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.