Comparative evaluation of sampling methods for early molecular detection of African swine fever virus in experimentally infected pigs

Abstract Background African swine fever (ASF) is a highly pathogenic viral disease characterized by severe hemorrhagic fever and mortality rates approaching 100% in acute phase. In the absence of effective vaccines, rapid and precise molecular diagnosis is essential for outbreak control. However, few studies have systematically compared the diagnostic efficiency of different specimen types, particularly minimally invasive blood sampling methods, to establish reliable strategies for early surveillance. This study evaluated the diagnostic efficiency and temporal dynamics of ASFV genomic detection across various specimen types including blood, swabs, and environmental samples to identify the most reliable diagnostic sampling strategy for early-stage surveillance. Results In pigs experimentally inoculated with a Korean ASFV isolate, viral DNA was first detected in both jugular and ear vein blood, prior to the onset of pyrexia by 1–2 days. Notably, blood samples exhibited the highest viral loads, approximately ten times higher than the maximum titers found in spleen tissue collected at necropsy. In contrast, non-invasive specimens – including oral, nasal, and rectal swabs – showed delayed detection and significantly lower viral loads. Environmental samples, such as rope-based oral fluids and feed bin swabs, also demonstrated late and intermittent detection, while floor and fecal samples remained negative for ASFV genomic DNA throughout the study period. Conclusions Our findings demonstrate that whole blood is the most sensitive and reliable specimen for early ASFV detection, aligning with the onset of viremia that precedes clinical signs. Specifically, small-volume ear vein sampling was validated as a highly efficient and less labor-intensive, and minimally invasive alternative to jugular vein collection, without compromising diagnostic sensitivity. Although tissue or environmental samples may confirm infection at later stages, they are suboptimal for rapid outbreak intervention. Consequently, ear vein blood sampling could serve as a primary diagnostic approach, though further validation under diverse field conditions and with multiple ASFV strains is warranted before broad application in both clinical and resource-limited field environments.