The gastrointestinal (GI) tract continues to undergo significant developmental changes in postnatal life. Environmental influences during this critical developmental period, including diet, stress and mucosal injury, have been shown to induce long-term changes in intestinal physiology and disease susceptibility in animal models( Reference McLamb, Gibson and Overman 1 – Reference Boudry, Douard and Mourot 4 ). Similarly in human subjects, increasing epidemiological evidence supports the concept that adverse early-life environmental factors, such as stress, are associated with subsequent GI diseases such as irritable bowel syndrome( Reference Agostini, Rizzello and Ravegnani 5 – Reference Apley and Hale 9 ). In the case of pigs, early (age < 21 d) weaning of piglets is a significant, early-life stress that has been shown to have deleterious impacts on GI function, including increased intestinal permeability( Reference Moeser, Klok and Ryan 10 , Reference Moeser, Ryan and Nighot 11 ), inflammation( Reference Pie, Blazy and Laffitte 12 ), hypersecretion( Reference Moeser, Klok and Ryan 10 ), reductions in the activity of brush-border digestive enzymes( Reference Makkink, Negulescu and Qin 13 ), altered nutrient transport mechanisms( Reference Boudry, Lalles and Malbert 14 , Reference Boudry, Peron and Le Huerou-Luron 15 ), and marked changes in villus and crypt morphology (reduced villus surface area and increased crypt depth)( Reference Nabuurs, Hoogendoorn and van der Molen 16 ). The mechanisms and factors associated with weaning stress (e.g. maternal and littermate separation, dietary changes, and transport stress) are not completely understood yet; however, it was demonstrated by Moeser et al. ( Reference Moeser, Klok and Ryan 10 ) that activation of the corticotropin-releasing factor (CRF) receptor system in the intestine, and subsequent activation of mast cells, were responsible for increased intestinal permeability and hypersecretion, demonstrating the role of stress signalling pathways in the intestine of the weaned pig. It is now evident that the deleterious effects of early weaning stress on the intestinal tract of the pig are seen well beyond the immediate post-weaning (PW) period. Smith et al. ( Reference Smith, Clark and Overman 17 ) demonstrated that early-weaned pigs (weaned between 15 and 21 d of age) exhibited greater intestinal permeability at 9 weeks PW, compared with late-weaned pigs (weaned between 23 and 28 d of age). In addition, McLamb et al. ( Reference McLamb, Gibson and Overman 1 ) showed that early-weaned pigs exhibited heightened clinical disease (increased severity of diarrhoea and reduced growth rate) and intestinal injury (increased intestinal permeability), in response to an enterotoxigenic Escherichia coli (ETEC) challenge at approximately 3 weeks PW. On the whole, results from the aforementioned studies provide strong evidence that PW intestinal injury can have lasting deleterious impacts on intestinal function. Therefore, therapeutic approaches to ameliorate GI injury during the PW period could have a positive impact on long-term barrier function and defence against subsequent pathogenic challenges.
Dietary inclusion of spray-dried plasma (SDP) proteins in nursery pig diets has proven to have a beneficial effect on PW gastrointestinal health and performance in young pigs( Reference Peace, Campbell and Polo 18 , Reference Van Dijk, Margry and Van Der Lee 19 ). Previous studies demonstrated that SDP not only promotes growth responses in young pigs, but also confers protective effects in GI infectious challenge models. Van Dijk et al. ( Reference Van Dijk, Enthoven and Van den Hoven 20 ) demonstrated that weaned pigs challenged with K88 ETEC, and fed a nursery diet containing 8 % SDP, exhibited reduced diarrhoea and increased average daily gain and average daily feed intake, compared with pigs fed control diets containing whey protein. In another experiment, weaned pigs fed with diets containing 6 % SDP exhibited reduced cytokine responses and intestinal inflammatory cell infiltrates, following a challenge with ETEC( Reference Bosi, Casini and Finamore 21 ). Similarly, diarrhoeal disease induced by an experimental rotavirus challenge has been observed to be reduced in neonatal piglets fed a diet containing 15 % SDP compared with control diets containing soya protein isolate( Reference Corl, Harrell and Moon 22 ). Peace et al. ( Reference Peace, Campbell and Polo 18 ) confirmed that inclusion of SDP at dietary levels of 2·5 and 5 % for 2 weeks PW reduced intestinal permeability, intestinal inflammatory cytokines and diarrhoea in early-weaned pigs. However, in the aforementioned previous experiments, growth responses and intestinal protective effects of SDP described were measured while SDP was in the diet; whether inclusion of SDP in early-life pig diets retains beneficial effects after its removal from the diet has not been investigated. Given that early-weaning stress induces short- and long-term deleterious changes in intestinal function and disease susceptibility, and that SDP has proven beneficial in reducing early changes in intestinal permeability and inflammatory responses in weaned pigs, we hypothesised that inclusion of SDP in PW pig diets would have sustained beneficial effects on intestinal responses to a later-life pathogenic challenge, even after the removal of SDP from the diet. The specific objective of the present study was to determine whether the inclusion of SDP during the first 2 weeks PW influenced intestinal epithelial barrier function, immune responses and clinical disease in response to a later-life challenge with Salmonella typhimurium.
Materials and methods
All procedures were approved by the North Carolina State University Institutional Animal Care and Use Committee (protocol no. 12-051-A).
Pigs and experimental design
A total of thirty-two Yorkshire–Large White piglets weaned from 16 to 17 d of age with a similar body weight of 5·49 (sem 0·1) kg were used in the present experiment. The weaned piglets were housed in four nursery pens (eight pigs per pen; 1·09 m2/pig) and were offered ad libitum access to water and one of three experimental nursery diets containing either 0 % SDP (fed to two pens, n 16 pigs), 2·5 % SDP (fed for 7 d PW; n 8 pigs) or 5 % SDP (fed for 14 d PW; n 8 pigs) (Fig. 1). Sex and litter origin were distributed equally across the experimental groups. The variable dietary levels of 2·5 and 5 % SDP along with feeding duration PW (7 v. 14 d PW) were selected to mimic the range of dietary level and feeding duration of SDP commonly utilised in commercial swine feeding. Diets were supplied in mashed form, and were formulated to contain identical levels of metabolisable energy and digestible lysine to meet nutrients requirements of the NRC (1998)( Reference Moeser, Ryan and Nighot 11 ). At 7 d PW, pigs fed with the 2·5 % SDP treatment were switched to control (0 % SDP) diets. At 14 d PW, all pigs were fed the same diet (0 % SDP), and maintained in the nursery for an additional 21 d.
Fig. 1 Experimental design. Piglets (n 8 per treatment) were weaned from their sows and offered nursery diets containing either 0 % spray-dried plasma (SDP), 2·5 % SDP for 7 d post-weaning (PW) or 5 % SDP for 14 d PW. SDP was removed from the experimental diets at indicated times and fed identical diets to the control pigs. At 34 d PW, pigs were challenged with Salmonella typhimurium. At 2 d post-challenge, tissues were harvested for analysis. P-1, phase 1 diet; P-2, phase 2 diet. (A colour version of this figure can be found online at http://www.journals.cambridge.org/bjn).
Salmonella typhimurium challenge
At 34 d PW, all pigs were transferred from the nursery to isolation rooms located in a nearby North Carolina State University research facility at the College of Veterinary Medicine campus. Upon arrival, the pigs were housed, by treatment, with eight pigs/pen (0·3 m2/pig). The pens were equipped with tenderfoot flooring, and pigs were allowed ad libitum access to feed and water. On the following day, eight pigs from each experimental group were inoculated orally with 3 × 109 colony-forming units of S. typhimurium in 4 ml of culture media as described previously( Reference Balaji, Wright and Hill 23 ). A non-challenged control group was housed in a separate, identical room within the facility, and was administered similarly with 4 ml sterile media. The S. typhimurium DT104 strain used in the present study exhibited antimicrobial resistance to ampicillin, chloramphenicol, sulfisoxazole, streptomycin and tetracycline. Salmonella cultures were grown overnight at 37°C on Luria broth agar, and then added to a sterile 0·7 % saline solution to obtain a final concentration of 7·5 × 108 colony-forming units per ml, and verified using a NanoDrop 2000c nanospectrometer (Thermo Fisher Scientific, Inc.). In the present study, we chose S. typhimurium as the challenging agent because it has dual relevance to human and swine diseases( Reference Bahnson, Kim and Weigel 24 ).
Growth rate and feed intake calculations and faecal scores
Body weight (BW) was recorded at days 0 and 14 during the PW nursery phases, and on days 0 and 2 of the S. typhimurium challenge study, and average daily gain was calculated. Given the short (2 d) challenge period, growth data were presented as the percentage of BW loss. Pen feed intakes were recorded during the PW and S. typhimurium challenge periods, and estimated feed intake per pig was calculated for each pen. Faecal scores were analysed by persons, who were blinded to the experimental treatments, according to a previously published scoring system by our group( Reference McLamb, Gibson and Overman 1 ) using a scale from 1 (no diarrhoea) to 4 (severe profuse diarrhoea).
Ussing chamber studies
On day 2 post-challenge, pigs were sedated with a TKX cocktail containing Telazol (500 mg), Ketamine (250 mg) and Xylazine (250 mg) that were administered intramuscularly at a dose of 0·025 ml/kg BW. Euthanasia was followed by the administration of an overdose (86 mg/kg BW) of sodium pentobarbital solution (Fatal-Plus; Virbac Animal Health) via a catheterised ear vein. The distal small intestine (ileum) was harvested from each pig immediately after euthanasia, and opened along the anti-mesenteric border. Intestinal mucosa was stripped from the seromuscular layer in oxygenated (95 % O2 and 5 % CO2) Ringer solution (in mmol/l: 154 Na+, 6·3 K+, 137 Cl−, 0·3 H2PO4, 1·2 Ca2+, 0·7 Mg2+ and 24 HCO3 −; pH 7·4), and mounted in 1·13 cm2 aperture Ussing chambers (World Precision Instruments, Inc.). Ileal mucosa was bathed on the serosal and mucosal sides with 10 ml Ringer solution. The serosal bathing solution contained 10 mm-glucose, which was osmotically balanced on the mucosal side with 10 mm-mannitol. Bathing solutions were oxygenated (95 % O2 and 5 % CO2) and circulated in water-jacketed reservoirs maintained at 37°C. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag–AgCl electrodes using a voltage clamp that corrected for fluid resistance. Tissues were maintained in the short-circuited state, except for brief intervals to record the open-circuit PD. Transepithelial electrical resistance (TER, measured as Ω·cm2) was calculated from the spontaneous PD and short-circuit current (I sc), as described previously( Reference Argenzio and Liacos 25 ). After a 30 min equilibration period on Ussing chambers, TER and I sc were recorded at 15 min intervals over a 1 h period, and then averaged to derive the basal TER and I sc values for a given animal.
Paracellular permeability to 4 kDa fluorescein isothiocyanate dextran
After a 30 min equilibration period on Ussing chambers, 4 kDa fluorescein isothiocyanate dextran (FD4, 100 mg/ml; Sigma) was added to the mucosal bathing reservoir of Ussing chambers. Standards were taken from the serosal side of each chamber 15 min after the addition of FD4, and a 60 min flux period was established by taking 0·5 ml samples in triplicate from the mucosal compartment. The quantity of FD4 was established by measuring fluorescence in mucosal reservoir fluid samples in a fluorescence plate reader at 540 nm. Data are presented as the rate of FD4 flux in mg FD4 flux/min per cm2.
Histological analyses of intestinal tissues
Ileum was fixed in 10 % neutral-buffered formalin and processed for paraffin embedding. Paraffin blocks were sectioned (5 μm thick) and stained with haematoxylin and eosin for histological analysis. A histological scoring system was applied to the tissue sections, and was performed by a board-certified veterinary pathologist (L. B. B.), who was blinded to the experimental treatments. The intestinal scoring system used was based on villus morphology and blunting (villus height and crypt depth), villus fusion, reduced lymphoid recruitment and neutrophil numbers. The detailed scoring criteria were designated as follows: villus blunting–0 = crypt:tip ratio of at least 1:4, 1 = crypt:tip ratio of 1:3, 2 = 1:2, 3 = 1:1 and 4 = complete tip loss; lymphoid depletion and villus fusion – 0 = normal, 1 = mild, 2 = moderate and 3 = severe; neutrophils – 0 = none to 10 neutrophils/40 × field, 1 = 11–20 neutrophils/40 × field, 2 = 21–30 neutrophils/40 × field and 3 = 31–40 neutrophils/40 × field. Neutrophils were identified based on nuclear and cytoplasmic morphology( Reference Wickramasinghe and Mills 26 ). Measurements for crypt depth and villus height were taken utilising the calibrated measurement caliper option, and villus measurements were taken from three well-oriented villi in five different fields/slide, such that fifteen villi/slide per pig were measured. Villi chosen for measurements were based on the criteria that (1) the entire crypt and villi be captured in the cross-section, and (2) the central lacteal be present. Villi overlying the gut-associated lymphoid tissue were excluded from the measurement. Photomicrographs were acquired with 20 × and 40 × magnifications at a resolution using imaging software (Infinity Analyze Software), running a high-resolution digital camera (Lumenera) equipped to a clinical light microscope (Model OMFL400; Meiji Microscope Solutions).
Ileal cytokine analysis
Ileal mucosa was homogenised in PBS containing protease inhibitors, and the supernatant was collected and analysed for protein content using a BCA assay( Reference Peace, Campbell and Polo 18 ). Samples were then diluted 1:10 in PBS and assayed for TNF, IL-8 and IL-6, using commercial porcine ELISA assays (R&D Systems). Concentrations of each cytokine are expressed on a per mg protein basis.
Myeloperoxidase assay
The distal ileum was obtained from each pig, opened lengthwise, and rinsed in cold Ringer solution. The epithelium and lamina propria were scraped from the seromuscular layers over ice using a glass slide, and then frozen in liquid N2 and stored at − 80°C. The ileal mucosal scrapings were thawed and homogenised in 0·5 % hexadecyltrimethylammonium bromide buffer (50 mm-phosphate buffer, pH 6), to release myeloperoxidase (MPO) from the primary granules of neutrophils. The homogenate was subjected to three cycles of freezing at − 80°C, thawed, and sonicated on ice. Samples were centrifuged at 21 000 g at 4°C for 15 min, and the supernatant assayed for MPO activity. An aliquot of the supernatant was allowed to react with a solution of tetramethylbenzidine in N-dimethylformamide and H2O2. Absorbance (655 nm) readings were taken at 30 s intervals over 15 min. MPO activity was determined based on a MPO standard curve, and is expressed as units per g (wet weight) mucosa (for ileum) or per ml of plasma( Reference Zadrozny, Stauffer and Armstrong 27 ).
Western blot analysis of corticotropin-releasing factor receptors in the porcine ileum
Ileal mucosal protein was extracted from mucosal scrapes using Mammalian Protein Extraction Reagent containing protease and phosphatase inhibitors (Fisher Scientific). Samples were sonicated and centrifuged at 14 000 rpm for 15 min at 4°C. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Fisher Scientific). Total protein was resolved by SDS–PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5 % (w/v) non-fat milk in Tris-buffered saline with 0·1 % Tween-20 (TBS-T) for 1 h at room temperature, washed in TBS-T, and incubated with CRF-RI/II antibody that detects both receptors (Santa Cruz Biotechnology). Subsequently, the membranes were washed and incubated with an appropriate secondary antibody for 1 h at room temperature, followed by washing with TBS-T and incubation with the SuperSignal West Pico Chemiluminescent Substrate (Fisher Scientific). As an internal loading control, the antibody was stripped from the membranes with Restore™ Western blot stripping buffer (Thermo Fisher Scientific, Inc.), and the membranes were reprobed with a β-actin antibody (Cell Signaling Technology). Bands were visualised with ChemiDoc™ MP Imaging System (Bio-Rad), densitometric analysis was performed using the Bio-Rad Image Lab software (version 4.1), and the CRF receptor band intensities were normalised to β-actin.
Statistical analysis
Data are presented as means with their standard errors based on the experimental sample number (n). With the exception of histological and faecal score data, all other data were analysed using a standard one-way ANOVA (SigmaStat; Jandel Scientific). A post hoc Tukey's test was used to determine the differences between treatments following ANOVA. Statistical significance was set at a level of P< 0·05. Histological and faecal scores were analysed using the non-parametric Kruskal–Wallis test (GraphPad Prism) with Dunn's post-test.
Results
Effects of early-life dietary spray-dried plasma on clinical responses to subsequent Salmonella typhimurium challenge
In the first 2 weeks PW, estimated feed intake for pigs fed diets containing 0, 2·5 % SDP (for 7 d) and 5 % SDP (for 14 d) was 0·221, 0·231 and 0·238 kg/d, respectively. The average daily gain (kg/d) during the first 2 weeks PW for pigs fed the 0, 2·5 and 5 % SDP diets was 0·121 (sem 0·015), 0·119 (sem 0·021) and 0·142 (sem 0·025), respectively. All pigs remained clinically normal throughout the nursery phase. During the 2 d S. typhimurium challenge study, at 34 d PW, the control (non-challenged, 0 % SDP) pigs gained 5 % of their BW, whereas growth responses in pigs challenged with S. typhimurium were significantly reduced and ranged between 0·5 and − 1 % BW gain (Fig. 2(A)). Compared with the non-challenged control pigs, the estimated feed intake of S. typhimurium-challenged pigs over the 2 d challenge period was 1·54, 1·20, 1·22 and 1·17 kg in pens from the control, 0 % SDP-, 2·5 % SDP- and 5 % SDP-challenged groups, respectively. All pigs challenged with S. typhimurium exhibited diarrhoea as indicated by higher (P< 0·05) faecal scores, compared with the non-challenged control pigs (Fig. 2(C)). Rectal body temperatures were also significantly elevated in challenged pigs compared with the control ones (P< 0·05; Fig. 2(B)). Dietary inclusion of SDP (2·5 or 5 % SDP) during the PW period had no significant effect on BW loss, faecal score or body temperature in response to S. typhimurium challenge in the present study.
Fig. 2 Body weight, body temperature and faecal scores in pigs challenged with Salmonella typhimurium. (A) Body-weight loss was calculated from body weights recorded on day 0 and day 2 post-challenge. (B) Rectal temperature and (C) faecal scores were recorded on day 2 post-challenge. Values are means, with their standard errors represented by vertical bars (n 8). a,bMean values with unlike letters were significantly different (P< 0·05; one-way ANOVA). SDP, spray-dried plasma. 
, Control; 
, S. typhimurium; 
, 2·5 % SDP+S. typhimurium; 
, 5 % SDP+S. typhimurium.
Effects of early-life dietary spray-dried plasma on histological injury responses to Salmonella typhimurium challenge
Compared with the non-challenged control group, S. typhimurium-challenged pigs exhibited elevated ileal histological injury scores (Fig. 3(A)). Histological scores from pigs fed with the 5 % SDP-14 d diet during the nursery period were lower compared with those from the challenged control pigs. Marked lymphoid depletion, an index of an overwhelming immune response, was observed in all S. typhimurium-challenged pigs, but was less severe in pigs fed with the 2·5 %-7 d and 5 % SDP-14 d PW diets. Extensive villus blunting (Fig. 3(A) and (B)) and fusion (adhesion) was observed in all pigs challenged with S. typhimurium; however, there were no effects of PW SDP treatments on these parameters. Crypt depth was found to be increased (P< 0·05) in the ileum from S. typhimurium-challenged pigs compared with the control ones (Fig. 3(C)). Ileum from pigs fed with the 5 % SDP-14 PW diet had increased (P< 0·05) crypt depth compared with all the other treatments. Increased numbers of ileal neutrophils (Fig. 3(A)) were observed in response to S. typhimurium challenge, which corresponded with higher activity of ileal MPO, a marker of neutrophil activation (Fig. 4(A)). MPO and neutrophil numbers were lower in the ileum from pigs fed with 5 % SDP-14 d diet in the PW period.
Fig. 3 Impact of early-life dietary spray-dried plasma (SDP) on histological damage caused by subsequent challenge with Salmonella
typhimurium. (A) Histological scores, (B) villus height, (C) crypt length and (D) histological appearance from pig ileal tissues, at 2 d post-S.
typhimurium challenge. Representative histological sections were taken at 4 × and 20 × magnification. Values are means, with their standard errors represented by vertical bars (n 8). a,b,cMean values with unlike letters were significantly different (P< 0·05; one-way ANOVA). 
, Control; 
, S. typhimurium; 
, 2·5 % SDP+S. typhimurium; 
, 5 % SDP+S. typhimurium. (A colour version of this figure can be found online at http://www.journals.cambridge.org/bjn).
Fig. 4 Impact of early-life dietary spray-dried plasma (SDP) on ileal and plasma immunological responses to subsequent Salmonella typhimurium challenge. (A) Ileal myeloperoxidase (MPO), (B) ileal IL-8, (C) ileal TNF, (D) plasma MPO, (E) plasma TNF and (F) plasma cortisol were measured at 2 d post-S.
typhimurium challenge. Values are means, with their standard errors represented by vertical bars (n 8). a,b,cMean values with unlike letters were significantly different (P< 0·05; one-way ANOVA). * At trend for ileal TNF between 5 % SDP and the other challenged treatments (P= 0·07) and for plasma cortisol between S.
typhimurium control and control (P= 0·09). 
, Control; 
, S. typhimurium; 
, 2·5 % SDP+S. typhimurium; 
, 5 % SDP+S. typhimurium.
Effects of spray-dried plasma on ileal and plasma cytokines in response to later-life Salmonella typhimurium challenge
TNF concentrations were found to be elevated in the ileum from all S. typhimurium-challenged groups compared with the non-challenged control group (Fig. 4(C)). There was a trend (P= 0·06) for elevated TNF levels in response to S. typhimurium challenge in pigs fed the 5 % SDP-14 d diet, compared with the other challenged treatments. Ileal IL-8 levels were increased in the S. typhimurium-challenged group (Fig. 4(B)); however, IL-8 levels were lower in the ileum from the challenged groups fed the 2·5 %-7 d or 5 % SDP-14 d diet in the PW period (Fig. 4(B)).
To assess the effects of S. typhimurium infection and SDP nursery feeding on systemic inflammatory responses, plasma levels of MPO (Fig. 4(D)), TNF (Fig. 4(E)) and cortisol (Fig. 4(F)) were assessed 2 d post-challenge. Plasma TNF was elevated in all pigs challenged with S. typhimurium. In line with responses observed in the ileum, pigs fed with the 5 % SDP-14 d nursery diet exhibited the greatest levels of plasma TNF in response to S. typhimurium challenge. Plasma cortisol levels tended to be elevated (P= 0·09) in the S. typhimurium-challenged control group, but were not different from the other experimental treatment groups.
Utilising an antibody that recognises both CRF receptor subtypes (CRF1 and CRF2), we found that the CRF1/2 antibody recognised three major protein bands in porcine ileal protein extracts at approximately 55, 37 and 28 kDa (Fig. 5) . These protein bands are consistent with the unprocessed form (55 kDa), the deglycosylated form (37 kDa) and the soluble CRF receptor forms (28 kDa)( Reference Slominski, Zbytek and Pisarchik 28 ), and have been reported previously in studies on the rodent intestine( Reference Lakshmanan, Magee and Richard 29 , Reference O'Malley, Dinan and Cryan 30 ). Based upon densitometric analysis, intestinal CRF1/2 receptor proteins (50 and 28 kDa bands) were markedly up-regulated (P< 0·05) in response to S. typhimurium challenge. However, the nursery SDP treatment did not appear to influence the level of ileal CRF receptor expression in the challenged pigs.
Fig. 5 (A) Western blot and (B) densitometric analysis of corticotropin-releasing factor (CRF1/2) receptors (CRFR) in porcine ileal mucosal scrapes from control and Salmonella typhimurium-challenged pigs. (B–D) Densitometry values for each protein band normalised to β-actin are shown. Values are means, with their standard errors represented by vertical bars (n 3). a,bMean values with unlike letters were significantly different (P< 0·05; one-way ANOVA). SDP, spray-dried plasma. 
, Control; 
, S. typhimurium; 
, 2·5 % SDP+S. typhimurium; 
, 5 % SDP+S. typhimurium.
Effects of spray-dried plasma on intestinal permeability in response to later-life Salmonella typhimurium challenge
At 2 d post-challenge, FD4 flux rates, an index of paracellular permeability, were elevated (P< 0·05) in the ileum from pigs challenged with S. typhimurium (P< 0·05) (Fig. 6(A)). Pigs fed with the 5 % SDP-14 d diet during the PW period exhibited lower FD4 flux rates at 2 d post-challenge, compared with the other challenged treatment groups. Ileal TER was higher in the challenged pigs compared with the non-challenged control pigs (P< 0·05; Fig. 6(B)). There was a trend (P= 0·06) for increased ileal TER in pigs fed the 2·5 % SDP-7 d and 5 % SDP-14 d diets during the PW period compared with the challenged control pigs. Transepithelial PD and short-circuit current (I sc) were reduced in S. typhimurium-challenged pigs compared with the non-challenged control pigs (Fig. 6(C) and (D), respectively). Pigs fed with the 5 % SDP-14 d diet exhibited greater ileal PD, compared with the challenged control pigs (P< 0·05). Ileal I sc was greater in pigs fed with the 2·5 % SDP-7 d and 5 % SDP-14 d diets compared with non-challenged control pigs.
Fig. 6 Impact of early-life dietary spray-dried plasma (SDP) on intestinal permeability and transepithelial short-circuit current (I
sc) following subsequent Salmonella
typhimurium challenge in pigs. (A) Fluorescein isothiocyanate dextran (FD4) flux rates, (B) ileal transepithelial electrical resistance (TER), (C) ileal transepithelial potential difference (PD), and (D) I
sc were measured using the ileum mounted on Ussing chambers on 2 d post-S. typhimurium challenge. Values are means, with their standard errors represented by vertical bars (n 8). a,b,cMean values with unlike letters were significantly different (P< 0·05; one-way ANOVA). 
, Control; 
, S. typhimurium; 
, 2·5 % SDP+S. typhimurium; 
, 5 % SDP+S. typhimurium.
Discussion
Inclusion of SDP into animal diets has been shown to promote growth responses( Reference Pierce, Cromwell and Lindemann 31 – Reference Coffey and Cromwell 34 ), and lessen inflammatory processes and clinical disease in pathogen challenge models( Reference Van Dijk, Enthoven and Van den Hoven 20 – Reference Corl, Harrell and Moon 22 , Reference Perez-Bosque, Amat and Polo 35 ). Previously, we demonstrated that dietary SDP, at 2·5 and 5 % of weaned pig diets, was beneficial in reducing intestinal permeability and inflammation induced in early-weaned pigs( Reference Peace, Campbell and Polo 18 ). While the aforementioned studies on rodents and pigs demonstrated the beneficial effects of SDP on growth and intestinal inflammatory responses to stress and pathogenic challenges, the response variables were measured when SDP was currently being included in the diet. Whether or not SDP confers beneficial effects on the intestine after its removal from the diet has not been investigated. Here, we showed that dietary inclusion of SDP during the first 2 weeks PW can modify subsequent intestinal and immunological responses to a pathogenic challenge with S. typhimurium in pigs.
In the present study, pigs fed diets containing SDP during the first 2 weeks PW exhibited differential intestinal and systemic immune responses following an S. typhimurium challenge at 50 d of age; compared with the challenged control pigs, those fed the 2·5 % SDP-7 d and 5 % SDP-14 d diets during the PW period exhibited reduced ileal IL-8 levels. IL-8 is a major inflammatory cytokine produced by intestinal epithelial cells during S. typhimurium infection, and it acts as a chemoattractant for the recruitment of circulating neutrophils into the intestine, resulting in classic intestinal inflammatory lesions associated with S. typhimurium enteritis( Reference McCormick, Colgan and Delp-Archer 36 , Reference McCormick, Parkos and Colgan 37 ). In line with this role, pigs fed with the 5 % SDP-14 d nursery dietary treatment had reduced neutrophil infiltration, MPO levels, and histological injury in response to S. typhimurium challenge. Bosi et al. ( Reference Bosi, Casini and Finamore 21 ) demonstrated earlier, in agreement with our findings, that piglets fed with 6 % SDP exhibited reduced ileal IL-8 concentrations, induced by ETEC challenge; however, unlike the present study, IL-8 levels were measured while SDP was included in the diet at the time of the challenge.
Interestingly, despite the dampened histopathological and inflammatory responses exhibited by the challenged pigs fed with the 5 % SDP-14 d nursery diet, ileal and plasma TNF levels were higher compared with the challenged control pigs. As mentioned above, S. typhimurium induces an intestinal inflammatory response mediated via the production of pro-inflammatory cytokines, including TNF and IL-8 and subsequent neutrophil recruitment and activation( Reference Moeser and Blikslager 38 ). TNF is best recognised as a pro-inflammatory cytokine that is central to the pathogenesis of a number of inflammatory disorders, and to stress-induced intestinal permeability( Reference Soderholm, Streutker and Yang 39 , Reference Overman, Rivier and Moeser 40 ).
Also, TNF is recognised as a critical and beneficial modulator of immune function and pathogen defence. For example, Nauciel & Espinasse-Maes( Reference Nauciel and Espinasse-Maes 41 ) demonstrated that administration of anti-TNF antibodies to mice exacerbated bacterial proliferation and mortality, following a sub-lethal dose of S. typhimurium. In similar studies carried out by Gulig et al. ( Reference Gulig, Doyle and Clare-Salzler 42 ) and Tite et al. ( Reference Tite, Dougan and Chatfield 43 ), anti-TNF antibodies increased the numbers of splenic colony-forming units of S. typhimurium following challenge.
Collectively, these studies have suggested that elevated TNF responses are critical for the control of infections. The present study is consistent with the study by Touchette et al. ( Reference Touchette, Carroll and Allee 44 ) showing that early-weaned pigs fed with a diet containing 7 % SDP for 7 d PW exhibited a 2-fold higher increase in serum TNF levels in response to systemic LPS challenge, compared with pigs that did not receive SDP. The authors also demonstrated a marked 110-fold increase in (interferon-α) in pigs, fed with the SDP-enriched diet, in contrast to a 16-fold increase in pigs fed a diet without SDP. Taken as a whole, the present study, along with the previous investigations, demonstrated that dietary SDP can modulate local and systemic immune responses to weaning and pathogen challenges. However, the present study provides, for the first time, evidence that the effects of SDP on immune responses can be retained even after the removal of SDP from the diet.
In addition to investigating the effects of early dietary SDP and subsequent S. typhimurium challenge on inflammatory signals, we also investigated stress signalling pathways. Specifically, we showed that plasma cortisol and intestinal expression of CRF receptors were increased 2 d post-challenge; however, there were no differences between pigs fed SDP. While elevations in plasma cortisol, following S. typhimurium challenge, have been shown previously in studies on pigs( Reference Balaji, Wright and Hill 23 ), our findings on the marked up-regulation of intestinal CRF1/2 receptors during an acute challenge with S. typhimurium are novel. Given the increasingly recognised role of the intestinal CRF system in inflammatory and stress-induced functional GI disorders, in human subjects and laboratory research animal models( Reference Smith, Clark and Overman 17 , Reference Gourcerol, Wu and Yuan 45 , Reference Chatzaki, Anton and Million 46 ), these findings warrant further investigation into the role of CRF in infectious inflammatory diseases.
In the present study, S. typhimurium challenge induced impairment in intestinal barrier function, which was indicated by increased ileal permeability to the paracellular probe FD4. The increase in ileal permeability in S. typhimurium-challenged pigs was attenuated in pigs fed with the 5 % SDP-14 d nursery diet, suggesting either an protective or reparative influence of early SDP dietary inclusion on intestinal barrier function.
It is not understood how early SDP feeding resulted in lasting protective effects on intestinal barrier during S. typhimurium infection in the present study. However, it is known that intestinal neutrophil infiltration in response to S. typhimurium infection is a central process, contributing to the breakdown of intestinal barrier function. Neutrophil-mediated disruption of intestinal barrier function involves a multi-step mechanism, including increased myosin light chain phosphorylation and myosin light chain kinase, up-regulation of tight junction phosphotyrosine and phopshoserine residues( Reference Edens, Parkos and Liang 47 ), and activation of epithelial protease-activated receptors( Reference Chin, Lee and Nusrat 48 ). Given that the 5 % SDP-14 d inclusion in nursery diets resulted in reduced neutrophil infiltration in response to S. typhimurium infection, it is plausible that this may represent an important mechanism, by which SDP led to a protective effect on the intestinal barrier in the present study.
In addition, we observed unexpected results with regard to ileal TER in the present study. Despite the elevated FD4 permeability in the ileum at 2 d post-challenge, ileal TER was significantly elevated in all the challenged groups. FD4 flux rates and TER measure two different aspects of intestinal epithelial barrier function: TER reflects changes in ion (predominantly Na+) permeability across the tight junction pores, while FD4 flux reflects large molecule fluxes across the leaky tight junctions. Another difference between the two measurements is that TER is calculated, on the basis of measured values of transepithelial voltage (PD) and current (I sc ), according to Ohm's law (V = IR), and expressed on the basis of the surface area of the tissue chamber aperture. Therefore, significant alterations in either PD or I sc could have a significant impact on calculated TER values. Further analysis of PD across S. typhimurium-infected ileum tissues revealed a significant reduction in PD, which indicates a compromised ability of the intestinal epithelium to resist ion flow through the paracellular space, and thus is in line with the elevated FD4 flux. However, in contrast to the FD4 flux data, PD was not significantly influenced by the early nursery 5 % SDP-14 d dietary treatment. S. typhimurium challenge also resulted in significant reductions in ileal I sc, which, in turn, might have contributed to the increased calculated TER values. Furthermore, I sc was greater in pigs fed the 2·5 %-7 d and 5 % SDP-14 d nursery diets, which accounted for the increased TER in pigs fed the SDP treatments. The basis for increased I sc observed in the challenged pigs fed SDP in the nursery is not clear. However, the suppressive influence of S. typhimurium on I sc has been demonstrated in previous investigations on pigs and mice( Reference Walsh, Rostagno and Gardiner 49 , Reference Aschenbach, Ahrens and Schwelberger 50 ). The mechanisms for reduced I sc in the ileum from S. typhimurium-challenged pigs could be due to reduced anion (Cl− or HCO3 −) secretion or electrogenic cation (e.g. Na+) absorption. In a recent study, it has been demonstrated that mice challenged with S. typhimurium exhibited reduced basal- and adenosine 3′,5′-cyclic monophosphate-mediated electrogenic I sc, an effect associated with reduced expression and/or localisation of colonic epithelial ion transporters including the Cl−/HCO3 − exchanger down-regulated in adenoma and the cystic fibrosis transmembrane regulator( Reference Marchelletta, Gareau and McCole 51 ). These suppressive effects were, in part, mediated by secreted S. typhimurium effector proteins. Therefore, in the light of these findings, it is plausible that the influence of early SDP on subsequent I sc responses to S. typhimurium challenge could be directly related to the effects of SDP treatments on subsequent S. typhimurium pathogenicity in the porcine intestine. The precise host intestinal pathways modulated by early SDP feeding in pigs that contribute to the I sc response remain to be elucidated.
Despite marked changes in immune and epithelial barrier responses, SDP had little influence on S. typhimurium-induced morphology of intestinal villi (villus blunting or villus fusion) and epithelium (denuded epithelium). Interestingly, increased crypt depths were observed in pig fed the 5 % SDP-14 d nursery diet. Increased crypt depth (crypt expansion) is a hallmark of intestinal injury, but at the same time is an index of epithelial repair processes, as increased proliferation of immature crypt enterocytes will migrate to the villus tip to replace damaged or denuded villus epithelial cells. Therefore, the increased crypt depths in pigs fed the 5 % SDP nursery diet could indicate increased epithelial renewal, which might potentially prove beneficial in later stages of recovery from S. typhimurium.
As mentioned previously, there are a number of studies in the literature that describe the beneficial impact of dietary SDP on growth responses. However, in the present study, there were no significant differences in growth and/or clinical responses in pigs observed either during the PW period or during the subsequent S. typhimurium post-challenge period. Despite the lack of measurable growth response to SDP in the present study, significant effects on immunological and intestinal responses were demonstrated.
There are several reasons that could explain the lack of SDP growth responses in the present study. First, the primary objective was not to measure growth performance, but to determine whether early dietary SDP influenced subsequent immunological and intestinal physiological responses to a later-life S. typhimurium challenge. Therefore, sufficient animal sample size needed to achieve the statistical power required to appropriately evaluate growth responses was not included in the experimental design.
Second, the experimental environment in which the pigs were raised might not have been ideal to demonstrate a SDP-dependent growth response. It has been shown previously that the effects of SDP on pig growth were observed in a commercial farm environment, but not in an experimental university research setting( Reference Coffey and Cromwell 34 ). A third reason for the lack of SDP growth response, specifically observed in the post-challenge phase of the experiment, is the short time period (2 d) in which BW changes were measured, which might have been inadequate to assess post-challenge growth responses during the peak challenge response. Given the beneficial effects of early SDP observed on intestinal physiological and immunological responses following S. typhimurium challenge, growth measurements over a longer post-challenge period (e.g. 7–14 d) could have a significant influence on the effects of SDP on growth responses in challenged pigs.
In conclusion, data from the present study demonstrate that early dietary inclusion of SDP have an impact on intestinal immunological and epithelial pathophysiological responses to S. typhimurium challenge, even after SDP has been removed from the diet. Given that stress and diet are increasingly recognised as key early-life factors that determine long-term health outcomes in human subjects and animals, a more fundamental understanding of biological mechanisms and optimal nutritional intervention strategies, such as dietary SDP, have potential to exert a positive impact on long-term intestinal health.
Acknowledgements
The authors thank the North Carolina State University Laboratory Animal Resources for their excellent technical assistance and advice during the entire period of the study.
The present study was funded by the American Proteins Company (APC), Inc. (to A. J. M.) and The National Institutes of Health (to A. J. M., K08 DK084313 and R01 HD072968).
The authors' contributions are as follows: J. M. C., J. D. C., J. P. and A. J. M. designed the research; A. J. M., L. L. E., P. E. B., E. S., S. T., M. M., S. D. and L. B. B. conducted the research; A. J. M., L. L. E., S. D. and L. B. B. analysed the data; P. E. B., S. D. and A. J. M. wrote the paper; A. J. M. had primary responsibility for the final content. All authors read and approved the final manuscript.
None of the authors has any conflict of interest to declare.
