A highly sensitive method for detecting African swine fever virus in oral fluids from naturally infected pigs in Northern Vietnam

Early detection and early slaughter through quarantine are essential to prevent the spread of the African swine fever virus (ASFV). Pooled oral fluids testing is a practical approach for pathogen monitoring, but compared to blood, the virus concentration is lower, increasing false negatives. We developed a highly sensitive method for ASFV detection in pig oral fluids using a pretreatment-based concentration protocol. We collected oral fluids from sub-clinical raised pigs in northern Vietnam and conducted a field evaluation using the developed method. A spike test showed up to 100-fold greater sensitivity than a reference method. For performance evaluation, 68 pooled oral fluid samples were collected, of which 63 originated from raised pigs in northern Vietnam and 5 were obtained from healthy pigs in Japan as negative controls. Using real-time PCR, 9/68 (13.2%) were positive by the reference method, while 23/68 (33.8%) were positive by the developed method. Real-time LAMP detected 1/68 (1.5%) and 6/68 (8.8%) respectively. The developed method therefore improved the diagnostic performance and enabled early diagnosis prior to disease onset. These results suggest enhanced sensitivity and feasibility for early ASF diagnosis, potentially contributing to more effective outbreak control. The developed method offers a simple and sensitive tool for rapid ASFV screening in both affected and at-risk farms.