Yamazaki, W. | Search bioRxiv

bioRxiv2025

Environmental and vector detection of African swine fever virus DNA in Vietnam: Evidence for potential transmission through wastewater and Amblyomma javanense ticks

African swine fever virus (ASFV) remains a major global threat to pig production, yet its environmental and vector reservoirs in Southeast Asia are poorly understood. This study provides the first evidence in Southeast Asia indicating that ASFV DNA can be detected in pig farm wastewater and that Amblyomma javanense ticks may act as potential mechanical carriers of the virus in natural settings. To elucidate part of the environmental dynamics and possible transmission routes of ASFV, we developed and applied a highly sensitive polyethylene glycol (PEG) precipitation-based real-time PCR method specifically for wastewater surveillance, as the viral load in aquatic environments is presumed to be substantially lower than that in arthropod vectors. A total of 40 tick samples and 93 wastewater samples were analyzed. ASFV DNA was identified in 3 of 40 ticks (7.5%) and 7 of 93 wastewater samples (7.5%). The developed method showed a 100-fold improvement in analytical sensitivity, lowering Ct values by 3-7 cycles and increasing the detection rate from 3.2% to 7.5% compared with the reference method. Diagnostic sensitivity and specificity for wastewater testing were 100.0% and 95.6%, respectively. Although infectious virus was not recovered from ASFV-positive wastewater samples, control experiments confirmed that PEG treatment did not interfere with viral infectivity. These findings highlight the potential role of environmental and tick-mediated transmission in ASF epidemiology and provide a practical tool for early warning and post-outbreak monitoring of ASFV in endemic regions.

microbiology↗

bioRxiv2024

Development of a simple and highly sensitive method for the concentration and detection of African swine fever virus in oral fluids and its feasibility assessment on raised pigs in Northern Vietnam

BackgroundEarly detection and early slaughter through quarantine are essential to prevent the spread of the African swine fever virus (ASFV). Highly accurate testing is effective for early detection, but it is still difficult to establish a system, especially in the Global South. Pooled oral fluids tests have been used for simple pathogen monitoring, but compared to blood tests, the virus concentration in oral fluids is low, resulting in false negative and missing true positive cases. We collected oral fluids from raised pigs in northern Vietnam and attempted a highly sensitive ASFV survey using a newly developed virus concentration and detection method. ResultsIn a spike test, the developed method showed up to 100 times greater sensitivity than a reference method. To compare and evaluate the performance of the developed method, a total of 68 pooled oral fluid samples were collected, 63 from northern Vietnam and 5 from southern Japan. Using real-time PCR, 9/68 (13.2%) were positive by the reference method, and 23/68 (33.8%) by the developed method. Using real-time LAMP, 1/68 (1.5%) were positive by the reference method and 6/68 (8.8%) by the developed method. ConclusionsThe developed method improved the sensitivity of ASFV detection from oral fluids and enabled early diagnosis of pigs before the onset of the disease. The developed method has the potential to enable simple and highly sensitive diagnosis of ASF, which is essential for its early diagnosis and effective surveillance. HighlightO_LIA method has been developed to detect ASFV in pig oral fluids with up to 100 times greater sensitivity than a reference method. C_LIO_LIThe developed method combines innovative pretreatment in less than 60 min with conventional nucleic acid extraction with a column kit followed by real-time PCR or LAMP. C_LIO_LIApplied to feasibility assessment for ASF diagnosis on raised pigs, the developed method showed higher diagnostic sensitivity than the reference method. C_LIO_LIThe developed method can be a useful tool that contributes to early detection and surveillance of ASFV. C_LI

microbiology↗