An overall summary of the statistically significant results (Table 1) illustrates that PEDV infection of gilts at different stages of gestation (Figure 1) affects multiple maternal systemic immune parameters prepartum, including natural killer (NK) cells, cytokines, B cells, and PEDV Abs and antibody secreting cells (ASCs). In addition, significant postpartum effects on lactogenic immune parameters in colostrum and milk were observed including significantly increased PEDV IgA (colostrum and milk) and IgG (colostrum only) ASCs and PEDV IgA Abs and VN Abs in PEDV-infected second trimester gilts (Table 1). Notably several gilt [PEDV IgA (colostrum and milk) and IgG (colostrum only) ASCs, Abs, and VN Abs] and piglet (serum PEDV IgA and IgG Abs) parameters were positively correlated with piglet survival rates (Table 2), demonstrating the association between IgA ASCs and Abs and VN Abs and lactogenic immune protection of suckling neonates. The detailed results for each parameter are described in the following sections.

Third trimester gilts had significantly higher PEDV RNA shedding titers at PID 2 compared with second trimester gilts (Figure 2A). Additionally, second trimester gilts had delayed onset of PEDV RNA shedding compared with first and third trimester gilts. Fecal consistency scores at PID 4 were significantly higher in third compared with second and first trimester gilts and third trimester gilts were the only treatment group with clinical diarrhea (mean fecal consistency score of >1) (Figure 2B).

Circulating NK cell frequencies were significantly higher in first trimester gilts at PID 0 compared with second or third trimester gilts (Figure 3A). At PID 6-8, peripheral NK cell activity was significantly higher in first compared with second and third trimester gilts (Figure 3B). Mean concentrations of serum IL-12, a cytokine essential for NK cell activation (29) and IL-22, a cytokine produced by subsets of T and NK cells (30–32), were numerically higher at PID 0 in first compared with second and third trimester gilts (Figures 3C,D). It is unlikely changes in serum cytokine concentrations were due to T cells as the frequency of circulating CD4⁺ or CD8⁺ cells did not significantly differ between treatment groups (data not shown). Third trimester gilts had numerically higher mean concentrations of serum proinflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-α and IL-17 at PID 12–17 compared with first and second trimester gilts (Figures S3A–C). This time point corresponds to gestation day (GD) 104–114 in third trimester gilts, suggesting innate, and proinflammatory cytokines increase immediately prior to parturition in swine, as demonstrated in human pregnancy (33–36). Additionally, third trimester gilts had numerically higher mean serum concentrations of T-helper cell type-1 (Th1) cytokine IFN-γ while first trimester gilts had numerically higher mean serum Th2 and T regulatory cytokines IL-4 and IL-10, respectively, compared with second and third trimester gilts at PID 6–8 (Figures 3SD–F).

Maternal B-cell immune responses were measured at PID 0, 6–8, and 12–17 in first, second and third trimester gilts. Second trimester gilts had significantly higher circulating PEDV IgA and IgG ASCs at PID 12–17 compared with first and third trimester gilts (Figures 4A,B). Additionally, numbers of circulating PEDV IgA ASCs were consistently higher than PEDV IgG ASCs in first, second and third trimester gilts at PID 6–8 and 12–17 (Figures 4A,B). PEDV IgA Ab titers were significantly higher in second trimester gilts at PID 6–8 compared with first trimester gilts (Figure 4C). Serum TGF-β, a cytokine important for IgA class-switching (37), was significantly higher at PID 0 and remained numerically higher at PID 6–8 and 12–17 in second compared with first and third trimester gilts (Figure 4D). Serum IL-6, a cytokine essential for Ab production (38), was numerically higher at PID 0 and 6–8 in second trimester compared with first and third trimester gilts (Figure 4E). No significant differences were observed for serum PEDV IgG or VN Ab titers within the first 2 weeks post-PEDV infection but there was a trend for higher mean titers in second trimester gilts (Figures S4A,B).

To standardize ASC and Ab responses among gilt treatment groups at uniform GDs, circulating PEDV IgA and IgG ASCs, Abs and VN Abs were compared at GD 82–95, 98–102, and 104–114 in first, second, and third trimester PEDV-infected gilts and third trimester mock gilts. Second trimester gilts had significantly higher circulating PEDV IgA and IgG ASCs at GD 82–95 and 98–102 and compared with first and third trimester gilts (Figures 5A,B). PEDV IgA ASCs were consistently higher than PEDV IgG ASCs in blood in first, second and third trimester gilts at GD 82–95, 98–102, and 104–114 (Figures 5A,B). Additionally, circulating PEDV IgA and IgG ASCs in first and second trimester gilts continually decreased in the 3 weeks prior to parturition, demonstrating parturition related or time post-infection effect on circulating PEDV IgA and IgG ASCs. This was not observed in third trimester gilts whose elevated numbers of PEDV IgA (numerically) and IgG (significantly) ASCs at GD 104–114 (Figures 5A,B) corresponded with PEDV RNA shedding in the feces at PID 6–8 and 12–17 (Figure 2A). Second trimester gilts had significantly higher serum PEDV IgA Ab titers compared with first trimester gilts at GD 82–95, 98–102, and 104–114 and third trimester gilts at GD 82–95 and 98–102 (Figure 5C). Serum PEDV IgG Ab titers were significantly higher in second compared with first trimester gilts at GD 82–95 and 104–114 and third trimester gilts at GD 82–95 and 98–102 (Figure 5D). Similarly, PEDV VN Ab titers were significantly higher in second compared with first trimester gilts at all time points but only significantly higher than third trimester gilts at GD 82–95 and 98–102 (Figure 5E).

Mean frequencies of circulating α4⁺β7⁺ (gut homing phenotype) B lymphocytes in second and third trimester gilts were significantly higher compared with first trimester gilts at PID 0 and 6–8 (Figure 6A). Additionally, first trimester gilts had a delayed increase in α4⁺β7⁺ B lymphocyte frequencies in blood post PEDV infection (Figure 6A). Frequencies of α4⁺β7⁺ B lymphocytes were standardized among gilt treatment groups at uniform GDs in late pregnancy (Figure 6B). Third trimester gilts had numerically elevated frequencies of circulating α4⁺β7⁺ B lymphocytes (Figure 6B) at GD 98–102 and 104–114 corresponding with PEDV RNA shedding in the feces at PID 6–8 and 12–17 (Figure 2A). The mean frequencies of circulating chemokine receptor type 10 (CCR10⁺) B lymphocytes were also measured post PEDV infection (Figure 6C) and standardized among gilt treatment groups at uniform GDs in late pregnancy (Figure 6D). No statistical differences among treatment groups were observed at PID 0, 6–8 or 12–17 (Figure 6C). However, third trimester gilts had significantly higher frequencies of circulating CCR10⁺ B lymphocytes at GD 98–102 (PID 6–8) compared with second and first trimester and mock gilts (Figure 6D). The increase in circulating CCR10⁺ B lymphocytes at GD 98–102 corresponded to peak PEDV RNA shedding titers at PID 6–8 (Figure 2A) in third trimester gilts. Additionally, significantly higher frequencies of activated B lymphocytes [CD2⁻CD21⁺ (39)] were observed in the blood of second and third compared with first trimester gilts at PID 0, 6–8, and 12–17 (Figure 6E).

To determine the effect of stage of gestation at time of PEDV infection on lactogenic immune protection, piglets were challenged with PEDV at 3–5 days of age. The number of viable piglets born were not statistically significant between treatment groups (Figure S5). Second trimester PEDV infection of gilts resulted in 100% survival of PEDV challenged piglets (Figure 7A). First trimester gilts provided intermediate protection (87.2% survival) while third trimester gilts provided the least amount of protection (55.9% survival) among PEDV-infected gilts. Mock piglet's survival rate (5.7%) was significantly lower than all other treatment groups. Comparison of piglet weight gain revealed second trimester litters gained significantly more weight than all other treatment groups starting at post challenge day (PCD) 4 and lasting throughout the experiment (Figure 7B). First trimester litters had significantly higher normalized weights than third trimester litters at PCD 6, 7, and 9, but were similar thereafter. Mock litters were stunted (decreased or no weight gain) from PCD 1–7 and had the lowest normalized weights throughout the study. Lactogenic immune protection coincided with decreased PEDV RNA shedding titers where mean peak titers were 6.1 ± 0.2 log₁₀ copies/ml, 7.8 ± 0.3 log₁₀ copies/ml, 9.1 ± 0.3 log₁₀ copies/ml, and 10.1 ±.2 log₁₀ copies/ml for second, first, third, and mock litters, respectively (Figure 7C). Corresponding to increased weight gain and decreased PEDV RNA shedding titers, diarrhea scores were significantly lower in second trimester litters throughout the study (Figure 7D). While first trimester litters had diarrhea, it was delayed and significantly lower than third trimester litters at PCD 2–5. Mock litters had diarrhea immediately at PCD 1, lasting through PCD 11 and diarrhea scores were significantly higher than all other treatment groups. Gilt mean PEDV RNA shedding titers and fecal consistency scores were measured post piglet challenge. Mock gilt PEDV RNA shedding titers were significantly higher at PCD 2–7 than first, second and third trimester PEDV-infected gilts and remained numerically higher until PCD 17 (Figure S6A). Additionally, first trimester gilt PEDV RNA shedding titers peaked at PCD 4, earlier compared with second and third trimester gilts (Figure S6A). Mock gilt fecal consistency scores were significantly higher at PCD 5–11 compared with first, second and third trimester PEDV-infected gilts (Figure S6B). Diarrhea was not observed in previously PEDV-infected gilts post piglet challenge (Figure S6B).

We evaluated the serum titers of piglet PEDV IgA and IgG Abs. Second trimester litters had significantly higher titers of circulating PEDV IgA Abs (Figure 8A) compared with first trimester litters at PCD 5–9 and PEDV IgG Abs at PCD 5–9 and 12–17 (Figure 8B). Additionally, compared with third trimester litters, second trimester litters had higher titers of PEDV IgA Abs at PCD 0 and 5–9 (Figure 8A) and PEDV IgG Abs at PCD 0 (Figure 8B). First trimester litters had significantly higher PEDV IgA and IgG Ab titers at PCD 0 compared with third trimester litters (Figures 8A,B). Lastly, circulating PEDV IgA and IgG Abs at PCD 0 were positively correlated with survival rates post piglet PEDV challenge (Figures 8C,D).

In colostrum, PEDV IgA and IgG ASCs were significantly higher in second compared with first and third trimester gilts (Figures 9A,B). However, in milk only PEDV IgA ASCs were significantly higher at postpartum day (PPD) 8–14/PCD 5–9 in second trimester gilts compared with all other treatment groups (Figure 9A). While PEDV IgA ASCs were maintained at high numbers in milk throughout lactation, milk PEDV IgG ASCs decreased significantly throughout the study (Figures 9A,B). Similar to ASCs, PEDV IgA and IgG Abs were significantly higher in the colostrum of second compared with first (IgA and IgG) and third trimester (IgA) gilts (Figures 9C,D). Additionally, PEDV VN Ab titers were significantly higher in colostrum in second compared with third trimester gilts (Figure 9E). After PEDV piglet challenge (PPD 8–14/PCD 5–9), PEDV IgA Ab titers in milk were significantly higher in second compared with third trimester gilts while PEDV IgG Ab titers were significantly higher in second compared with first trimester gilts (Figures 9C,D). Lastly, PEDV VN Ab titers were significantly higher in milk at PPD 8–14/PCD 5–9 compared with first and third trimester gilts (Figure 9E). We observed a significant correlation between PEDV IgA and IgG ASCs, and IgA and VN Abs in colostrum and piglet survival (Table 2). However, in mid and late lactation milk, only PEDV IgA ASCs, and IgA and VN Abs were significantly correlated with piglet survival. No significant (P < 0.05) correlations were observed between milk IgG ASCs or Abs and piglet survival (Table 2). These correlations are consistent with our hypothesis that IgA ASC and Ab titers in milk are responsible for lactogenic immune protection in neonatal piglets against PEDV challenge.

Third trimester gilts had lower mean numbers of PEDV Ab⁺ cells in the MG (2.7 ± 0.7) per microscopic field (30×) compared with second (6.1 ± 3.2) and first (6.9 ± 1.4) trimester gilts at GD 104–114 (Figure S7). There were no PEDV Ab⁺ cells in the MG of mock gilts prepartum (GD 104–114). This suggests that while not significantly different, the mean numbers of PEDV Ab⁺ cells in the MG may be reflective of levels of PEDV ASCs and Abs in colostrum. For example, third trimester gilts had the lowest mean number of PEDV Ab⁺ cells per microscopic field in the MG prepartum coinciding with the lowest mean numbers of IgA and IgG ASCs and mean titers of PEDV IgA and VN Abs in colostrum. PEDV Ab⁺ cells were also observed in the lactating MG at PCD 5–9. First, second and third trimester PEDV-infected gilts had significantly higher numbers of PEDV Ab⁺ cells per microscopic field in the MG compared with mock gilts post piglet challenge (Figure S7).

The highest mean numbers of PEDV IgA ASCs were in the ileum while the highest mean numbers of PEDV IgG ASCs were in spleen and ileum (Figures S8A,B). Second trimester gilt mean numbers of IgA and IgG ASCs were significantly higher in the ileum than third and first trimester gilts, respectively. However, mock gilts had similar or higher mean numbers of IgA and IgG ASC in the spleen, mesenteric lymph node (MLN), and ileum compared with first, second, and third trimester gilts (Figures S8A,B), corresponding to higher PEDV RNA shedding titers in feces and PEDV-induced diarrhea of mock gilts (Figures S6A,B). Due to the high mortality rate of mock litters (Figure 7A), the MGs of mock gilts regressed rapidly post piglet challenge (40). There was not enough MG tissue left at PCD 21–29 to collect MG MNCs for ASC analysis.

The wild-type PC22A strain of PEDV was used for gilt infection and piglet challenge at a dose of 1 × 10⁵ plaque forming units (PFU) diluted in Minimal Essential Media [MEM (Life Technologies, Carlsbad, CA)]. Briefly, PC22A was isolated and cultured in Vero cells as described previously (75, 76). Cells were grown in growth medium containing Dulbecco's Modified Eagle's Medium [DMEM (Life Technologies, Carlsbad, CA)] supplemented with 5% fetal bovine serum (Life Technologies, Carlsbad, CA) and 1% antibiotic-antimycotic (Life Technologies, Carlsbad, CA). Virus was grown in Vero cells in maintenance medium containing DMEM supplemented with 10 μg/ml trypsin (Life Technologies, Carlsbad, CA), 0.3% tryptose phosphate broth (Sigma Aldrich, St. Louis, MO), and 1% antibiotic-antimycotic. Cells were kept in a humidified incubator at 37°C and 5% CO₂. PC22A was passaged three times in Vero cells before passaging once for generation of inoculum in a gnotobiotic pig. The virulence of pig passaged PC22A was confirmed in adult and neonatal pigs (4, 77, 78). Cell-culture adapted PC22A was used as a positive control in the VN Ab assay.

All animal experiments were approved by the Institutional Animal Care and Use Committee at The Ohio State University. All pigs were maintained, sampled, and euthanized humanely. First parity PEDV, porcine deltacoronavirus (PDCoV) and TGEV seronegative pregnant gilts (Landrace × Yorkshire × Duroc cross-bred) were acquired from either Wilson Farms (Elkhorn, WI) or The Ohio State University swine center facility and randomly assigned to one of four treatment groups: PEDV-infected (1) first trimester (GD 19–22) (n = 10); (2) second trimester (GD 57–59) (n = 7); or (3) third trimester (GD 96–97) (n = 6) or MEM-infected third trimester control [GD 96–97 (n = 4)] (Figure 1A). Second and third trimester gilts arrived at our facilities at GD 49–51 and 89–92, respectively, and acclimated for 1 week prior to PEDV infection. First trimester gilts were housed and PEDV-infected at the National Animal Disease Center, USDA-ARS, in Ames, Iowa and then four gilts were shipped to our facilities at GD 93 after they were determined to be fecal shedding negative by real-time quantitative polymerase chain reaction (RT-qPCR). Gilt fecal samples were collected, and clinical signs observed on PID 0, 2, 4, 6, 10, and 14. Fecal consistency was scored as follows: 0, solid; 1, pasty; 2, semi-liquid; 3, liquid, respectively. A fecal consistency score of >1 was considered as diarrhea (4, 77). Blood samples were collected post-PEDV infection at PID 0, 6–8 and 12–17 and also prior to parturition at GD 82–95, 98–102, and 104–114 for serum and mononuclear cell (MNC) isolation. All gilts farrowed naturally in our facilities at GD 114 (± 3) and colostrum was collected within 12 h of parturition. Piglets were orally PEDV-challenged at 3–5 days of age (mean ± SD challenge day for first trimester litters = 4.0 ± 0.82, second trimester litters = 4.4 ± 0.96, third trimester litters = 4.2 ± 0.82 and mock litters = 3.7 ± 0.96). Gilt and piglet serum were collected on PCD 0, 5–9, 12–17, and 21–29. All colostrum and milk samples were collected after administration of 2cc oxytocin intramuscularly (IM) at PPD 0, 3–5, 8–14, and 15–22 (Figure 1B). Gilt MG biopsies were collected at GD 104–114 and PPD 8–14. Piglet fecal samples were collected and clinical signs and body weights were recorded daily on PCD 0–7 and every other day through PCD 15–17. All animals were euthanized at PCD 21–29. Upon euthanasia, gilt blood, ileum, MG, MLN, and spleen tissues were collected for MNC isolation. Piglet blood was collected and the serum separated for immunologic assays.

To determine PEDV RNA shedding titers, two rectal swabs were suspended in 4 mL MEM as described previously (78). Viral RNA was extracted from 50 μl of fecal supernatants following centrifugation (2,000 × g for 30 min at 4°C) using the MagMAX Viral RNA Isolation Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Titers of viral RNA shed in feces were determined by TaqMan RT-qPCR using the Onestep RT-PCR Kit (QIAGEN, Valencia, CA) (78). The detection limit was 10 copies per 20 μL of reaction, corresponding to 4.8 log₁₀ copies/mL of original fecal samples.

Blood, spleen, MLN, and ileum were collected aseptically at euthanasia and processed for MNC isolation as described previously (79). The isolated cells were resuspended in enriched RPMI [E-RPMI (Roswell Park Memorial Institute)] medium containing 8% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 20 mM HEPES (N-2-hydroxy- ethylpiperazine-N−2-ethanesulfonic acid), and 1% antibiotic-antimycotic (Life Technologies, Carlsbad, CA) and used for assays. The viability of MNCs was determined by trypan blue exclusion. Briefly, MNCs were diluted two-fold in 0.4% trypan blue before visualizing using an automated cell counter (Cellometer, Nexcelom, Lawrence, MA). The viability (%) was calculated as [1.00–(number of blue cells/numbers of total cells)] × 100.

The MG was collected aseptically at euthanasia and placed in ice-cold wash medium (RPMI 1640 with 10 mM HEPES, 200 g of gentamicin per ml, and 20 g of ampicillin per ml [Life Technologies, Carlsbad, CA]). Tissue was minced and pressed through stainless steel 80-mesh screens of a cell collector (Cellecter; E-C Apparatus Corp., St. Petersburg, FL.) to obtain single-cell suspensions and then pooled. A 90% Percoll solution (Sigma Aldrich, St. Louis, MO) was added to the MG cell suspensions and centrifuged at 1,200 × g for 30 min at 4°C. Cell pellets were resuspended in 43% Percoll, underlaid with 70% Percoll, and centrifuged at 1,200 × g for 30 min at 4°C. The MNC were collected from the 43-to-70% interface and washed with wash medium. Cells were filtered through a 70 μM pore filter and resuspended in E-RPMI. The viability of MNCs was determined by trypan blue exclusion.

Serum samples were processed and analyzed for proinflammatory (TNF-α, IL-6, IL-17, IL-22), innate (IFN-α) and Th1 (IL-12, IFN-γ), Th2 (IL-4), and T regulatory (IL-10 and TGF-β) cytokines as described previously with some modifications (80, 81). Briefly, Nunc Maxisorp 96-well plates were coated with anti-porcine IL-4 (2 μg/ml, clone A155B16F2), anti-porcine IL-10 (4 μg/ml, clone 945A4C437B1), anti-porcine IFN-γ (1.5 μg/ml, clone A151D5B8), anti-porcine TGF-β (1.5 μg/ml, clone 55B16F2) (Thermo Fisher Scientific, Waltham, MA), anti-porcine IL-6 (0.75 μg/ml, goat polyclonal Ab), anti-porcine IL-12 (0.75 μg/ml, goat polyclonal Ab), anti-porcine IFN-α (2.5 μg/ml, clone K9) (R&D systems, Minneapolis, MN), anti-porcine TNF-α (1.5 μg/ml, goat polyclonal Ab), anti-porcine IL-17 (1.5 μg/ml, rabbit polyclonal Ab), and anti-porcine IL-22 (1.5 μg/ml, rabbit polyclonal Ab) (Kingfisher Biotech, Saint Paul, MN) overnight at 37°C for IFN-α or 4°C for all other cytokines. Biotinylated anti-porcine IL-4 (0.5 μg/ml, clone A155B15C6), anti-porcine IL-10 (1 μg/ml, clone 945A1A926C2), anti-porcine IFN-γ (0.5 μg/ml, clone A151D13C5), anti-TGF-β (0.4 μg/ml, TGF-β-1 Multispecies Ab Pair CHC1683) (Thermo Fisher Scientific, Waltham, MA), anti-porcine IL-6 (0.1 μg/ml, goat polyclonal IgG), anti-porcine IL-12 (0.2 μg/ml, goat polyclonal IgG), anti-porcine IFN-α (3.75 μg/ml, clone F17) (R&D systems, Minneapolis, MN), anti-porcine TNF-α (0.4 μg/ml, goat polyclonal Ab), anti-porcine IL-17 (1 μg/ml, rabbit polyclonal Ab) or anti-porcine IL-22 (1 μg/ml, rabbit polyclonal Ab) (Kingfisher Biotech, Saint Paul, MN) were used for detection. Porcine IFN-α detection Ab was biotinylated using a commercial kit as described previously (81). Plates were developed and cytokine concentrations were calculated as described previously (80). Sensitivities for these cytokine ELISA assays were 1 pg/ml for IL-4, IL-12, and IFN-α, 4 pg/ml for TNF-α, IL-17, and IL-22, 8 pg/ml for TGF-β, and 16 pg/ml for IL-6, IL-10, and IFN-γ.

Colostrum/milk was collected aseptically after gilts were given 2cc oxytocin (VetOne, Boise, ID) IM to facilitate collection of mammary secretions. Colostrum/milk was placed immediately on ice. Samples were filtered through a 70 μM pore filter and centrifuged at 1,800 × g for 30 min at 4°C to separate fat, skim milk, and cell pellet portions. Fat was removed utilizing sterile plain-tipped applicators (Fisher Scientific, Hampton, NH). Skim milk was collected, centrifuged at 28,000 × g for 1 h at 4°C to separate the whey that was then stored at −20°C until tested. The cell pellet portion was resuspended in a 90% Percoll solution and centrifuged at 1,200 × g for 30 min at 4°C. Cell pellets were resuspended in 43% Percoll, underlaid with 70% Percoll, and centrifuged at 1,200 × g for 30 min at 4°C. The MNC were collected from the 43-to-70% interface and washed with wash medium. Cells were filtered through a 70 μM pore filter and resuspended in E-RPMI. The viability of the MNC preparation was determined by trypan blue exclusion.

Blood MNC and K562 cells were used as effector and target cells, respectively. Effector: target cell ratios of 10:1, 5:1, and 1:1 were used and the assay was done as described previously (82).

To determine the frequencies of NK cells, CD3⁻, CD172⁻, CD8⁺ MNCs were identified. Briefly, 100 μl of peripheral blood mononuclear cells (PBMCs) at 1 × 10⁷ cells/ml were stained with anti-porcine CD3 (clone PPT3, Southern Biotech), anti-porcine SWC3a [CD172 human analog (clone 74-22-15, Southern Biotech)] and anti-porcine CD8 (clone 76-2-11, Southern Biotech) monoclonal Abs (mAbs) for 15 min at 4°C. Subsequently cells were washed and incubated with streptavidin APC (BD Biosciences, San Jose, CA, USA) secondary Ab. Appropriate isotype matched control Abs were included. Acquisition of 50,000 events and analyses were done using the Accuri C6 flow cytometer (BD Biosciences). The gating strategy is depicted in Figure S1.

A plaque reduction VN assay was performed as described previously (83) with modifications. Serum and whey were inactivated at 56°C for 30 min prior to testing for PEDV neutralizing Abs. Serial 4-fold dilutions of test sera or whey were mixed with 70 PFU and 140 PFU PEDV PC22A cell-culture adapted strain, respectively. The serum/whey-virus mixtures (290 μl/well) were incubated for 1.5 h at 37°C with gentle rocking and then duplicate samples were infected onto 2–3 day confluent Vero cell monolayers in 6-well plates. Plates were incubated at 56°C for 1 h, lightly rocking every 15 min. Subsequently, the inoculum was removed, cells were washed twice with sterile PBS [1X pH 7.4 (Sigma Aldrich, St. Louis, MO)] and overlaid with 0.75% low melting point agarose (SeaPlaque, Lonza, Riverside, PA) in serum free media supplemented with tryptose phosphate broth and trypsin as described for PEDV cultivation (75). Plates were incubated at 37°C for 3 days and then stained with 0.001% neutral red solution (Sigma Aldrich, St. Louis, MO). Plaques were counted and the VN titers were determined by calculating the reciprocal of the highest dilution of a serum/whey sample showing an 80% reduction in the number of plaques compared with seronegative control serum/whey.

Cell culture adapted PC22A was propagated on 2–3-day-old Vero cells in polystyrene roller bottles (Fischer Scientific, Hampton, NH) until the cells demonstrated 90–95% cytopathic effects (CPE). Roller bottles were subjected to two freeze-thaw cycles at −80°C before collecting the supernatant and centrifuging at 3,000 × g for 10 min at 4°C to remove Vero cell debris. The supernatant was overlayed onto 3 ml sucrose [35% in TNC buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 10 mM CaCl₂, 0.02% NaN₃)] and centrifuged at 112,700 × g for 2 h at 4°C. The viral pellet was resuspended in TNC buffer, centrifuged at 107,200 × g for 2 h at 4°C and washed twice with sterile PBS (1X pH 7.4). Then the viral pellet was inactivated by using binary ethylenimine (BEI) as previously described (84). The semipurified inactivated virus was resuspended in PBS (1X pH 7.4) at a dilution of 1:3 of the original supernatant volume and stored at −80°C. At the time of testing, PEDV or mock antigen solution was further diluted 1:4 and added (60 μl per well) to uncoated polystyrene plates (Fisher Scientific, Hampton, NH) in alternating wells. Plates were incubated for 4 h at 37°C or overnight at 4°C. Plates were washed 5X with PBS-T wash solution (PBS 1X, 0.1% Tween-20 [Fisher Scientific, Hampton NH], pH 7.4). General Block ELISA blocking solution (Immunochemistry Technologies, Bloomington, MN) was added (200 μl per well) and plates were stored at 4°C overnight. After incubation, plates were washed twice with PBS-T and samples were added. All samples were serially diluted starting at 1:4 and added (50 μl per well) in duplicate to coated plates. Positive and negative controls (Ab-positive and -negative experimental samples) were included per plate for each sample type. Plates were incubated at room temperature (RT) for 1.5 h and washed 5X with PBS-T. Primary Abs were added (100 μl per well) diluted in PBS-T to detect IgA Ab [peroxidase-conjugated goat anti-pig IgA (Bio-Rad, Hercules, CA)] and IgG [biotin-conjugated goat anti-pig IgG (Seracare, Milford, MA)] in serum (1:4,000 and 1:20,000, respectively) and milk/intestinal samples (1:20,000). IgA plates were incubated at RT for 1.5 h and IgG at 37°C for 1 hr. For IgG plates, peroxidase-conjugated streptavidin [1:10,000 (Roche, Basel, Switzerland)] was added (100 μl per well) and incubated at RT for 1 h. For both IgA and IgG, plates were washed twice with PBS-T and the reaction was visualized for all plates by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate with H₂O₂ membrane peroxidase substrate system (Seracare, Milford, MA). Plates were incubated at RT for 5 min and stopped by addition of 100 μl stop solution (1 M sulfuric acid) to each well. Reactions were measured as optical density at 450 nm using an ELISA plate reader (Spectramax 340pc, Molecular Devices, San Jose, CA). The ELISA Ab titer was expressed as the reciprocal of the highest dilution that had a corrected A₄₅₀ value (sample absorbance in the virus-coated well minus sample absorbance in the mock antigen-coated well) greater than the cut-off value (mean corrected A₄₅₀ value of negative controls plus 3 standard deviations). Samples negative at a dilution of 1:4 were assigned a titer of 1:2 for the calculation of geometric mean titers (GMTs).

To detect PEDV ASCs, PEDV PC22A, and mock-infected, acetone-fixed Vero cells in 96-well plates were used similar to methods used to detect TGEV ASC (85, 86). Briefly, after infected Vero cells (at 95–100% confluency) showed 90–95% CPE, cells were fixed with 80% acetone for 15 min, allowed to dry for 1–2 h and stored at −20°C. Mock-infected cell monolayers served as negative controls. Plates were thawed and rehydrated in sterile PBS for 15 min at RT. Serial dilutions of MNCs (5 × 10⁵, 5 × 10⁴, 5 × 10³, and 5 × 10²) were added to duplicate wells (100 μl per well) of fixed PEDV PC22A and mock-infected cell monolayers. Plates were centrifuged at 120 × g, for 5 min at RT and incubated at 37°C with 5% CO₂ for 12–14 h. After incubation, the plates were washed 5X with PBS-T. ASCs were detected by incubating plates with HRP-conjugated anti-pig IgA [1:4,000 (Bio-Rad, Hercules)] or biotinylated anti-pig IgG [1:20,000 (Seracare, Milford, MA)]. All Abs were added at 100 μl per well. Plates incubated with Abs to IgA were incubated at RT for 1.5 h and for Abs to IgG at 37°C for 1 h. For IgG plates, peroxidase-conjugated streptavidin [1:10,000 (Roche, Basel, Switzerland)] was added (100 μl per well) and incubated at RT for 1 h. The reaction was visualized for all plates by adding 100 μl TMB substrate with H₂O₂ membrane peroxidase substrate system (Seracare, Milford, MA). Spots were detected and counted using a light microscope. Counts were averaged from duplicate wells and expressed relative to 5 × 10⁵ MNC.

To evaluate PEDV Ab⁺ cells in the MG, 5 cm biopsies were collected using a 12 gauge × 10 cm semi-automatic needle (Bard Peripheral Vascular, Tempe, AZ) and fixed in 10% neutral buffered formalin. Sections were trimmed, processed, and embedded in paraffin. Sections were cut (3.5 μm thick) and processed for antigen retrieval (0.05% pronase E treatment [Sigma Aldrich, St. Louis, MO]). For detection of PEDV Ab⁺ cells, a PEDV viral suspension sandwich immunohistochemistry (IHC) method was developed. Semipurified inactivated PEDV antigen, as described previously for PEDV Ab ELISA plates, was added to tissue sections and incubated overnight at 4°C. A mAb to the N protein of a highly virulent US PEDV strain [PC22A-like (PEDV 72-111-25 IgM G-14, kindly provided by Steven Lawson and Eric Nelson, Department of Veterinary and Biomedical Sciences, South Dakota State University)] was added to tissue sections and incubated overnight at 4°C. Supersensitive Polymer-HRP IHC Detection System (Biogenex, Fremont, CA) was used as a secondary Ab and substrate prior to hematoxylin and eosin (H&E) staining. Microscopic imagines (30 × magnification) were obtained using a fluorescence microscope (Olympus IX70-S1F2). Mean numbers of PEDV Ab⁺ cells were evaluated by measuring at least 3 different microscopic fields at (30 × magnification) for each sample time point (GD 104–114 and PCD 5–9) from first, second and third PEDV-infected or mock gilts.

Procedures for flow cytometry staining (including buffers used) were performed as described previously with minor modifications (87). Briefly, 100 μl of MNCs at 1 × 10⁷ cells/ml were stained with anti-porcine CD21-PE (clone BB6-11C9.6, Southern Biotech) and anti-porcine CD2 (clone MSA4, VMRD) monoclonal Abs (mAbs) to determine B cell subsets (88). To determine expression of α4 integrin, β7 integrin and CCR10, cells were stained with porcine cross-reactive anti-human α4 integrin (clone HP2/1, Abcam, Cambridge, MA), anti-mouse β7 integrin (clone FIB27, BD Biosciences), and anti-mouse CCR10 (clone 248918, R&D Systems) mAbs. Additionally, to determine expression of IgA, cells were stained with anti-porcine IgA (clone K61 1B4, Bio-Rad) mAb. After washing, cells were stained with appropriate secondary antibodies. For intracellular CD79β staining, stained cells were permeabilized with Cytofix/Cytoperm (BD Biosciences), washed with Perm/Wash Buffer (BD Biosciences), and stained with porcine cross-reactive anti-mouse CD79β-FITC Ab (clone AT1072, Bio-Rad) mAb. Additionally, CD4⁺ (anti-porcine CD4, clone 74-12-4, Southern Biotech) and CD8⁺ (anti-porcine CD8, clone 76-2-11, Southern Biotech) T cells were assessed within the CD3⁺ (anti-porcine CD3, clone PPT3, Southern Biotech) MNC population (T lymphocytes). Appropriate isotype matched control antibodies were included. Acquisition of 50,000 events and analyses were done using the Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The gating strategy for T and B cell phenotypes are depicted in Figures S1, S2.

PEDV RNA shedding titers, fecal consistency scores, normalized weights, frequencies of blood MNC populations in flow cytometry, mean concentrations of serum cytokines, PEDV IgA and IgG ASCs, log-transformed PEDV IgA, IgG and VN Ab titers and PEDV Ab⁺ cells in the MG were analyzed by a two-way analysis of variance (ANOVA-general linear model), followed by Bonferroni's posttest. NK cell activity in blood and PEDV IgA and IgG ASCs in MG, spleen, MLN, and ileum tissues were compared among groups with the Mann-Whitney (non-parametric) test. The log-rank (Mantel-Cox) test was used for comparison of survival curves amongst treatment groups. Statistical significance was assessed at P ≤ 0.05 for all comparisons. All statistical analyses were performed with GraphPad Prism 5 (GraphPad Software, Inc., CA).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.