Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies. A recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6–9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. Well-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

This study describes a model developed to evaluate the transboundary risk of PEDV-contaminated swine feed ingredients and the effect of two mitigation strategies during a simulated transport event from China to the US. Ingredients imported to the USA from China, including organic & conventional soybeans and meal, lysine hydrochloride, D-L methionine, tryptophan, Vitamins A, D & E, choline, carriers (rice hulls, corn cobs) and feed grade tetracycline, were inoculated with PEDV. Control ingredients, and treatments (ingredients plus a liquid antimicrobial (SalCURB, Kemin Industries (LA) or a 2 % custom medium chain fatty acid blend (MCFA)) were tested. The model ran for 37 days, simulating transport of cargo from Beijing, China to Des Moines, IA, US from December 23, 2012 to January 28, 2013. To mimic conditions on land and sea, historical temperature and percent relative humidity (% RH) data were programmed into an environmental chamber which stored all containers. To evaluate PEDV viability over time, ingredients were organized into 1 of 4 batches of samples, each batch representing a specific segment of transport. Batch 1 (segment 1) simulated transport of contaminated ingredients from manufacturing plants in Beijing (day 1 post-contamination (PC)). Batch 2 (segments 1 and 2) simulated manufacturing and delivery to Shanghai, including time in Anquing terminal awaiting shipment (days 1–8 PC). Batch 3 (segments 1, 2 and 3) represented time in China, the crossing of the Pacific and entry to the US at the San Francisco, CA terminal (day 1–27 PC). Batch 4 (segments 1–4) represented the previous events, including transport to Des Moines, IA (days 1–37 PC). Across control (non-treated) ingredients, viable PEDV was detected in soybean meal (organic and conventional), Vitamin D, lysine hydrochloride and choline chloride. In contrast, viable PEDV was not detected in any samples treated with LA or MCFA. These results demonstrate the ability of PEDV to survive in a subset of feed ingredients using a model simulating shipment from China to the US. This is proof of concept suggesting that contaminated feed ingredients could serve as transboundary risk factors for PEDV, along with the identification of effective mitigation options.

Contaminated complete feed and porcine plasma are risk factors for PEDV introduction to farms and a liquid antimicrobial has been proven useful for reducing risk. This study provides information on the survivability of PEDV across common swine feed ingredients in the presence or absence of the liquid antimicrobial. Eighteen ingredients commonly included in commercial swine diets were selected, including 3 grain sources (corn, soybean meal (SBM), dried distillers grains with solubles (DDGS)), 5 porcine by-products (spray-dried plasma, purified plasma, intestinal mucosa, meat and bone meal and red blood cells (RBCs)), 3 vitamin/trace mineral (VTM) mixes (sow, nursery, finishing), 2 fat sources (choice white grease and soy oil), 3 synthetic amino acids (lysine HCL, D/L methionine, threonine), as well as limestone and dry choline chloride. Complete feed and stock PEDV served as controls. Thirty grams of each ingredient were inoculated with 2 mL PEDV. A matched set of samples were treated with the formaldehyde-based liquid antimicrobial SalCURB® (LA). All samples (n = 320) were stored outdoors under winter time ambient conditions for 30 days. Samples were submitted on 1, 7, 14 and 30 days post-inoculation (DPI) and tested by PCR and virus isolation (VI). All VI-negative samples were tested by swine bioassay. Viable PEDV was detected by VI or swine bioassay at 1, 7, 14 and 30 DPI from SBM, DDGS, meat & bone meal, RBCs, lysine HCL, D/L methionine, choice white grease, choline chloride, complete feed and stock virus control and at 7 DPI in limestone and at 14 DPI in threonine. Supplementary testing of complete feed and SBM indicated viable virus out to 45 and 180 DPI, respectively. All other samples were negative by VI and bioassay. In contrast, treatment with LA inactivated PEDV across all ingredients on 1 DPI and induced RNA reduction over time. Under the conditions of this study, PEDV viability in feed was influenced by ingredient with extended survival in SBM. Furthermore, LA treatment rendered virus inactive, independent of ingredient type.

Since its initial detection in May 2013, porcine epidemic diarrhea virus (PEDV) has spread rapidly throughout the US swine industry. Recently, contaminated feed was confirmed as a vehicle for PEDV infection of naïve piglets. This research provides in vivo data supporting the ability of a liquid antimicrobial product to reduce this risk. Sal CURB® (Kemin Industries, Des Moines, IA, USA) is a FDA-approved liquid antimicrobial used to control Salmonella contamination in poultry and swine diets. To test its effect against PEDV, Sal CURB®-treated feed was spiked with a stock isolate of PEDV (Ct = 25.22), which PEDV-naïve piglets were allowed to ingest via natural feeding behavior (ad libitum) for a 14-day period. For the purpose of a positive control, a separate group of piglets was allowed to ingest non-treated (Sal CURB®-free) feed also spiked with stock PEDV (Ct = 25.22). A negative control group received PEDV-free feed. Clinical signs of PEDV infection (vomiting and diarrhea) and viral shedding in feces were observed in the positive control group 2–3 days post-consumption of non-treated feed. In contrast, no evidence of infection was observed in pigs fed Sal CURB®-treated feed or in the negative controls throughout the 14-day study period. In addition, the Sal CURB®-treated feed samples had higher (p< 0.0001) mean PEDV Ct values than samples from the positive control group. These data provide proof of concept that feed treated with Sal CURB® can serve as a means to reduce the risk of PEDV infection through contaminated feed. Furthermore, the results from the positive control group provide additional proof of concept regarding the ability of contaminated feed to serve as a risk factor for PEDV infection of naïve piglets.