Yamkasem Jidapa, Tattiyapong Puntanat, Gardner Ian A, Surachetpong Win
Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A4P3, Canada.
Viruses. 2025 Jun 22;17(7):877. doi: 10.3390/v17070877.
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.
对鱼类群体中的病毒性疾病进行有效监测对于疾病控制和全球水产养殖的可持续发展至关重要。在此，我们评估了基于重组第4节段蛋白的间接ELISA法在检测罗非鱼感染罗非鱼湖病毒（TiLV）时群体血清样本的应用及性能，以评估养殖场层面的免疫力。TiLV - S4 ELISA法采用重组核蛋白（第4节段）抗原开发，通过棋盘滴定法进行优化，并对其重复性和再现性进行了验证，批内和批间变异系数均低于10%。在使用酶联免疫吸附测定（ELISA）检测TiLV特异性抗体之前，采用合并策略将多个血清样本合并。我们的结果表明，合并五个血清样本对于检测TiLV特异性抗体是有效的，特别是当合并样本中有多个血清阳性个体时，这支持了其在群体水平监测中的应用。然而，由于稀释效应，当合并样本中仅包含一个血清阳性样本时，ELISA的敏感性可能会降低。尽管存在这一局限性，但合并检测仍产生了较高比例的阳性结果，表明在许多情况下具有相似的检测性能。总体而言，合并策略为大规模监测罗非鱼群体的免疫状态提供了一种经济高效且省时的方法。
