Wataru Yamazaki

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.

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.

Background Controlling novel coronavirus pandemic infection (COVID-19) is a global challenge, and highly sensitive testing is essential for effective control. The saliva is a promising sample for high-sensitivity testing because it is easier to collect than nasopharyngeal swab samples and allows large-volume testing. Results We developed a simple SARS-CoV-2 concentration method from saliva samples that can be completed in less than 60 min. We performed a spike test using 12 ml of saliva samples obtained from healthy volunteer people, and the developed method performance was evaluated by comparison using a combination of automatic nucleic acid extraction followed by RT-qPCR detection. In saliva spike tests using a 10-fold dilution series of SARS-CoV-2, the developed method was consistently 100-fold more sensitive than the conventional method. Conclusions The developed method can improve the sensitivity of the SARS-CoV-2 test using saliva and speed up and save labor in screening tests by pooling many samples. Furthermore, the developed method has the potential to contribute to the highly sensitive detection of various human and animal viral pathogens from the saliva and various clinical samples. Highlight A method has been developed to detect SARS-CoV-2 from human saliva with 100 times higher sensitivity than conventional methods. The developed method combines simple pretreatment within 60 min with conventional nucleic acid extraction and RT-qPCR. This method can be applied for more sensitive virus testing from individual saliva. This method can potentially be applied to screening more than 100 saliva samples while maintaining the equivalent detection power of conventional methods. The method can be adapted to improve the sensitivity of detecting various pathogens from human and animal saliva.

Background Early 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. Results In 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. Conclusions The 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. A method has been developed to detect ASFV in pig oral fluids with up to 100 times greater sensitivity than a reference method. The 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. Applied to feasibility assessment for ASF diagnosis on raised pigs, the developed method showed higher diagnostic sensitivity than the reference method. The developed method can be a useful tool that contributes to early detection and surveillance of ASFV.

Background: While early detection and early containment are key to controlling the African swine fever (ASF) pandemic, the lack of practical testing methods for use in the field are a major barrier to achieving this feat.Objectives: To describe the development of a rapid and sensitive point‐of‐care test (POCT) for ASF, and its evaluation using swine whole blood samples for field settings.Methods: In total, 89 swine whole blood samples were collected from Vietnamese swine farms and were performed the POCT using a combination of crude DNA extraction and LAMP (loop‐mediated isothermal amplification) amplification.Results: The POCT enabled crude DNA to be extracted from swine whole blood samples within 10 min at extremely low cost and with relative ease. The entire POCT required a maximum of 50 min from the beginning of DNA extraction to final judgment. Compared to a conventional real‐time PCR detection, the POCT showed a 1 log reduction in detection sensitivity, but comparable diagnostic sensitivity of 100% (56/56) and diagnostic specificity of 100% (33/33). The POCT was quicker and easier to perform and did not require special equipment.Conclusions: This POCT is expected to facilitate early diagnosis and containment of ASF invasion into both regions in which it is endemic and eradicated.