Thomas E. Burkey

BOARD-INVITED REVIEW: Porcine mucosal immunity of the gastrointestinal tract

The gastrointestinal tract (GIT) constitutes one of the largest immunological organs of the body. The GIT must permit absorption of nutrients while also maintaining the ability to respond appropriately to a diverse milieu of dietary and microbial antigenic components. Because of the diverse population of antigenic components within the GIT, a sophisticated mucosal immune system has evolved that relies on collaboration between the innate and adaptive arms of immunity. The collaborative, mucosal immune effort offers protection from harmful pathogens while also being tolerant of dietary antigens and normal microbial flora. Knowledge with respect to porcine mucosal immunity is important as we strive to understand the interrelationships among GIT physiology, immunology, and the resident microbiota. The aim of this review is to provide a descriptive overview of GIT immunity and components of the mucosal immune system and to highlight differences that exist between the porcine species and other mammals.

In Vivo Selection To Identify Bacterial Strains with Enhanced Ecological Performance in Synbiotic Applications

ABSTRACT One strategy for enhancing the establishment of probiotic bacteria in the human intestinal tract is via the parallel administration of a prebiotic, which is referred to as a synbiotic. Here we present a novel method that allows a rational selection of putative probiotic strains to be used in synbiotic applications: in vivo selection (IVS). This method consists of isolating candidate probiotic strains from fecal samples following enrichment with the respective prebiotic. To test the potential of IVS, we isolated bifidobacteria from human subjects who consumed increasing doses of galactooligosaccharides (GOS) for 9 weeks. A retrospective analysis of the fecal microbiota of one subject revealed an 8-fold enrichment in Bifidobacterium adolescentis strain IVS-1 during GOS administration. The functionality of GOS to support the establishment of IVS-1 in the gastrointestinal tract was then evaluated in rats administered the bacterial strain alone, the prebiotic alone, or the synbiotic combination. Strain-specific quantitative real-time PCR showed that the addition of GOS increased B. adolescentis IVS-1 abundance in the distal intestine by nearly 2 logs compared to rats receiving only the probiotic. Illumina 16S rRNA sequencing not only confirmed the increased establishment of IVS-1 in the intestine but also revealed that the strain was able to outcompete the resident Bifidobacterium population when provided with GOS. In conclusion, this study demonstrated that IVS can be used to successfully formulate a synergistic synbiotic that can substantially enhance the establishment and competitiveness of a putative probiotic strain in the gastrointestinal tract.

Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species

ABSTRACT The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs. IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

Interaction of Bacillus species and Salmonella enterica serovar Typhimurium in immune or inflammatory signaling from swine intestinal epithelial cells.

Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted antiinflammatory effects on Salmonella enterica serovar Typhimurium (Sal)-induced secretion of IL-8. The current investigation evaluated the antiinflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, 2 Bacillus subtilis (BS1 and BS3) and 1 BL (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without Sal, and the same Bacillus treatments but with 10(8) cfu of Sal added in the final hour of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (control) and positive control wells receiving only Sal (10 total treatments). After bacterial incubation, wells were washed and fresh medium containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for determination of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received Sal were lysed and lysates were cultured to determine treatment effects on Sal invasion. Exposure to Sal alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared with control wells (P < 0.001 for both the apical and basolateral directions). Pretreatment with each Bacillus isolate followed by challenge with Sal reduced Sal-induced IL-8 secretion in both the apical and basolateral compartments compared with wells receiving only Sal (P < 0.001; except for BS3 apical, P < 0.01). The residual presence of bacitracin could be detected only in BL2 and BL2+Sal. Fewer Sal colonies could be cultured from lysates of BL2+Sal than from the Sal, BS1+Sal, and BS3+Sal treatments (P < 0.001). Results indicate that B. subtilis and BL have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by reduced invasion of epithelial cells by Sal.

Identification and characterization of intestinal lactobacilli strains capable of degrading immunotoxic peptides present in gluten

Identify and characterize bacteria from the proximal gastrointestinal tract of pigs capable of degrading immunogenic gluten peptides.

Effects of spray-dried porcine plasma on growth performance, immune response, total antioxidant capacity, and gut morphology of nursery pigs

Two experiments were conducted to evaluate the effects of spray-dried porcine plasma (SDPP) on growth performance, immunity, antioxidant capacity, and gut morphology of nursery pigs. In Exp. 1, 96 weaned pigs (Nebraska female × Danbred sire; 20 ± 1 d of age; initial BW = 6.06 ± 0.02 kg) were assigned to 16 pens and randomly allotted to the control (CTL; no SDPP) or the CTL + SDPP treatment in 2 phases (phase 1: d 0 to 14, 5% SDPP; phase 2: d 14 to 28, 2.5% SDPP). Blood samples were collected on d 0 and weekly thereafter to quantify IgG, IgA, and total antioxidant capacity. On d 14, pigs (n = 16; 8 pigs/treatment) were selected and euthanized for small intestine tissue and alveolar macrophage collection. On d 7, pigs fed SDPP had greater ADG, ADFI (P = 0.001), and G:F (P = 0.019) compared with CTL pigs. On d 28, pigs fed SDPP had greater BW (P = 0.024) and tended to have greater ADG (P = 0.074) and ADFI (P = 0.062) compared with CTL pigs. There were no differences between treatments for serum IgG, IgA, and total antioxidant capacity. On d 14, greater villus height (P = 0.011) and villus:crypt (P = 0.008) were observed in duodenal tissue sections obtained from SDPP-fed pigs compared with CTL pigs. To evaluate effects of SDPP on immune biomarkers, alveolar macrophages collected from 3 pigs/treatment on d 14 were cultured in vitro and challenged with lipopolysaccharide (LPS; 10 ng/mL). Therefore, 4 treatments included 1) CTL diet with no LPS, 2) CTL diet with LPS (CTL+), 3) SDPP diet with no LPS, and 4) SDPP diet with LPS. There were no diet effects on tumor necrosis factor-α gene expression or secretion by alveolar macrophages. For IL-10 gene expression, a diet × LPS interaction (P = 0.009) was observed where CTL+ had greater (P < 0.05) IL-10 mRNA abundance compared with other treatments. A second experiment was conducted to evaluate the in vitro effects of porcine plasma using model porcine jejunal epithelial cells (IPEC-J2). The treatments applied to the IPEC-J2 cells were 1) CTL, 2) CTL + 10% porcine plasma, 3) CTL + 5% porcine plasma, 4) CTL + 2.5% porcine plasma, or 5) CTL + 5% fetal bovine serum. The addition of porcine plasma increased (P < 0.001) proliferation of IPEC-J2 cells at 24 and 48 h following exposure to the respective treatments. In conclusion, dietary SDPP increased growth performance of nursery pigs during the first week postweaning and provide benefits to gut morphology possibly via increased proliferation of enterocytes.

Effect of corn distillers dried grains with solubles on growth performance and health status indicators in weanling pigs.

Two experiments were conducted to evaluate effects of corn distillers dried grains with solubles (DDGS) on growth performance and health status of weanling pigs. Experiment 1 evaluated effects of increasing concentrations of DDGS on growth performance and health of weanling pigs. Dietary treatments included 1) control (CTL), 2) 0% DDGS (0% DDGS in phase 2 and 30% DDGS in phase 3), 3) 5% DDGS (5% DDGS in phase 2 and 30% DDGS in phase 3), and 4) 30% DDGS (phases 2 and 3). Overall, pigs fed 30% DDGS during phases 2 and 3 had decreased (22.1 vs. 25.1 and 24.0 kg; P = 0.003) BW compared with CTL pigs and pigs that only received DDGS during phase 3. In addition, pigs fed 5 or 30% DDGS in phase 2 had decreased (422.7 or 390.0 vs. 468.2 g; P = 0.003) ADG compared with CTL pigs. However, pigs fed 0% DDGS during phase 2 had similar BW, ADG, and ADFI compared with CTL pigs. Experiment 2 was conducted to evaluate effects of DDGS, lactose, and their interaction on growth performance and health of weanling pigs. Dietary treatments included 1) CTL, 2) lactose (20%), 3) DDGS (15%), and 4) lactose + DDGS. Diets of interest were fed during phase 1 (d 0 to 14), and a common diet was fed during phase 2 (d 14 to 28). Pigs receiving DDGS in phase 1 had greater ADG (576.2 vs. 534.6 g; P = 0.01) and ADFI (814.9 vs. 751.6 g; P = 0.01) during phase 2 compared with non-DDGS-fed pigs. Pigs receiving lactose during phase 1 had greater ADG (214.7 vs. 177.2 g; P = 0.01) and G:F (741.0 vs. 660.3 g/kg; P = 0.01) and tended to have greater ADFI (289.3 vs. 267.6 g; P = 0.07) during phase 1 but decreased (537.7 vs. 573.1 g; P = 0.09) ADG during phase 2. Serum immunoglobulin analyses and fecal microbial profiling were conducted in both experiments as indicators of health status. No effects of dietary treatment were observed for serum immunoglobulin in either experiment. Fecal microbial profiling resulted in statistically significant effects of dietary treatment with respect to microbial similarity and diversity indices (Exp. 1) and lactic acid-producing bacteria (Exp. 2), where main effects of both lactose and DDGS were observed with respect to putative Lactobacillus reuteri (P < 0.05). Results from Exp. 1 indicate that decreased concentrations of DDGS early in the nursery phase may negatively affect growth performance; however, growth performance may be maintained when inclusion of high concentrations (30%) of DDGS is delayed until the late nursery period. Results from Exp. 2 indicate that lactose may be incorporated in nursery diets containing DDGS to help maintain growth performance, and DDGS and lactose may affect fecal microbial profiles.

Oral inoculation with Salmonella enterica serovar Typhimurium or Choleraesuis promotes divergent responses in the somatotropic growth axis of swine.

Enteric disease and immune challenge are processes that have detrimental effects on the growth performance of young swine. The current study tested the hypothesis that salmonella-induced enteric disease would perturb the endocrine growth axis in a serovar-dependent fashion. Specifically, we evaluated the effects of Salmonella enterica serovar Typhimurium (Typhimurium) and serovar Choleraesuis (Choleraesuis) on critical regulatory components of growth in young swine. Weaned pigs were housed 2 per pen with ad libitum access to feed and water in a 14-d experiment. Pigs were then repeatedly fed 10(8) cfu of either Choleraesuis or Typhimurium in dough balls, with control pigs receiving dough without bacteria. Bacteria were refed twice weekly. Rectal temperatures were monitored daily from d 0 to 7 and ADFI was measured through d 14. Pigs were weighed and samples of serum were obtained for circulating IGF-I on d 0, 7, and 14. At the conclusion of the study, samples of semitendinosus muscle and liver were obtained and subsequently assayed for IGF-I, IGFBP-3, and IGFBP-5 mRNA. Rectal temperatures were elevated in pigs given Choleraesuis from d 2 through 7 (P < 0.05) when compared with control pigs and pigs fed Typhimurium. Pigs receiving Choleraesuis had a substantially decreased feed intake on d 2, 3, 4, 7, 8, 9, and 10 (P < 0.01), with a trend for a reduction on d 5 (P = 0.08), and they experienced an approximately 25% reduction in BW compared with control pigs and pigs given Typhimurium by the conclusion of the study. Pigs given Choleraesuis also experienced marked reductions in circulating IGF-I on d 7 (P < 0.01 vs. control and Typhimurium), with reductions of lesser magnitude on d 14 (P = 0.07 vs. control and P < 0.05 vs. Typhimurium). Inoculation tended to affect liver IGFBP-3 mRNA (P = 0.08), for which expression tended to be elevated in pigs given Typhimurium and Choleraesuis. In contrast, IGFBP-3 mRNA relative abundance was increased (P < 0.03) in pigs given Typhimurium compared with control pigs. Muscle IGF-I mRNA was reduced in pigs given Choleraesuis compared with control pigs and pigs given Typhimurium (P < 0.05). Treatment tended to affect muscle IGFBP-3 mRNA (P = 0.10). Oral inoculation of growing pigs with Choleraesuis disrupted feed intake and BW gain, and this was accompanied by decreases in circulating IGF-I and reduced muscle expression of mRNA for IGF-I and IGFBP-3.

Effects of a yeast-dried milk product in creep and phase-1 nursery diets on growth performance, circulating immunoglobulin A, and fecal microbiota of nursing and nursery pigs1

Four experiments were conducted to evaluate effects of yeast-dried milk (YDM) product in creep and phase-1 nursery diets. In Exp. 1, 24 parity-4 litters were allotted to 3 dietary treatments (8 litters/treatment) including no creep (NC), control creep (CTL), and experimental creep (EC; 10% YDM). Creep diets were fed twice daily from d 7 after birth until weaning (23.6 ± 1.8 d). In Exp. 2, 108 weaned pigs were selected based on mean BW of pigs from the respective treatments in Exp. 1. For phase 1 (d 0 to 7 postweaning) of Exp. 2, NC and CTL pigs were fed the CTL diet and EC pigs continued to receive the EC diet. For phase 2 and 3 (d 7 to 28 postweaning) of Exp. 2, all pigs received a common diet containing antibiotics. Blood and fecal samples were collected on d 0, 7, 14, and 21 postweaning to evaluate serum IgA and fecal microbiota. In Exp. 1, pigs fed EC and CTL tended to have greater (P < 0.10) weaning BW compared with NC pigs. Pigs fed EC had greater (P < 0.05) ADFI compared with CTL pigs. In Exp. 2, pigs fed EC tended to have greater BW (P < 0.10) and greater ADG (P < 0.05) and ADFI (P < 0.01) compared with CTL and NC pigs (d 0 to 28). For serum IgA, EC and CTL pigs tended (P < 0.10) to have greater IgA compared with NC pigs. For microbial data, EC pigs had greater (P < 0.01) microbial diversity compared with CTL (d 7 postweaning). On d 7 and 21 postweaning, microbial similarity decreased (P < 0.01) in pigs fed EC compared with NC and CTL. Overall (d 0 to 14), lactobacilli gene copy numbers tended (P < 0.10) to be greater in EC (7.3 log10) compared with NC pigs (6.9 log10). In Exp. 3, 23 parity-1 litters were allotted to 3 dietary treatments as described for Exp. 1. In Exp. 4, 108 weaned pigs were selected based on mean BW of all pigs from Exp. 3 and dietary treatments were the same as described for Exp. 2 except no antibiotic was included in phase-2 diet. In Exp. 3, there were no treatment effects on litter ADFI, ADG, and serum IgA, but a tendency (P < 0.10) for lower Lactobacillus reuteri in the CTL (5.1 log10) compared with NC pigs (5.6 log10) was observed. Overall (d 0 to 21) in Exp. 4, pigs fed EC had greater ADG (P < 0.01) and G:F (P < 0.05) and tended to exhibit greater ADFI (P < 0.10) compared with the CTL. For microbial data, pigs fed CTL (4.8 log10) and EC (4.8 log10) had lower (P < 0.01) fecal Lactobacillus johnsonii compared with NC (5.2 log10). Microbial ecology and immune parameters are affected by YDM.

Effects of spray-dried porcine plasma on fecal microbiota in nursery pigs

Spray-dried porcine plasma (SDPP) has been considered as an alternative for in-feed antibiotics to improve pig growth performance; however, the effect of SDPP on gut microbiota is unknown. The objective of this study was to evaluate effects of feeding SDPP on fecal microbial communities of nursery pigs. Ninety-six weaned pigs were assigned to 16 pens, which were allotted to two dietary treatments, including the control or the control + SDPP (5% and 2.5% SDPP inclusion in phase 1 and 2, respectively) diet. Fecal samples were collected at d 0, 7, 14, 21, and 28. Multiplex sequencing of V3 region of the 16S rRNA gene was used to characterize the bacterial community structure of fecal samples. Pearsons correlation tests were performed in Calypso to identify bacterial taxa that were either positively or negatively associated with overall growth performance. Feeding SDPP altered microbial structure at family, genus, and operational taxonomic unit (OTU) classifications; however, fecal microbes shifted with time. At the family level, Clostridiaceae increased (P < 0.001) on d 14, but decreased (P < 0.05) on d 28 in SDPP-fed pigs compared with control pigs. Decreased Veillonellaceae (P < 0.05; d 14) and Lachnospiraceae (P = 0.001; overall) were observed in SDPP-fed pigs compared with control pigs. Feeding SDPP increased lactic acid-producing bacteria (Lactobacillus delbrueckii, d 7) and cellulolytic bacteria (Ruminococcus albus, d 7; Clostridium thermocellum, d 7 and 14; and Clostridium saccharoperbutylacetonicum/beijerinckii, d 14; and Megasphaera elsdenii, d 21). On d 28, feeding SDPP decreased (P < 0.05) Clostridium difficile compared with control pigs. In conclusion, feeding SDPP altered fecal microbial communities in nursery pigs. The results of this study may provide information to help explain the positive effects associated with feeding SDPP on nutrient digestibility and gut health of nursery pigs.