Development and Validation of a Population Assay for the Seroprevalence of Lumpy Skin Disease

Lumpy Skin Disease (LSD) is a transboundary bovine viral disease. It has a significant economic impact and is caused by the Lumpy Skin Disease Virus (LSDV). Effective surveillance tools are essential for the early detection of infection, outbreak control, and assessment of vaccination coverage in endemic regions such as India. In this study, an in-house ELISA based on inactivated whole-virus antigen (WVA) was developed, optimized, and validated for the detection of LSDV antibodies in cattle. Its field applicability was assessed through a cross-sectional seroprevalence survey conducted across five Indian states. A local field isolate of LSDV (strain 5-Chitra) was cultured in MDBK cells, inactivated using binary ethylenimine (BEI), and used as the antigen source. The assay was optimized by checkerboard titration and evaluated against the Serum Neutralization Test (SNT). Diagnostic sensitivity and specificity were evaluated using the receiver operating characteristic (ROC) curve and area under the curve (AUC) analyses, while cross-reactivity was assessed using sera positive for HS, IBR, BQ, MCF, GTP, SPP, CE, FMD, and Brucellosis. Assay reproducibility was confirmed through inter- and intra-laboratory validation. For the seroprevalence study, 3230 cattle serum samples were collected using a stratified random sampling design across five Indian states, and logistic regression analysis of a subset of 1302 samples was performed to assess the influence of age and sex on LSDV seropositivity. Checkerboard titration identified optimal ELISA conditions at 50 ng of antigen per well, a 1:150 serum dilution, and a 1:10,000 dilution of anti-bovine HRP-conjugated secondary antibody. The WVA-ELISA demonstrated excellent diagnostic performance, with 100% sensitivity, 95% specificity, and no cross-reactivity with other ruminant bacterial or viral pathogens, and showed high laboratory reproducibility (κ > 0.96). Seroprevalence ranged from 50.6% to 71.1% across the five states, indicating widespread exposure to LSDV. Risk factor analysis revealed significantly higher odds of seropositivity among calves (≤1 year old) and female cattle, suggesting age- and sex-dependent susceptibility.