Bovine leukemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus (Hossain et al., 2025), which belongs to the Retroviridae family. BLV infects cattle and is the etiological agent of enzootic bovine leukosis (EBL), a condition characterized by lymphoproliferation and B-cells tumors. Currently, there is no approved treatment or vaccine for EBL (Aida et al., 2013, Burny et al., 1988). BLV infection poses a significant animal health problem worldwide, particularly in the Americas and Asia, leading to economic losses attributed to reduced milk production (Ott et al., 2003), increased morbidity (EFSA, 2015), commercial barriers to semen export (Miller and van der Maaten, 1982), and reduced longevity of animals (Bartlett et al., 2013, Erskine et al., 2012), among other factors. Even subclinical infected animals contribute to economic losses (Rhodes et al., 2003, Yang et al., 2016). Furthermore, lymphosarcoma resulting from BLV infection represent the most common neoplastic condition identified in cattle slaughtered in the United States (Bartlett et al., 2014). Consequently, WOAH recommends the development and implementation of efficient serological diagnostic tools to carry out effective control programs for this infectious disease (EFSA, 2015).

BLV infection presents three different clinical expressions, which are not always detected as successive stages but rather as distinct forms of the disease: asymptomatic carrier (affecting 60 % of infected cattle), persistent lymphocytosis (PL) (affecting 30 % of BLV-positive animals), and B-cell leukemia/lymphoma or lymphosarcoma (affecting 5–10 % of infected cows) (Aida et al., 2013, EFSA, 2015). Due to these variable clinical manifestations, the identification of positive animals relies on various diagnostic tests, ranging from clinical examination to molecular methods, including hematological and serological tests. In fact, agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) are the major techniques recognized by the WOAH and most governmental organizations for the diagnosis of BLV infection (OIE, 2018). In recent years, more sensitive methods such as the amplification of the gp51 gene coding region by polymerase chain reaction (PCR) have been incorporated as a direct diagnostic method (Jaworski et al., 2018, OIE, 2018), although it is usually used as confirmatory complementary test (Martin et al., 2001).

One of the most useful proteins for the serological diagnosis of BLV is the envelope glycoprotein (Env), which is anchored to the viral envelope forming trimers and is cleaved by host furin proteases into two subunits: the heavily glycosylated gp51 (SU), which binds to the cellular receptor in the target cell, and the transmembrane gp30 (TM). Both subunits are covalently associated by disulfide bonds in a metastable prefusion complex (Johnston and Radke, 2000). While SU is highly immunogenic, the extracellular fraction of TM subunit preserves several immunogenic sites, and a full-length Env enctodomain produced in S2 Drosophila cells has been extensively characterized and shown to be antigenic, supporting its potential use in diagnostic and/or vaccine development (Tome-Poderti et al., 2024). On the other hand, the p24 capsid protein (CA) is the main component of the viral capsid, located in the interior of the particle and has been successfully produced in E. coli (Obal et al., 2015). Although less immunogenic (Gillet et al., 2007), CA is also a target of host immune system and can induce a specific antibody response. Thus, diagnostic tests based on the serological detection of specific antibodies against the envelope glycoprotein gp51 or the capsid protein p24 are among the most commonly used methods for identifying infected animals (Martin et al., 2001).

There are many commercially available BLV-ELISA kits (Kuczewski et al., 2018), some of them used for EBL diagnosis in Uruguay. For example, the Bovine Leukemia Virus Antibody Test Kit (VMRD, Inc.) can identify specific serum antibodies against the gp51 protein of the BLV. However, the high costs associated with these kits limit their application in our country, thereby hindering the accurate estimation of EBL prevalence, which has been estimated at approximately 75 % of dairy cows with estimates reaching 95 % in animals older than 4 years (Zaffaroni NA et al., 2007). This critical situation results in significant production losses, emphasizing the urgent need for the implementation of BLV control programs (Rhodes et al., 2003). Undoubtedly, achieving this goal requires the availability of reproducible, simple, and reliable diagnostic tools at a low cost. Additionally, it’s important to note that most of these commercial tests are designed for serum diagnosis, with only a few suitable for diagnosis using more readily obtainable bovine milk samples (Evermann et al., 2019). Several reports have demonstrated the production of gp51 and p24 recombinant proteins using heterologous expression systems such as Escherichia coli (E. coli), Saccharomyces cerevisiae, and baculovirus for diagnostic purposes (Bai et al., 2019, Bicka et al., 2001, De Giuseppe et al., 2004, Gutierrez et al., 2009, Legrain et al., 1989). Despite the widespread use of bacteria and yeast expression systems, they have limitations regarding correct protein processing and post-translational modifications such as glycosylation (Dell et al., 2010, Mattanovich et al., 2012). In this regard, the expression of Env in Drosophila melanogaster (D. melanogaster) Schneider 2 (S2) eukaryotic cells represents a reasonable and efficient strategy (Astray et al., 2016). Although the glycosylation pattern of insect cells differs from that of mammalian cells, S2 cells share several key features with them, such as proper protein folding and efficient secretion. In parallel, they offer additional advantages including higher production yields, easy propagation, minimal risk of contamination, high scalability, and reduced production time and overall purification costs (Tripathi and Shrivastava, 2019).

The aim of this study is to develop and optimize three different indirect ELISAs for detecting anti-BLV antibodies in serum and milk. We utilize the Env ectodomain (ectoEnv: gp51 + extracellular domain of gp30) and p24 recombinant proteins, expressed in D. melanogaster S2 cells and E. coli systems, respectively. For completeness, we further compare their performance with a widely used commercial ELISA, the Bovine Leukemia Virus Antibody Test Kit (VMRD, Inc).