Lumpy Skin Disease (LSD) is characterized by fever, swollen lymph nodes, and the formation of firm, round skin nodules that can grow up to 5 cm in diameter (Kumar et al., 2021, Mebratu et al., 1984, Pandita et al., 2023, Yadav et al., 2024). These nodules may progress to ulcers, which can then become scabs and lead to secondary bacterial infections (Woods, 1988). Affected animals often suffer from weight loss, decreased milk production, infertility, and in severe cases, death (Tuppurainen et al., 2021). While the overall mortality rate is generally low, it can rise dramatically during outbreaks, with some instances reaching up to 60% (Kumar et al., 2024).

The LSD virus (LSDV) belongs to the Capripoxvirus genus within the Poxviridae family (Tuppurainen and Oura, 2012). Historically, LSDV has been endemic to parts of Africa and the Middle East, but it has spread rapidly to Asia, including India, in recent years (Bianchini et al., 2023, Kumar et al., 2023c). This spread has heightened concerns over the global impact of LSDV on the livestock industry. In India, the economic consequences of LSD are considerable, as the country is one of the largest producers of milk and a major exporter of livestock products (Kumar and Tripathi, 2022). The disease significantly impacts milk production, fertility, and animal health, leading to direct financial losses for farmers, particularly in regions where cattle and buffalo farming is a vital source of income (Dhanda et al., 2024, Kumar and Tripathi, 2022). Furthermore, the spread of LSDV has prompted stringent trade restrictions, affecting both domestic and international markets for livestock. The costs associated with control measures, such as vaccination campaigns, vector control, and quarantine protocols, further burden the agricultural economy (Manjunathareddy et al., 2024).

The Capripoxvirus genus, which includes LSDV, sheep pox virus (SPV), and goatpox virus (GTPV), shares a degree of genetic similarity, leading to potential cross-reactivity among them (Abutarbush and Tuppurainen, 2018). In the absence of a homologous LSDV vaccine, some countries have used heterologous vaccines derived from SPV or GTPV (Abutarbush and Tuppurainen, 2018, Kitching, 2003, Teffera and Babiuk, 2019, Tuppurainen et al., 2014, Tuppurainen et al., 2017). However, the WOAH recommends independent evaluation of any heterologous LSD vaccine before its use in cattle (WOAH, 2023a, WOAH, 2023b). In India, due to the unavailability of a homologous LSDV vaccine, a GTPV- Uttarkashi strain based vaccine authorized, however, it was later found to be partially effective against LSD in cattle

(Kumar et al., 2025).

LSDV strains are globally categorized into three main genetic lineages (Breman et al., 2023). Clade 1.1 comprises the classical Neethling-type strains, which are confined to Africa. Clade 1.2 includes the Kenyan-type strains, which have a broad geographic distribution across parts of Asia, Africa, and Europe (Breman et al., 2023). The third clade consists of recombinant strains, predominantly found in Kazakhstan, Russia, China, Taiwan, and Vietnam (Breman et al., 2023, Kononov et al., 2019, Vandenbussche et al., 2022). Three LSDV strains have been utilized in the development of commercially available vaccines against LSD. These include the South African Neethling strain, the Kenyan sheep and goatpox (KSGP or KS-1) strain, and the recently approved an Indian vaccine based on the Ranchi strain (Kumar et al., 2025).

While the widespread use of live-attenuated LSDV vaccines has been effective in controlling outbreaks, it poses a challenge for disease surveillance (Kumar et al., 2023a). Vaccination triggers an immune response similar to natural infection, making it difficult to differentiate between infected and vaccinated animals using conventional serological methods (Kumar et al., 2023a, Kumar et al., 2023b). This challenge emphasizes the urgent need for a serological test capable of distinguishing between LSDV infected and vaccinated animals (DIVA) (Kumar et al., 2023a, Kumar et al., 2023b). Such a tool would be crucial for accurate disease monitoring, outbreak management, and the implementation of control programs.

Serological testing plays a crucial role in confirming the success of disease control programs, particularly when vaccination has been employed (Gil et al., 2020, Shaukat et al., 2020, van Loon et al., 2019). However, differentiating between animals that are infected with LSDV and those that have been vaccinated using the same strain can be challenging (Panyasing et al., 2023). This differentiation is particularly important when determining whether a region is free from infection, as required by the World Organization for Animal Health (WOAH) guidelines (Kumar et al., 2023a, Kumar et al., 2023b, Wang et al., 2011, Wang et al., 2022). With the LSDV vaccines, such as those based on the Neethling and KSGP strains, it is not possible to differentiate the infected and vaccinated animals (DIVA). This issue arises because the immune responses generated by these vaccines are similar to those produced by natural infection (Moller et al., 2019).

The LSDV genome comprises 156 open reading frames (ORFs) organized within a central coding region, flanked by variable inverted terminal repeat (ITR) regions of approximately 2.4 kbp (Tulman et al., 2001). The ITR includes ORF1 to ORF4 at the 5′ end and ORF153 to ORF156 at the 3′ end. ORF1 is an inverted repeat of ORF156, ORF2 corresponds to ORF155, ORF3 to ORF154, and ORF4 to ORF153 (Tulman et al., 2001). The Ranchi LSDV strain-based vaccine offers a distinct advantage due to its unique genetic makeup. Compared to LSDV field strains, the Ranchi LSDV vaccine strain has an 801 bp deletion in the ITR region, encompassing ORF3/ORF154. As a result, the protein encoded by ORF3/ORF154 of LSDV Ranchi strain becomes non-functional. Although the precise nature of the protein encoded by ORF3/ORF154 is not known, it is a homolog of Myxoma virus M004 which codes for host immunomodulatory protein (Virokine) that modulates apoptosis and inflammatory response to virus infection (Hnatiuk et al., 1999).

The 801 bp deletion in the ITR region of the Ranchi strain prevents vaccinated animals from producing antibodies against ORF3/ORF154 proteins, whereas those infected with a field strain of LSDV will develop them (Kumar et al., 2023a, Kumar et al., 2023b). To exploit this difference, we developed an enzyme-linked immunosorbent assay (ELISA) that uses a recombinant protein derived from the ORF3/ORF154 region of LSDV field strain. This recombinant protein acts as a capture antigen in the ELISA test. When sera from infected animals were tested, they react strongly with the recombinant protein. However, sera from animals vaccinated with the Ranchi LSDV strain do not show this reaction, allowing for a clear serological distinction between infected and vaccinated animals. This DIVA test offers a reliable way to confirm the success of vaccination campaigns and ensures that vaccinated animals are not misidentified as being infected.