Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7 of sensitivity, 98.1 of specificity, 93 of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohens kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

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.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

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.