use of animals. The in vitro and in vivo antigens are evaluated within serological diagnostic methods for Dourine, using Complement Fixation Test and Indirect Fluorescent Antibody Test. These methods are validated according to the principles and procedures outlined the WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, in order to assess their diagnostic performance and ethical sustainability. The results show the sensitivity and specificity of the in vivo antigen is comparable to the in vitro antigen. Precision parameters confirmed the robustness of the assays, with 100% concordance in repeatability and reproducibility. The IFAT showed excellent concordance with the CFT, suggesting its potential as a confirmatory test in eradication programs. The in vitro production system significantly reduces biosafety risks and logistical constraints associated with animal housing and infection. This approach is consistent with the 3Rs (Replacement, Refinement, and Reduction), offering a sustainable alternative to antigen production and supporting more ethical animal management in research, promoting animal welfare and sustainable veterinary diagnostic practices.