Soy isoflavones and viral infection in weanling pigs

Transcription

1 Final Report Soy isoflavones and viral infection in weanling pigs Project # Submitted by: Dr. Ryan N. Dilger, Associate Professor, PI Department of Animal Science, University of Illinois Phone: (217) rdilger2@illinois.edu Brooke N. Smith, PhD student Department of Animal Science, University of Illinois bnmurph2@illinois.edu To: United Soybean Board - and - Hamlet Protein January 17, 2017

2 Summary An experiment was conducted to determine the effects of supplemental dietary soy isoflavones (ISF) on the growth performance and immune response of pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Results from a previous trial conducted at the University of Illinois indicated that an increased dietary SBM inclusion resulted in improved growth and immune performance of pigs during a PRRSV infection. Therefore, the current trial was designed to attempt to identify a potential mode of action for these benefits, specifically the ability for soy-derived isoflavones to confer immunological benefits to young pigs infected with PRRSV. Sixty weanling pigs were fed diets containing either soy protein concentrate (Arcon AF; ADM, Decatur, IL) or a further processed soy ingredient (HP300; Hamlet Protein, Findlay, OH) as a protein source without or with supplemental soy isoflavones (Novasoy 400; ADM; Decatur, IL). Following a 1- week adjustment period on their experimental diets, pigs received either a sham inoculation or a % tissue culture infective dose of PRRSV at 28 d of age. Growth performance was recorded weekly, and blood was collected at 0, 3, 6, and 14 days post-inoculation (DPI) for determination of differential complete blood cell counts, serum PRRSV load, serum anti-prrsv antibodies, and serum cytokine concentrations. Additional blood was collected on 8 and 12 DPI for cytokine recall analysis and immunophenotyping, respectively. As evidence of a successful PRRSV inoculation, uninfected pigs exhibited greater average daily gain (ADG) and feed efficiency (i.e., gain:feed, G:F) throughout the infective period (0-14 DPI), while differences in average daily feed intake (ADFI) were not as consistently evident between uninfected and infected groups. There were no differences in any growth parameters between PRRSV-infected pigs receiving different soy protein sources or supplemental ISF. With currently available data, it appears that the immune response to PRRSV infection in pigs fed supplemental ISF was unaffected compared with non-isf-supplemented groups, with the exception of serum viral load at 3 and 6 DPI. Materials and Methods Animal Husbandry and Allotment Sixty weanling pigs (60 barrows; 5.71 ± 0.44 kg) were obtained from Pinnacle Genetics, LLC (Carthage, IL) and housed in disease containment chambers in Edward R. Madigan Laboratory at the University of Illinois (Urbana, IL). A single hallway was used, with access to 8 containment chambers each housing 4 individual pigs, and the total number of pigs was split into two separate cohorts (n=28 and 32 in corhorts 1 and 2, respectively) that were conducted in successive months. Each chamber (3.34 m 2 ) was divided into 4 individual pens (0.84 m 2 ) and was equipped with 1 nipple waterer and 1 feeder. Experimental diets that met or exceeded NRC (2012) nutrient requirements for weanling pigs were provided beginning at the time of allotment using pigs at approximately 21 days of age. One intramuscular injection of enrofloxacin (7.5 mg/kg BW; Baytril 100; Bayer, Shawnee Mission, KS) was administered on the day they arrived as a precautionary measure against bacterial infections during transition of the pigs to the new rearing environment. Pigs were provided their assigned experimental diet and allowed to adjust to housing conditions for 7 d prior to initiating inoculation procedures described below. Lights were maintained on a 12 h light cycle throughout the study, with light provided from 0600 to 1800 h, and temperature was maintained at approximately 26ºC. Pigs were weighed upon arrival for allotment into 5 experimental treatment groups. Pigs were allocated so that body weights were similar within each individual chamber across all treatment groups. Furthermore, litter of origin was taken into account, and pigs from each litter were stratified across treatment groups as

3 evenly as possible. This allotment resulted in 12 pigs for each treatment group, with each chamber having 1 pig per dietary treatment (1 replicate of each dietary treatment per chamber). Experimental Treatments Five experimental treatments were used in this study, with 4 different diets and 2 states of infection. A 2 2 factorial arrangement of dietary soy protein sources (Arcon AF vs HP300) and supplemental isoflavones (none vs. Novasoy400) constituted the total of dietary treatments (Table 1). The control diet contained soy protein concentrate (Arcon AF, Decatur, IL) as a protein source with no addition of soy isoflavones, and this diet was fed to both sham-inoculated and PRRSV-infected groups. Isoflavone and saponin concentrations of ingredients and experimental diets were quantified at the USDA-ARS National Center for Agricultural Utilization Research (Peoria, IL; courtesy of Dr. Mark Berhow), and amino acid concentrations were determined at the University of Missouri Agricultural Experiment Station (Columbia, MO; Table 2). Diets were formulated on a standardized ileal digestible (SID) amino acid basis, with identical concentrations across all diets (Table 3). Experimental diets were isocaloric, and with the exceptions of corn and protein source, identical in ingredient composition. All diets met or exceeded NRC (2012) nutrient requirements for weanling pigs, and analyzed dietary concentrations are presented in Table 4. Following a 1-week adaptation period to experimental diets, blood was collected from each pig to establish 0 DPI baseline measurements and to ensure that all pigs were PRRSV-free at study initiation. Immediately following blood collection, pigs were intranasally inoculated with either 2 ml of a 2% fetal bovine serum + phosphate-buffered saline solution (sham-control) or % tissue culture infective dose of PRRS virus (strain NADC20, Federico Zuckermann, University of Illinois, Urbana, IL) Response Variables Growth Performance and Rectal Temperatures Individual pig and feeder weights were recorded weekly throughout the trial to allow for calculation of average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (gain to feed, G:F). Growth performance data are reported in reference to the inoculation schedule (-7 to 14 DPI). Rectal temperatures of all pigs were measured on 0, 3, 6, 8, 12, and 14 DPI using a digital thermometer. Plasma and serum analyses Blood was collected from each pig at 0, 3, 6, 8, 12, and 14 DPI. Serum was analyzed for PRRSV load at 0, 3, 6, and 14 DPI by RT-PCR, and differential complete blood counts of plasma were determined at those same time-points. Serum concentrations of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-1 beta (IL-1β), and anti-inflammatory cytokine interleukin-10 (IL- 10) will be measured at 0, 3, 6, and 14 DPI with the Swine Cytokine Magnetic 7-Plex Panel according to manufacturer instructions (Novex by Life Technologies, Frederick, MD). Cytokine recall analysis performed with cultured peripheral blood mononuclear cells (PBMC) from blood collected on 8 DPI will also be analyzed using the 7-Plex Panel. Blood from 12 DPI was used to perform T cell immunophenotyping via flow cytometry.

4 Statistical Analysis Statistical analysis was dependent on whether outcomes were measured at a single time-point or at multiple time-points for the same subject. In all cases, individual pig was considered the experimental unit, with 12 replicate pigs for each of the 5 experimental treatments. A total of 4 different diets were fed to PRRSVinfected pigs, while a single group of uninfected pigs received only the control diet. Thus, the 2- or 3-way analyses of variance described below involve effects between diets and within PRRSV-infected pigs, while a separate analysis was used to compare uninfected and infected groups receiving the control diet. For all single time-point outcomes, a 2-way ANOVA was conducted using the Mixed procedure of SAS 9.3 (SAS Institute, Inc., Cary, NC) with factors including the dietary soy source (soy protein concentrate vs. HP300) and addition of supplemental soy isoflavones (no vs. yes) with the following statistical model: Yijk = µ + αi + βj + αβij + γk +εijk where μ = the general mean, αi = the fixed effect of soy source, βj = the fixed effect of isoflavone supplementation, αβij = the interaction between soy source and isoflavone supplementation, γk = the random effect of cohort of pigs, and εijk = experimental error. A repeated measures, 3-way ANOVA was conducted for all outcomes involving samples collected from the same subject at multiple time-points with the following statistical model: Yijkl = µ + αi + βj + αβij + δk + αβδijk + γl + εijkl where μ = the general mean, αi = the fixed effect of soy source, βj = the fixed effect of isoflavone supplementation, αβij = the interaction between soy source and isoflavone supplementation, δk = the fixed effect of DPI, αβδijk = the 3-way interactive effect of soy source, isoflavone supplementation, and DPI, γl = the random effect of cohort of pigs, and εijkl = experimental error. No 2-way interactive effects involving DPI were significant, so they were removed from the model to preserve degrees of freedom. Comparison of uninfected and infected groups each fed the control diet was made using a single degree of freedom contrast between these experimental treatments. In all cases, statistical significance was considered at P 0.05, with trends accepted at 0.05 < P < Results Growth Performance Growth performance results are presented in Table 5. Within the PRRSV-infected groups, no main or interactive effects were noted during the pre-inoculation period (P > 0.05; -7 to 0 DPI). However, significant decreases (P < 0.01) in ADG were observed between uninfected and infected groups fed the control diet throughout the post-inoculation period, thereby signifying a successful PRRSV infection. During the infective period, there were main effects of isoflavone supplementation on G:F between 6 and 14 DPI and main effects of soy source on ADG and G:F over the entire 2-week infection period (P < 0.05; 0 to 14 DPI). From 6 to 14 DPI, pigs receiving the HP300+ISF diet experienced less efficient growth compared to pigs receiving only HP300 (P < 0.05). Over the 2-week infection period, pigs receiving the HP300 diet maintained a higher ADG compared to pigs fed the Control diet, regardless of isoflavone supplementation (P < 0.05). Additionally, pigs receiving

5 HP300, regardless of isoflavone supplementation, had greater feed efficiency compared to pigs receiving the Control+ISF diet over the 2-week infection period (P < 0.05). Overall, PRRSV-infection reduced ADG and G:F during the 2-week infection period (0 to 14 DPI) resulting in a lower final body weight (P < ) for PRRSV-infected pigs compared to uninfected pigs fed the control diet. Rectal Temperatures Rectal temperature data were collected frequently over the entire infection period (0 to 14 DPI; Figure 1 and Appendix A). Infection with PRRSV increased (P < 0.05) rectal temperatures of pigs at 0, 3, and 8 DPI, but there was no influence (P > 0.05) of dietary soy protein source or ISF supplementation on this outcome. Serum Viral Load Analysis of serum by RT-PCR indicated that all pigs were PRRSV-negative at 0 DPI (Table 6). At 3, 6, and 14 DPI, all pigs in the uninfected group remained free of PRRSV, while all pigs inoculated with PRRSV tested positive for PRRSV DNA. Among the PRRSV-infected group, cycle threshold (Ct) values, the number PCR cycles required to detect presence the viral genome, was lower (i.e., more viral genome present, P < 0.05) in pigs fed the HP300+ISF diet compared with pigs in all other groups at 3 DPI. Pigs fed the HP300+ISF diet also had lower (P < 0.05) Ct values compared with pigs fed the unsupplemented HP300 diet at 6 DPI. T Cell Population Immunophenotyping Analysis of PBMC using flow cytometry indicated that PRRSV-infection increased (P < 0.05) the relative proportion of cytotoxic T cells (i.e., positive for the CD3 and CD8 cell-surface markers) in the total lymphocyte population, when compared to uninfected pigs receiving the control diet (Table 7). Relative proportions of total T cells (i.e., positive for the CD3 cell-surface marker) and helper T cells (i.e., positive for the CD3 and CD4 cell-surface markers) did not differ between uninfected and infected animals. However, PRRSV-infection increased the proportion of memory T cells (i.e., positive for the CD3, CD4 and CD8 cellsurface markers), but showed a reduction (P < 0.05) in the percentage of cells functioning as effector memory T cells (i.e., memory T cells also expressing intracellular maker interferon-gamma). Among PRRSV-infected pigs, there was a main effect (P = 0.011) of isoflavone supplementation on the relative proportion of helper T cell populations, with ISF supplementation increasing the proportion of helper T cells. PRRSV-infected pigs receiving the HP300+ISF diet maintained the largest proportion of helper T cells (26.4%), which was higher (P < 0.05) than pigs receiving the control or HP300 diets without supplemental isoflavones. Pigs receiving the control+isf diet maintained the second largest proportion (23.1%), which was not different from other treatment groups. There were no main effects for isoflavone supplementation on any other T cell population parameter. Across all parameters, there were no main effects of soy protein source or interaction between soy source and isoflavone supplementation. Red Blood Cell Measurements Whole blood was submitted to the University of Illinois College of Veterinary Medicine Clinical Pathology Lab for total and differential blood cell counts. Time-dependent effects were observed for all outcomes, and main effects (P < 0.01) for ISF supplementation were observed for red blood cell (RBC) counts, hemoglobin (HGB, g/dl), and hematocrit (HCT, %), regardless of study time-point (Table 8). No main effect of soy protein source or interaction between soy source and isoflavone supplementation were observed for those

6 same parameters. PRRSV-infected pigs receiving the control diet had lower (P < 0.05) hemoglobin concentrations and hematocrit ratios compared to uninfected pigs at 14 DPI, but no other time-points were affected. Main effects (P 0.05) for soy protein source were observed for mean cell volume (MCV, fl) and mean corpuscular hemoglobin (MCH, pg). No main effect of ISF supplementation or interaction between soy source and ISF supplementation were observed for those same parameters. PRRSV-infected pigs receiving the control diet had lower (P < 0.05) MCH measurements compared with uninfected pigs at 14 DPI, but this was not observed at any other time-points. Additionally, PRRSV-infected pigs receiving the HP300+ISF diet had lower (P < 0.05) MCHC compared with infected pigs receiving the control diet at 3 DPI, but not when compared to the other two dietary treatments. Leukocytes Counts Whole blood was submitted to the University of Illinois College of Veterinary Medicine Clinical Pathology Lab for total and differential blood cell counts. The only cell population for which there were significant main effects (P < 0.05) of soy source and ISF supplementation on total white blood cell (WBC) count were lymphocytes (Table 9). However, no interaction between soy source and ISF supplementation was observed. PRRSV-infected pigs receiving the control diet had lower (P < 0.05) total WBC counts on 3, 6, and 14 DPI compared with uninfected pigs. In regards to relative proportions of individual WBC populations, there was a main effect of ISF supplementation for neutrophil and lymphocyte populations (P < 0.05) (Table 9). There was a main effect of soy protein source only for relative proportion of eosinophil populations (P < 0.05). PRRSV-infected pigs receiving the control diet maintained higher (P < 0.05) relative populations of neutrophils at 6 and 14 DPI, naïve neutrophils (aka. band cells) on 6 DPI, and monocyte populations on 0 DPI as compared with uninfected pigs. These same pigs, however, maintained lower (P < 0.05) relative populations of lymphocytes on 3,6, and 14 DPI compared to uninfected controls. Histological Scoring of Lung Tissue Histological interpretation and scoring of lung tissue for both cohorts was not possible due to concurrent and confirmed Mycoplasma hyopneumoniae infection in pigs enrolled in cohort 2 of the study. Tissue disruption associated with M. hyopneumoniae infection was severe and obscured quantification of PRRSV-associated lesions present in lung tissue. For these reasons, outcomes from histological samples have been omitted from this report. Serum Cytokine Concentrations Serum cytokine concentrations, quantified using the Swine Cytokine Magnetic 7-Plex Panel (Novex, Life Technologies, Frederick, MD), are presented in (Table 10). Results of peripheral cytokine analysis showed a main effect of isoflavone supplementation for interleukin-1β (IL-1β) concentration (P < 0.05). Pigs receiving diets supplemented with isoflavones had detectable concentrations of IL-1β at 6 DPI, with a greater than 5-fold increase by 14 DPI for the Control+ISF group (335 pg/ml to 2,449 pg/ml) and a greater than 30-fold increase by 14 DPI for the HP300+ISF group (347 pg/ml to 12,790 pg/ml). There were no main effects of soy source or interaction between soy source and isoflavone supplementation at any time point for any cytokine analyzed.

7 PRRSV-infected pigs had higher circulating concentrations of IFNα and TNFα 3 and 4 DPI, respectively, compared with uninfected controls. No other cytokines or time-points were influenced by infection status. PRRSV-infected pigs receiving the control diet had higher (P < 0.05) circulating IFNγ concentrations compared to other PRRSV-infected treatments at 3 DPI, but not at any othertime-points. PRRSV-infected pigs receiving supplemental ISF maintained detectable IL-10 concentrations at 14 DPI while non-supplemented groups did not, with the HP300+ISF group maintaining concentrations over 4-times higher compared with pigs receiving the Control+ISF diet (154 pg/ml vs pg/ml, respectively). For circulating IFNα concentrations, PRRSV-infected pigs exhibited the lowest (P < 0.05) concentrations at 3 DPI compared to all other infected treatments, with the HP300-fed groups maintaining higher (P < 0.05) concentrations compared with pigs fed the control diet. Cytokine Recall Analysis Samples were analyzed using the Swine Cytokine Magnetic 7-Plex Panel (Novex by Life Technologies, Frederick, MD). Results for the first composite of cytokine recall samples showed 90% of samples reporting below the detectable range of the 7-plex panel. This suggests that no or little stimulation by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) in vitro was achieved by this model. Based on these results, further analysis for the remaining cytokine recall samples was discontinued. Missing Data Points Appendix B contains information about missing data points associated with this study. This table includes metadata, which outcomes were affected, and reasoning for absence of data in the final dataset. Conclusions A primary aim of this trial was to determine whether increased dietary soy isoflavones would support the immune response of PRRSV-infected pigs over those that were not supplemented. Our results suggest that in a carefully-controlled experimental setting, there were minimal effects of soy protein source and ISF on growth performance or immunological outcomes in PRRSV-infected pigs. ISF supplementation during 6 to 14 DPI reduced individual efficiency (G:F), but this was not maintained over the entire course of infection. Over the entire infection period, pigs receiving HP300 as a soy protein source did maintain higher ADG than those fed the Control basal diet, regardless of ISF supplementation, and were more efficient (G:F) than pigs receiving the Control+ISF diet. Contrary to our hypothesis, dietary ISF concentration had no influence on clinical (e.g., serum PRRSV load, rectal temperatures) or immunological (e.g., T cell phenotypes, with the exception of helper T cells) outcomes in PRRSV-infected pigs. Among PRRSV-infected groups, it does appear that pigs fed the HP300 + ISF diet maintained a higher serum viral load at 3 DPI compared with other experimental diets, and while it maintained this higher serum viral load compared to pigs fed the HP300 diet at 6 DPI, this difference diminished as the infective period progressed. Additionally, isoflavone supplementation increased peripheral circulating of pro-inflammatory cytokine IL-1β in PRRSV-infected pigs on 6 and 14 DPI, but this effect was not observed in other cytokines analyzed. Though PRRSV-infection does have an influence on the proportion of peripheral T cell populations and certain subpopulations as well as some inflammatory cytokine concentrations, we observed no influence of dietary ISF concentration between PRRSV-infected groups.

8 Table 1. Experimental treatments 1 Treatment Dietary treatment Infection status Control Soy protein concentrate Uninfected Control Soy protein concentrate PRRSV-infected Control + ISF Soy protein concentrate + ISF PRRSV-infected HP300 HP300 PRRSV-infected HP300 + ISF HP300 + ISF PRRSV-infected 1 Abbreviations: ISF, soy isoflavones; PRRSV, porcine reproductive and respiratory syndrome virus.

9 Table 2. Analyzed isoflavone, saponin, and amino acid concentrations of experimental ingredients (as-fed basis) Ingredient Item Arcon AF 1 HP300 2 Novasoy 3 Isoflavones, mg/kg Total genistein ,317 Total daidzein , ,902 Total glycitein ,983 Total isoflavones , ,203 Total saponins, mg/kg 1,313 3,656 - Total amino acids, % Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine Arcon AF is a soy protein concentrate (referred as soy protein concentrate in subsequent tables) manufactured by traditional process to remove soluble sugars and reduce anti-nutritional factors; ADM Foods & Wellness, Decatur, IL. 2 HP300 is a gentle soya-yeast (10% yeast components) supplement for piglet feed with a low content of anti-nutritional factors (trypsin inhibitors, antigens, and flatulent oligosaccharides); Hamlet Protein, Findlay, OH. 3 Novasoy 400 Soy Isoflavone Concentrate has a guaranteed 40% minimum total isoflavone concentration (referred to as suppl. ISF in subsequent tables); ADM, Decatur, IL.

10 Table 3. Ingredient and calculated composition of experimental diets (as-fed basis) 1 Soy source Soy protein concentrate HP300 Item Suppl. ISF No Yes No Yes Ingredient, % Corn HP Arcon AF Dried whey Poultry by-product meal Choice white grease Ground limestone Monocalcium phosphate Sodium chloride Vitamin and mineral premix Choline chloride L-Lys HCl DL-Met L-Trp L-Thr L-Val Novasoy Calculated composition ME, kcal/kg 4 3,472 3,458 3,465 3,453 CP, % SID amino acids, % 4 Lys Met + Cys Trp Thr Val Isoflavones, mg/kg Total geinistein Total daidzein Total glycitin Total isoflavones 3 1, ,500 Total saponins, mg/kg 664 1, ,128 1 All pigs received allotted treatment diet upon starting -7 DPI. Abbreviations: DPI, day postinoculation; ISF, isoflavones; SID, standardized ileal digestible. 2 Low ash pet-food-grade poultry by-product meal, American Proteins, Inc., Hanceville, AL 3 Vitamin-mineral premix (JBS United, Sheradin, IN) included the following per kilogram of complete diet: Vitamin A (retinyl acetate), 11,128 IU; Vitamin D3 (cholecalciferol), 2,204 IU; Vitamin E (dl-α tocopheryl acetate), 66 IU; Vitamin K (menadione nicotinamide bisulfite), 1.42 mg; Thiamine (thiamine mononitrate), 0.24 mg; Riboflavin, 6.58 mg; Pyridoxine (pyridoxine hydrochloride), 0.24 mg; Vitamin B12, 0.03 mg; d-pantothenic acid (d-calcium pantothenate), 23.5 mg; Niacin (nicotinamide and nicotinic acid), 44 mg; Folic acid, 1.58 mg; Biotin, 0.44 mg; Cu (copper sulfate), 10 mg; Fe (iron sulfate), 125 mg; I (potassium iodate), 1.26 mg; Mn (manganese sulfate), 60 mg; Se (sodium selenite), 0.3 mg; and Zn (zinc oxide), 100 mg. 4 Metablizable energy and standardized ileal digestible (SID) amino acid values were calculated using NRC (2012). Analyzed crude protein determined as CP = (N 6.25).

11 Table 4. Analyzed composition of experimental diets (as-fed basis) 1 Soy source Soy protein concentrate HP300 Item Suppl. ISF No Yes No Yes Dry matter, % Organic matter, % Crude protein 2, % Lactose, % Total dietary fiber, % Isoflavones, ppm Total geinistein Total daidzein Total glycitin Total isoflavones , ,041 Total saponins, ppm 1,336 1,316 1,382 1,252 Total amino acids, % Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine All pigs received allotted treatment diet upon starting -7 DPI. Abbreviations: DPI, day postinoculation; ISF, isoflavones. 2 Analyzed crude protein determined as % total nitrogen 6.25.

12 Table 5. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on growth performance of weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF BW, kg initial final * < to 0 DPI 4 ADG, g/d ADFI, g/d * G:F, g/kg to 6 DPI ADG, g/d * ADFI, g/d 5 1,236 1,381 1,188 1,128 1, G:F, g/kg * to 14 DPI ADG, g/d * ADFI, g/d G:F, g/kg ab 268 ab 484 b 173 a to 14 DPI 4 ADG, g/d *a 103 a 219 b 165 ab ADFI, g/d G:F, g/kg *ab 171 a 378 b 361 b * Difference (P < 0.05) between uninfected and infected groups fed the control diet. 1 Values represent least square means of 6 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model. 4 Feed intake data and G:F values for -7-0 DPI from the first cohort of pigs (n=28) were omitted due to a mechanical error with the feeders that led to incorrect feed intake measurements. 5 ADFI was omitted if there was a net weight loss over measured period; G:F data was omitted if calculated value was less than 0 g/kg or greater than 1,000 g/kg

13 Table 6. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on serum viral load and presence of anti-prrsv antibodies in weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF Ct Value 4 0 DPI DPI b* b b a DPI ab* ab b a 1.60 < DPI * ELISA S/P Ratio 5 6 DPI < DPI * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without common superscript letter do differ (P < 0.05) 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones; ELISA = enzyme-linked immunosorbent assay. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model. 4 Cycle threshold (Ct) values represent the mean number (± SEM) of PCR cycles required to detect the presence of PRSV DNA. A higher CT value indicates less PRRSV DNA in the serum. No PRRSV DNA was detected in uninfected pigs. 5 IDEXX-ELISA S/P Ratio of 0.4 or greater is considered positive result. IDEXX ELISA assays were not performed on samples confirmed negative by qrt-pcr.

14 Table 7. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on peripheral blood T cell immunophenotypes of weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM Soy Source ISF Soy Source ISF T Cell, % (CD3+) Helper T Cell, % (CD3+CD4+) a ab a b Cytotoxic T Cell, % (CD3+CD8+) * Memory T Cell, % (CD3+CD4+CD8+) * Memory T Cell Expressing IFNγ, % (CD3+CD4+CD8+IFNγ+) * Memory T Cell Expressing IFNγ MFI (CD3+CD4+CD8+IFNγ+) * * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without a common superscript letter differ (P < 0.05). 1 Values represent least square means of 10 to 12 pigs with collection of blood occurring at 12 DPI. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model. 4 Percent of total lymphocytes that are positive for cell-surface marker CD3. 5 Percent of CD3-positive lymphocytes that are also positive for cell-surface markers CD4, CD8, CD4/CD8, or CD4/CD8 and intercellular IFN γ 6 Median fluorescence intensity (MFI) is a measure of the fluorescence intensity in a fluorescence channel being measured. It provides an alternative measurement to percent positive for comparison of cell populations between individuals. It is less sensitive to outliers, which is important for very small cell populations like memory T cells.

15 Table 8. Effects of dietary soy isoflavone levels and porcine reproductive and respiratory virus (PRRSV) infection on red blood cell measures in weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Soy Source Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF ISF RBC, 10 6 /µl 0 DPI DPI DPI < DPI HGB, g/dl 0 DPI DPI DPI < DPI * HCT, % 0 DPI DPI DPI < DPI * MCV, fl 0 DPI DPI DPI DPI * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without common superscript letter do differ (P < 0.05) 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones; RBC = red blood cells; HGB = hemoglobin; HCT = packed cell volume; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

16 Table 8. (cont.) P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF MCH, pg 0 DPI DPI DPI DPI * MCHC, g/dl 0 DPI DPI b 31.9 ab 31.9 ab 29.3 a DPI DPI * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without common superscript letter do differ (P < 0.05) 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days postinoculation; ISF = isoflavones; RBC = red blood cells; HGB = hemoglobin; HCT = packed cell volume; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

17 Table 9. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on differential leukocyte counts and population proportions in weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF WBC, 10 3 /µl 0 DPI DPI * DPI a* 12.7 a 24.4 b 14.5 a 3.24 < DPI * NEU, % of WBC 0 DPI DPI DPI * DPI * BAND, % of WBC 0 DPI DPI DPI * < DPI LYM, % of WBC 0 DPI DPI * DPI * < DPI * * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without common superscript letter do differ (P < 0.05) 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days postinoculation; ISF = isoflavones; NEU = neutrophils; BAND = immature neutrophils aka. band cells; LYM = lymphocytes; MONO = monocytes; EOSIN = eosinophils; BASO = basophils. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

18 Table 9. (cont.) P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF MONO, % of WBC 0 DPI * DPI DPI < DPI EOSIN, % of WBC 0 DPI DPI DPI bc 2.49 b 2.25 ac 3.40 c DPI BASO, % of WBC 0 DPI DPI DPI DPI * Difference (P < 0.05) between uninfected and infected groups fed the control diet. ab Means without common superscript letter do differ (P < 0.05) 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days postinoculation; ISF = isoflavones; WBC = white blood cells; NEU = neutrophils; BAND = immature neutrophils aka. band cells; LYM = lymphocytes; MONO = monocytes; EOSIN = eosinophils; BASO = basophils. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

19 Table 10. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on serum cytokine concentrations (pg/ml) in weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF TNFα 0 DPI DPI DPI DPI *ab 113 a 187 ab 353 b 92.9 IL-1β 0 DPI BDL BDL BDL BDL BDL -- 3 DPI BDL 131 BDL BDL BDL 1,843 6 DPI BDL BDL 335 BDL 347 1, DPI BDL 953 a 2,449 a 175 a 12,790 b 1,844 IFNγ 0 DPI BDL BDL BDL BDL BDL -- 3 DPI BDL 39.4 b 3.39 a 4.96 b 3.10 b DPI BDL DPI IL-10 0 DPI BDL BDL 12.8 BDL BDL DPI BDL BDL DPI BDL DPI BDL BDL 33.6 a BDL 154 b Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones; TNFα = tumor necrosis factor alpha; IL-1β = interleukin 1 beta; IFNγ = interferon gamma; IL-6 = interleukin 6; BDL = below detectable limits. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

20 Table 10. (cont.) P-value Uninfected PRRSV-infected Main effects Interaction Item, pg/ml Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF IL-8 0 DPI DPI DPI DPI IFNα 0 DPI DPI *a 1483 ab 1643 bc 2156 bc DPI < DPI Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones; TNFα = tumor necrosis factor alpha; IL-1β = interleukin 1 beta; IFNγ = interferon gamma; IL-6 = interleukin 6; BDL = below detectable limits. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Main effect of day post-inoculation (DPI); no 2- or 3-way interactive effects involving DPI were significant, so they were not included in the statistical model.

21 Appendix A. Effects of dietary soy isoflavones level and porcine reproductive and respiratory virus (PRRSV) infection on daily rectal temperatures of weanling pigs 1 P-value Uninfected PRRSV-infected Main effects Interaction Item, ºC Control 2 Control 2 Control + ISF 2 HP300 2 HP300 + ISF 2 SEM DPI 3 Soy Source ISF Soy Source ISF DPI * * * < * Difference (P < 0.05) between uninfected and infected groups fed the control diet. 1 Values represent least square means of 10 to 12 pigs. All pigs received allotted treatment diet starting -7 DPI. Abbreviations: DPI = days post-inoculation; ISF = isoflavones; WBC = White blood cells; NEU = neutrophils; LYM = lymphocytes; MONO = monocytes. 2 The following diet names have been assigned to a respective experimental diet groups: Control: soy protein concentrate + no supplemented ISF, fed to both uninfected and PRRSV-infected control pigs; Control + ISF: soy protein concentrate + supplemented ISF, fed to PRRSV-infected pigs only; HP300: HP300 + no supplemented ISF, fed to PRRSV-infected pigs only; HP300 + ISF: HP300 + supplemented ISF, fed to PRRSV-infected pigs only. 3 Single degree of freedom contrast comparing uninfected and infected groups receiving the control diet.

22 Appendix B. Missing data points and affected outcomes 1 Animal ID Treatment Cohort Affected Outcomes 2 Cause 284 UC 1 G:F (0-6 DPI) Immunophenotyping (12 DPI) G:F value generated > 1,000 g/kg Poor sample quality (Immunophenotyping) UC 1 All Became PRRSV+ by end of study; omitted from data set 275 UC 1 All Became PRRSV+ by end of study; omitted from data set 256 UC 2 Cytokine Recall Analysis (8 DPI) No sample collected 405 UC 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 265 UC 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 386 A 1 G:F (0-6 DPI) rt-pcr PRRSV Viral Load (3 DPI) Total and Differential Blood Cell Counts (3 DPI) Serum Cytokine Analysis (3 DPI) Immunophenotyping (12 DPI) 290 A 1 ADG (6-14 DPI, 0-14 PDI) G:F (6-14 DPI, 0-14DPI) Immunophenotyping (12 DPI) G:F value generated > 1,000 g/kg No samples collected No samples collected 152 A 1 Total and Differential Blood Cell Counts (6 DPI) No samples collected 281 A 1 Immunophenotyping (12 DPI) rt-pcr PRRSV Viral Load (14 DPI) IDEXX-ELISA (14 DPI) IFA (14 DPI) Total and Differential Blood Cell Counts (14 DPI) Serum Cytokine Analysis (14 DPI) Cranial Lung Cytokine Gene Analysis (14 DPI) 409 A 2 ADG (6-14 DPI) G:F (6-14 DPI) No samples collected Animal perished on 12 DPI of study No net weight gain during reported range No samples collected Cytokine Recall Analysis (8 DPI) 260 A 2 Cytokine Recall Analysis (8 DPI) No samples collected 402 A 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 418 A 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 412 A 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 423 A 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 172 B 1 All Never became PRRSV+; omitted from data set 378 B 1 G:F (0-6 DPI) Total and Differential Blood Cell Counts (0 DPI) 1 Experiement split into cohort 1 (n=28 pigs) and cohort 2 (n=32 pigs), ran in succession. 2 Abbreviations: DPI = days post-inoculation; rt-pcr = real-time polymerase chain reaction; IFA = immunofluorescent assay. 3 Sample dropped from data set due to low cells counts (<1,000 CD3+ cells) or irregular cell distribution. G:F value generated > 1,000 g/kg No samples collected

23 Appendix B. (cont.) Animal ID Treatment Cohort Affected Outcomes 2 Cause 298 B 1 Immunophenotyping (12 DPI) rt-pcr PRRSV Viral Load (14 DPI) No samples collected Animal perished on 13 DPI of study IDEXX-ELISA (14 DPI) IFA (14 DPI) Total and Differential Blood Cell Counts (14 DPI) Serum Cytokine Analysis (14 DPI) Lung histological scoring (14 DPI) Cranial Lung Cytokine Gene Analysis (14 DPI) 253 B 2 Cytokine Recall Analysis (8 DPI) No samples collected 407 B 2 ADG (0-6 DPI) No net weight gain during reported range G:F (0-6 DPI) 417 B 2 ADG (0-6 DPI, 6-14 DPI, 0-14 DPI) No net weight gain during reported range G:F (0-6 DPI, 6-14 DPI, 0-14 DPI) 403 B 2 ADG (6-14 DPI) No net weight gain during reported range G:F (6-14 PDI) 415 B 2 ADG (6-14 DPI) No net weight gain during reported range G:F (6-14 PDI) 254 B 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 390 C 1 Total and Differential Blood Cell Counts (0 DPI) No samples collected Immunophenotyping (12 DPI) 292 C 1 ADG (6-14 DPI, 0-14 DPI) G:F (6-14 DPI, 0-14 DPI) Cytokine Recall Analysis (8 DPI) Immunophenotyping (12 DPI) No net weight gain during reported range No samples collected Poor sample quality (Immunophenotyping) C 1 Total and Differential Blood Cell Counts (14 DPI) No samples collected 170 C 1 Total and Differential Blood Cell Counts (14 DPI) No samples collected 392 C 1 ADG (6-14 DPI, 0-14 DPI) No net weight gain during reported range G:F (6-14 DPI, 0-14 DPI) 406 C 2 G:F (0-6 DPI) G:F value generated > 1,000 g/kg 398 C 2 G:F (6-14 DPI) G:F value generated > 1,000 g/kg 250 C 2 G:F (6-14 DPI) G:F value generated > 1,000 g/kg 1 Experiement split into cohort 1 (n=28 pigs) and cohort 2 (n=32 pigs), ran in succession. 2 Abbreviations: DPI = days post-inoculation; IFA = immunofluorescent assay. 3 Sample dropped from data set due to low cells counts (<1,000 CD3+ cells) or irregular cell distribution.