Contagious ecthyma is a highly contagious viral disease with zoonotic significance caused by orf virus (ORFV) that affects domestic, ruminants and humans. Live attenuated virus and attenuated tissue culture vaccines are widely used in the fight against ORFV, however, the conventional attenuated vaccine strains have many drawbacks. The aim of this project was to construct a promising contagious ecthyma vaccine strain with safety, high protection efficacy and accessibility by genetic manipulation to against the disease. Using a natural ORFV-GS14 strain as the parental virus, recombinant virus, rGS14-ΔCBP-ΔGIF, with double deletions in the genes encoding the chemokine binding protein (CBP) and granulocyte/macrophage colony-stimulating factor inhibitory factor (GIF) was generated and characterized in vitro and in vivo. Results showed that the growth kinetics curve of rGS14-ΔCBP-ΔGIF and parental virus was consistent, both reaching plateau phase at 48 h post infection, which indicated that the double deletion of cbp and gif genes had little impact on the replication properties of the recombinant virus in primary goat testis (PGT) cell cultures compared with the parental virus. The safety of the double gene-deleted virus was evaluated in lambs. The lambs were monitored for 21 days post infection of the recombinant virus and no ORFV associated symptoms were observed in 21 days post-infection except for slight fever and anorexia in 5 days post-infection, and all lambs inoculated with either recombinant virus or PBS exhibited no clinical signs. To assess the protection efficacy of the rGS14-ΔCBP-ΔGIF, groups of four lambs each were inoculated with rGS14-ΔCBP-ΔGIF, rGS14-ΔCBP, rGS14-ΔGIF or PBS and challenged by a wild type virulent ORFV strain that was isolated from proliferative scabby lesions tissues of infected goat at 21-day post-inoculation. During 14 days post-challenging, lambs inoculated with rGS14-ΔCBP-ΔGIF all remained healthy with unimmunized group all infected, while the single gene-deleted viruses only protected 40% to 50% animals. These results indicated that the double gene-deleted recombinant virus could provide complete protection against virulent ORFV challenging. In conclusion, the double gene-deleted recombinant virus strain, rGS14-ΔCBP-ΔGIF, would be a promising candidate vaccine strains with safety, high protection efficacy and availability.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

Contagious ecthyma is a zoonotic disease caused by the orf virus (ORFV). Since there is no specific therapeutic drug available, vaccine immunization is the main tool to prevent and control the disease. Previously, we have reported the construction of a double-gene deletion mutant of ORFV (rGS14ΔCBPΔGIF) and evaluated it as a vaccine candidate. Building on this previous work, the current study reports the construction of a new vaccine candidate, generated by deleting a third gene (gene 121) to generate ORFV rGS14ΔCBPΔGIFΔ121. The in vitro growth characteristics, as well as the in vivo safety, immunogenicity, and protective efficacy, were evaluated. RESULTS: There was a minor difference in viral replication and proliferation between ORFV rGS14ΔCBPΔGIFΔ121 and the other two strains. ORFV rGS14ΔCBPΔGIFΔ121 induced continuous differentiation of PBMC to CD4⁺T cells, CD8⁺T cells and CD80⁺CD86⁺ cells and caused mainly Th1-like cell-mediated immunity. By comparing the triple-gene deletion mutant with the parental strain and the double-gene deletion mutant, we found that the safety of both the triple-gene deletion mutant and the double-gene deletion mutant could reach 100% in goats, while the safety of parental virus was only 50% after continually observing immunized animals for 14 days. A virulent field strain of ORFV from an ORF scab was used in the challenge experiment by inoculating the virus to the hairless area of the inner thigh of immunized animals. The result showed that the immune protection rate of triple-gene deletion mutant, double-gene mutant, and the parental virus was 100%, 66.7%, and 28.6%, respectively. In conclusion, the safety, immunogenicity, and immune-protectivity of the triple-gene deletion mutant were greatly improved to 100%, making it an excellent vaccine candidate.

(1) Background: Contagious ecthyma, also known as orf, is an epitheliotropic zoonotic disease caused by the orf virus (ORFV), primarily affecting the skin and mucous membranes of ruminants such as goats and sheep, leading to the formation of papules and pustules. Vaccination is the most effective way to prevent this disease in susceptible animals; however, traditional attenuated vaccines carry the potential risk of reversion to virulence. Therefore, there is an urgent need to develop safe and effective vaccines for the prevention and control of orf. (2) Methods: In this study, building upon the previously constructed ORFV three-gene deletion strain rGS14-TrypMut, we employed homologous recombination to knock out the VIL-10 gene and successfully constructed a four-gene deletion strain, rGS14-QuadMut. We evaluated its in vitro growth characteristics, safety, and protective efficacy in a challenge model. (3) Results: The in vitro results show that rGS14-QuadMut had a replication ability similar to that of other two-gene deletion strains, with good genetic stability. In in vivo experiments, compared to rGS14-TrypMut, rGS14-QuadMut caused only mild redness and swelling at the inoculation site, with a faster healing rate, indicating better safety. Additionally, rGS14-QuadMut induced strong differentiation of CD4⁺ and CD8⁺ T cells, increased the CD4⁺/CD8⁺ ratio, and primarily stimulated a Th1-type immune response, with significant changes in cytokine levels, including IL-8, IFN-γ, and IL-2. In the challenge protection experiment, both rGS14-QuadMut and rGS14-TrypMut provided 100% protective efficacy. In conclusion, rGS14-QuadMut demonstrated enhanced safety without compromising immune protection efficacy and is a promising candidate for an orf live vaccine strain.