Abstract
Effective surveillance of viral disease in fish populations is critical for disease control and the sustainable development of global aquaculture. Here, we evaluated the application and performance of pooled serum samples using an indirect ELISA based on recombinant segment 4 protein to assess farm-level immunity in tilapia infected with Tilapia lake virus (TiLV). The TiLV-S4 ELISA was developed using a recombinant nucleoprotein (segment 4) antigen, optimized through checkerboard titration, and validated for repeatability and reproducibility, with intra- and inter-assay coefficients of variation below 10%. A pooling strategy was used to combine multiple serum samples before testing for the presence of TiLV-specific antibodies using an enzyme-linked immunosorbent assay (ELISA). Our results showed that pooling five serum samples was effective for detecting TiLV-specific antibodies, particularly when multiple seropositive individuals were presented in the pool, supporting its application for population-level surveillance. However, ELISA sensitivity may be reduced when only one seropositive sample is included in the pool, due to the dilution effects. Despite this limitation, pooled testing yielded a high proportion of positive results, suggesting similar detection performance in many cases. Overall, the pooling strategy provides a cost-effective and time-efficient approach for large-scale monitoring of immune status in tilapia populations.