Determining the minimum infectious dose of porcine epidemic diarrhea virus in a feed matrix

Abstract Text: Feed has been confirmed as a potential vehicle for porcine epidemic diarrhea virus (PEDV) transfer. In order to determine the overall magnitude of risk and provide the necessary information for future research studies, a study was conducted to determine the minimum dose of PEDV in feed required to induce infectivity using a 10-d-old pig bioassay model. An initial source of PEDV isolate (USA/IN/2013/19338 P7) was serially diluted in 1 log increments to form 9 different PEDV doses ranging from 5.6x10⁵ to 5.6x10^-3 TCID50/ml with corresponding PCR cycle thresholds (Ct) of 14.0 to >45, respectively. Aliquots (500 ml) of the PEDV dilutions were mixed in 4.5 kg of a corn soybean meal based swine gestation diet to form 9 experimental treatments that were compared to a control that contained PEDV-negative tissue culture medium. The inoculated feed (100 g) was then mixed with 400 ml of PBS and refrigerated (4°C) overnight before extraction of the supernatant, which was subsequently used for the bioassay (10 ml of supernatant/pig and 3 pigs/dose). The 4 highest feed doses had detectable PEDV RNA (5.6x10⁴, 10³, 10², and 10¹ TCID50/g corresponding to a Ct of 27, 30, 33, and 37, respectively) and Ct increased linearly (P < 0.01, R² 0.98) as PEDV dose decreased. Every 1 log reduction in PEDV concentration resulted in an increase in 3.4±0.21 Ct in feed with detectable PEDV RNA. When the PEDV was added to the feed, an increase of 9.6±0.4 SEM Ct was observed compared to the tissue culture PEDV concentration for those feed samples that had detectable PEDV RNA. When the supernatant was used to inoculate 10-d-old pigs, fecal sample Ct's ranged from 16 to 27 on d 4 and 6 after inoculation for pigs inoculated with the 4 highest feed doses. No detectable PEDV RNA  (Ct >45) was noted in pigs inoculated with the other doses. Infection with PEDV was further confirmed in pigs from these 4 doses by histopathology and PEDV specific immunohistochemistry. In conclusion, these data suggest that PEDV infectivity was correlated with a positive feed PEDV PCR analysis. The minimum infective dose of PEDV in a feed matrix was demonstrated to be 5.6x10¹ TCID50/g and had an equivalent feed PCR Ct of 37. Overall, these data confirm that feed can be a vehicle for PEDV transfer and that a Ct of 37 can lead to infectivity in 10-d-old pigs.

Keywords: PEDV, feed, bioassay, minimum infectious dose