Lumpy skin disease (LSD) is an infectious disease affecting bovine with severe symptomatology. The implementation of effective control strategies to prevent infection outbreak requires rapid diagnostic tools. Two monoclonal antibodies (mAbs), targeting different epitopes of the LSDV structural protein p32, and gold nanoparticles (AuNPs) were used to set up a colorimetric sandwich-type lateral flow immunoassay (LFIA). Combinations including one or two mAbs, used either as the capture or detection reagent, were explored to investigate the hook effect due to antigen saturation by the detector antibody. The mAb-AuNP preparations were optimized by a full-factorial design of experiment to achieve maximum sensitivity. Opposite optimal conditions were selected when one Mab was used for capture and detection instead of two mAbs; thus, two rational routes for developing a highly sensitive LFIA according to Mab availability were outlined. The optimal LFIA for LSDV showed a low limit of detection (10^3.4 TCID₅₀/mL), high inter- and intra-assay repeatability (CV% < 5.3%), and specificity (no cross-reaction towards 12 other viruses was observed), thus proving to be a good candidate as a useful tool for the point-of-need diagnosis of LSD.

Lumpy skin disease (LSD) is a notifiable, transboundary cattle disease that spreads rapidly and has a relevant economic impact. The etiological agent is the LSD virus (LSDV), genus Capripoxvirus, and family Poxviridae. To date, LSDV is widely present in Africa, Asia, and in transcontinental regions like Russia, Turkey, and the Middle East, thus representing a continuous threat to free neighbours. Appropriate serosurveillance programs can complement disease control, inform about its spread, and enable the assessment of vaccination campaigns. Since reliable and practical diagnostic tools could improve serological surveillance, this study aimed to produce and characterize monoclonal antibodies (MAbs) that allowed us to develop ELISA tests for the serological detection of LSD. Four MAbs recognizing a 35kDa viral protein were selected and used to develop and optimize competitive and indirect ELISAs. Both assays detected seroconversion within 14 days postinfection (dpi) in 18 cattle experimentally infected with LSDV and sequentially sampled for up to 4 weeks. The two novel ELISAs detected also antibodies raised by other capripoxviruses: as observed in cattle, both assays revealed seroconversion within 14dpi in all nine sheep experimentally infected with sheeppox virus (SPPV), while in eight goats infected with goatpox virus (GTPV) competitive ELISA identified seropositivity earlier and in more animals compared to indirect ELISA. Overall, the sensitivity performance of both developed ELISAs resulted comparatively superior to those of virus neutralization test and the commercial Id.Vet ELISA. Testing of about 200 negative sera from each species recorded a single false-positive cattle in the indirect ELISA, which gave a specificity of 99.5, whereas for the competitive ELISA, the diagnostic specificity was 100 irrespective of the species tested. The results enable concluding that both new tests correctly detect anti-LSDV antibodies in cattle and can also be reliable tools to recognize antibodies to SPPV and GTPV.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018–2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Introduction: Lumpy skin disease virus (LSDV), a member of the Capripoxvirus genus, poses a significant threat to livestock health and productivity in both endemic and newly affected regions. The disease is primarily transmitted by blood-feeding insects, leading to fever, cutaneous nodules, lymphadenopathy, and substantial economic losses. While vaccination remains the cornerstone of control efforts, effective surveillance—especially in high-risk areas—relies on robust and scalable diagnostic tools. Although the virus neutralization test is considered the reference standard among serological assays for detecting neutralising antibodies, it is labor-intensive and requires high-containment laboratories. Methods: In this study, we produced and evaluated two recombinant LSDV antigens: ORF074 (p32), a well-known immunodominant protein, and ORF060 (homologous to the Vaccinia virus L1R), a myristoylated membrane protein identified as a promising immunogenic target. Both proteins were expressed in E. coli . Recombinant p32 was purified under native conditions, whereas recombinant L1R required denaturation and refolding. The antigens were used to develop two indirect ELISAs, and were evaluated using sera from experimentally infected cattle, as well as both vaccinated and infected field samples from Albania and Serbia. Results and discussion: Both assays demonstrated high immunoreactivity and strong concordance with the VNT. These results support the suitability of both antigens for use in serological assays and suggest that a combined, multi-target ELISA approach could enhance diagnostic sensitivity. Once validated for routine use, these novel tools may significantly improve large-scale, cost-effective serological surveillance of LSDV in endemic and at-risk regions.

Lumpy skin disease (LSD), caused by the LSD virus (LSDV) from the Capripoxvirus genus, affects cattle, water buffalo, and wild bovines, leading to significant economic losses. Characterised by fever, skin nodules, and mucosal lesions, LSD raises global concerns due to vector-borne transmission. The World Organisation for Animal Health (WOAH) classifies LSD as a notifiable disease, emphasising the need for rapid diagnostic methods for timely disease confirmation and control. This study evaluates the performance of two previously developed ELISA tests - competitive and indirect. The validation involved 450 field sera from infected and vaccinated herds in Albania (collected in 2016, during the LSD outbreak), 332 sera from vaccinated cattle in Serbia (collected in 2017 from farms with no prior history of LSD detection), 90 sera from experimental infections at Friedrich-Loeffler-Institut, and 412 field negative sera from a Capripox-free country. The comparison with the virus neutralisation test - the gold standard - demonstrated high specificity (≥0.95) and significant sensitivity (0.87-0.94), with 8-9 % of sera showing discordant results. The results diverged more in sera from animals with a single vaccination or sampled five months post-vaccination, indicating reduced antibody detectability over time. The study confirms the ELISAs' efficacy for large-scale LSDV serological surveillance, highlighting their potential to provide a cost-effective and rapid solution for monitoring and controlling LSD in endemic regions.