Detection of African Swine Fever Virus (ASFV) in Pork Meat Products by PCR Assay

African swine fever (ASF) is a highly contagious viral disease causing high mortality in members of the Suidae family. Besides animal health, the disease also imposes serious economic consequences, including trade barriers. African swine fever virus (ASFV) is known for its long-term survival in infected animals, including their carcasses and environment. Because of such stability of ASFV, importing pig-derived materials and feedstuffs from ASF-endemic countries would be potential sources of ASFV introduction to the US. ASF PCR validation for meat products is important for the quality control of meat sources and business continuity of domestic products in case ASF occurs in the US. The following study was to address this. The study consisted of two parts. The first part was an in-vitro study to evaluate and validate PCR assays to detect ASFV in pork meat products spiked with a synthetic DNA (hereafter, plasmid) constructed to contain the ASFV p72 gene (hereafter ASFV plasmid). The second part was an in-vivo study to assess ASFV distribution in blood, muscle, and other organs/tissues of pigs over time after an experimental exposure. For the first part, the test performance of two OIE recommended PCR assays (hereafter, OIE PCR#1 and OIE PCR#2) for ASFV detection and one of the commercially available ASFV PCR kits (hereafter, commercial PCR) was evaluated using various concentrations of the ASFV plasmid and pork products (loin, sausage, ground pork) spiked with various copy numbers of the ASFV plasmid or viral or bacterial pathogens commonly found in US swine farms. Although all PCR assays were specific for ASFV and could be used to detect ASFV DNA in pork products, OIE PCR#1 was less sensitive than OIE PCR#2 and commercial PCR, suggesting an appropriate PCR test should be selected for the testing purpose. For the second part, a group of growing pigs at 7 to 8 weeks of age were inoculated oronasally with a field isolate of ASFV and periodically euthanized to collect various internal and external organs and tissues, including muscle. Blood samples were also collected from the pigs daily. The serum and tissue samples were then tested for ASFV using the OIE PCR#2 and commercial PCR. Some pigs became viremic on 3 to 5 days post-inoculation (DPI), while clinical signs, such as fever, anorexia, and/or lethargy, started to be noted in some pigs on 5 to 6 DPI. Not all of the inoculated pigs were clinical. The onset of viremia also varied among the pigs. ASFV DNA was detectable in tonsil at as early as 2 DPI. The viral DNA was detected in all major internal organs (brain, lung, kidney, liver, spleen, heart, intestine, regional lymph nodes, and tonsil), muscle, and other tissues, such as diaphragm, feet, and ear, from all pigs necropsied on 5, 7, 10 and 14 DPI, demonstrating systemic infection. All or majority of the pigs necropsied on 21 DPI still harbored ASFV DNA in their lungs, hearts, tonsils, kidneys, livers, ears, and lymph nodes, suggesting that ASFV can persist in infected pigs for a long time as expected, and unprocessed pork and offal from those pigs may harbor the virus. This should be considered for procuring pork meats from an ASF-positive country or herd and developing biosecurity/control measures.