Detection of African swine fever virus in feed dust collected from experimentally inoculated complete feed using quantitative PCR and virus titration assays

Abstract

African swine fever virus (ASFV) is a current threat to global pork production due to its high case fatality rate, lack of efficacious vaccine and recent transboundary spread into new regions of the world. Preventing introduction and further spread of ASFV is critical for countries currently negative for the virus. ASFV is stable in feed ingredients subjected to transoceanic conditions and transmission occurs through the natural consumption of contaminated feed. In this study, we investigated the use of feed dust collected from experimentally inoculated feed as a novel diagnostic sample type for ASFV detection. Moist swabs were used to collect dust from creep feeders after natural consumption of feed inoculated with 3.1-5.4 log 10 TCID 50 /g ASFV Georgia 2007 in the presence and absence of antimicrobial feed additives. Results validate the potential use of feed dust swabs as a novel diagnostic surveillance tool for detection and quantification of viral nucleic acid and infectious virus titre in ASFV-contaminated feed.

Stability of African swine fever virus in feed during environmental storage

“…Conserved regions of ASFV p72 were amplified using two PCR protocols. First, PCR primers and probe were designed (King et al., 2003) and utilized as previously described (Khanal et al., 2021). Second, the PCR protocol was designed (Zsak et al., 2005) and utilized according to the manufacturer's instructions ( VetAlert TM African Swine Fever Virus DNA Test Kit, Tetracore ® ).…”

“…Conserved regions of ASFV p72 were amplified using two PCR protocols. First, PCR primers and probe were designed (King et al, 2003) and utilized as previously described (Khanal et al, 2021). Second, the PCR protocol was designed (Zsak et al, 2005) TCID 50 /ml).…”

Diagnostic sensitivity of porcine biological samples for detecting African swine fever virus infection after natural consumption in feed and liquid

“…All biological samples were tested for the presence of ASFV using quantitative polymerase chain reaction (PCR) and virus isolation (VI). Nucleic acid was extracted from each sample using the MagMAX 96 Viral RNA Isolation Kit (ThermoFisher) as previously described (Khanal et al., 2022; Niederwerder et al., 2021; Stoian et al., 2020). Paramagnetic beads were mixed with a bead enhancer solution, and 20 μl of the bead mix was added to wells on a U‐bottom 96‐well plate.…”

“…Viral RNA Isolation Kit (ThermoFisher) as previously described (Khanal et al, 2022;Niederwerder et al, 2021;Stoian et al, 2020 PCR primers and probe were designed to amplify a conserved region of ASFV p72 (King et al, 2003) as previously described (Niederwerder et al, 2019). The PCR master mix included 10 µl SsoAdvanced Universal Probes Supermix (Bio-Rad Laboratories), 4 µl nuclease-free water,…”

Feed: A new pathway for the domestic and transboundary spread of viral pathogens of veterinary significance

“…These topics are timely, as ASFV DNA has been detected in commercial feed systems in Asia, with a range of 0.5-2.0% of samples collected from dust from complete feed and grain-based ingredients, such as soybean meal, testing positive by PCR. Extending the concept of ASFV risk in feed, this Special Issue describes the complexities of ASFV contamination of the feed milling environment, including an evaluation of feed dust sampling for the detection of the virus at the level of the feeder (Khanal et al, 2021), the feed mill and the swine production facility (Gebhardt et al, 2021). This Special Issue also covers the field of feed risk mitigation, studying the effect of mixing and feed batch sequencing on the distribution of ASFV in feed batches (Elijah et al, 2021), and an evaluation of a novel monoglyceride feed additive designed to reduce the risk of the original viral nemesis in feed, PEDV (Phillips et al, 2021).…”