Effects of ß-glucan on Salmonella enterica serovar Typhimurium swine colonization and microbiota alterations.

BACKGROUND: The 2017 Veterinary Feed Directive eliminated the use of medically important antibiotics for growth promotion of food animals; thus, alternative growth promoters are highly desirable by food animal producers to enhance animal health and reduce pathogen colonization, including the human foodborne pathogen Salmonella. ß(1-3)(1-6)-D-glucan (ß-glucan) is a soluble fiber with prebiotic characteristics; it has been shown to modulate immune and intestinal functions that strengthen swine resistance to health challenges such as bacterial infections when supplemented in the diets of growing pigs. The current study evaluated the effects of a ß-glucan product on gut microbial community structure as well as Salmonella shedding and intestinal colonization. RESULTS: Five-week-old pigs were fed a ß-glucan amended diet at 500 g/ton (n = 13) or a non-amended control diet (n = 14) for three weeks, followed by inoculation of the 27 pigs with 1 × 109 colony forming units of Salmonella enterica serovar Typhimurium strain UK1. While remaining on the respective diets, fecal samples collected at 2, 4, 7, and 16 days post-inoculation (dpi) were similar for Salmonella shedding counts between the two diets. At 16 dpi, Salmonella counts were significantly lower in the cecal contents of the ß-glucan-fed pigs (P = 0.0339) and a trend towards a reduction was observed in the Peyer's patches region of the ileum (P = 0.0790) compared to the control pigs. Pigs fed ß-glucan for three weeks exhibited an increase in members of the Clostridia class in their fecal microbial communities, and after inoculation with Salmonella, several potentially beneficial microorganisms were enriched in the microbiota of ß-glucan-fed pigs (Lactobacillus, Ruminococcaceae, Prevotellaceae, Veillonellaceae, Bifidobacterium and Olsenella). CONCLUSION: Administration of ß-glucan altered the swine gut microbiome and reduced Salmonella colonization in the cecal contents.

Effect of dietary ß-glucan on intestinal microbial diversity and Salmonella vaccine immunogenicity and efficacy in pigs.

Alternatives to antibiotics to improve animal performance, limit the negative impact of infectious disease, and/or reduce colonization with foodborne pathogens is a major focus of animal agricultural research. ß-glucans, a generally-recognized-as-safe (GRAS) product derived from various sources, are used in swine and can serve as both a prebiotic and/or stimulant of the immune system given the expression of ß-glucan receptors on immune cells. When supplied in the diet of nursery pigs, it is unclear how dietary additives, particularly those known to modulate immune status, impact immunogenicity and efficacy of mucosal-delivered vaccines. Salmonellosis is one of the most common bacterial foodborne infections in the United States, and consumption of contaminated pork is a major source of human infection. Reduction of foodborne Salmonella in pigs via vaccination is one strategy to reduce contamination risk and subsequently reduce human disease. We examined the ability of dietary ß-glucan to modulate fecal microbial diversity, and immunogenicity and efficacy of a mucosally-delivered, live-attenuated Salmonella vaccine during the nursery period. While dietaryß-glucan did modulate fecal alpha diversity, it did not alter the induction of peripheral Salmonella-specific IFN-γ secreting Tcells or Salmonella-specific IgA in oral fluids. In addition, vaccination reduced Salmonella enterica serovar Typhimurium fecal shedding and tissue colonization. Overall, addition of ß-glucan to the nursery diet of pigs impacted the microbiota but did not alter mucosal vaccine immunogenicity and efficacy.

Effects of dietary live yeast supplementation on growth performance and biomarkers of metabolism and inflammation in heat-stressed and nutrient-restricted pigs.

Study objectives were to determine the effects of dietary live yeast (Saccharomyces cerevisiae strain CNCM I-4407; ActisafHR+; 0.25g/kg of feed; Phileo by Lesaffre, Milwaukee, WI) on growth performance and biomarkers of metabolism and inflammation in heat-stressed and nutrient-restricted pigs. Crossbred barrows (n = 96; 79 ± 1 kg body weight [BW]) were blocked by initial BW and randomly assigned to one of six dietary-environmental treatments: 1) thermoneutral (TN) and fed ad libitum the control diet (TNCon), 2) TN and fed ad libitum a yeast containing diet (TNYeast), 3) TN and pair-fed (PF) the control diet (PFCon), 4) TN and PF the yeast containing diet (PFYeast), 5) heat stress (HS) and fed ad libitum the control diet (HSCon), or 6) HS and fed ad libitum the yeast diet (HSYeast). Following 5 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (7 d), pigs were housed in TN conditions (20 °C) and fed their respective dietary treatments ad libitum. During P2 (28 d), HSCon and HSYeast pigs were fed ad libitum and exposed to progressive cyclical HS (28-33 °C) while TN and PF pigs remained in TN conditions and were fed ad libitum or PF to their HSCon and HSYeast counterparts. Pigs exposed to HS had an overall increase in rectal temperature, skin temperature, and respiration rate compared to TN pigs (0.3 °C, 5.5 °C, and 23 breaths per minute, respectively; P < 0.01). During P2, average daily feed intake (ADFI) decreased in HS compared to TN pigs (30%; P < 0.01). Average daily gain and final BW decreased in HS relative to TN pigs (P < 0.01); however, no differences in feed efficiency (G:F) were observed between HS and TN treatments (P > 0.16). A tendency for decreased ADFI and increased G:F was observed in TNYeast relative to TNCon pigs (P < 0.10). Circulating insulin was similar between HS and TN pigs (P > 0.42). Triiodothyronine and thyroxine levels decreased in HS compared to TN treatments (~19% and 20%, respectively; P < 0.05). Plasma tumor necrosis factor-alpha (TNF-α) did not differ across treatments (P > 0.57) but tended to decrease in HSYeast relative to HSCon pigs (P = 0.09). In summary, dietary live yeast did not affect body temperature indices or growth performance and had minimal effects on biomarkers of metabolism; however, it tended to improve G:F under TN conditions and tended to reduce the proinflammatory mediator TNF-α during HS. Further research on the potential role of dietary live yeast in pigs during HS or nutrient restriction scenarios is warranted.

Effect of live yeast Saccharomyces cerevisiae supplementation on the performance and cecum microbial profile of suckling piglets.

One mechanism through which S. cerevisiae may improve the performance of pigs is by altering the composition of the gut microbiota, a response that may be enhanced by early postnatal supplementation of probiotics. To test this hypothesis, newborn piglets (16 piglets/group) were treated with either S. cerevisiae yeast (5 x 109 cfu/pig: Low) or (2.5 x 1010 cfu/piglet: High) or equivalent volume of sterile water (Control) by oral gavage every other day starting from day 1 of age until weaning (28±1 days of age). Piglet body weight was recorded on days 1, 3, 7, 10, 17, 24 and 28 and average daily gain (ADG) calculated for the total period. At weaning, piglets were euthanized to collect cecum content for microbial profiling by sequencing of the 16S rRNA gene. ADG was higher in both Low and High yeast groups than in Control group (P<0.05). Alpha diversity analyses indicated a more diverse microbiota in the Control group compared with Low yeast group; the High yeast being intermediate (P < 0.01). Similarly, Beta diversity analyses indicated differences among treatments (P = 0.03), mainly between Low yeast and Control groups (P = 0.02). The sparse Partial Least Squares Discriminant Analysis (sPLS-DA) indicated that Control group was discriminated by a higher abundance of Veillonella, Dorea, Oscillospira and Clostridium; Low yeast treated pigs by higher Blautia, Collinsella and Eubacterium; and High yeast treated pigs by higher Eubacterium, Anaerostipes, Parabacteroides, Mogibacterium and Phascolarctobacterium. Partial Least Squares (PLS) analysis showed that piglet ADG was positively correlated with genus Prevotella in High yeast group. Yeast supplementation significantly affected microbial diversity in cecal contents of suckling piglets associated with an improvement of short chain fatty acid producing bacteria in a dose-dependent manner. In conclusion, yeast treatment improved piglet performance and shaped the piglet cecum microbiota composition in a dose dependent way.

Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated from PBMC, thymus, and lung lymphocytes of young pigs.

Interleukin-17 (IL-17) producing cells, referred to as Th17, have recently emerged as a third subset of the T helper (Th) cell family. Studies in mice have demonstrated that Th17 cells and their associated cytokines are involved in several autoimmune diseases and host defense against infection. Murine Th17 cells differentiate from naïve CD4(+) T-cells in the presence of TGFß and IL-6, however, there are contradicting reports as to the role of TGFß in the differentiation of human Th17 cells and very little is known about these cells in other animals. We report here the presence of IL-17 secreting lymphocytes in the lung and peripheral blood of pigs. The cDNA of porcine IL-17 gene was cloned and sequenced from activated lung lymphocytes and PBMC from piglets. A 17kDa recombinant protein was expressed and purified both under denaturing and native conditions from E. coli BL21 cells. Furthermore, we demonstrate that TGFß in the presence of IL-6 and/or IL-1ß induces in vitro differentiation of Th17 cells from naïve porcine CD4(+) thymocytes.

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis.

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel(®) immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.

Immunization of newborn and adult mice with low numbers of BCG leads to Th1 responses, Th1 imprints and enhanced protection upon BCG challenge.

Neonatal bacille Calmette-Guerin (BCG) vaccination is widely employed to protect against tuberculosis. Predominant Th1 but not mixed Th1/Th2 responses are thought to be protective. If so, effective vaccination must cause Th1 imprints. The immune system of infants differs from that of adults and such differences could critically affect neonatal vaccination. We demonstrate that BCG infection of infant and adult mice produces similar responses. Infection with low and high numbers of BCG, respectively, leads to sustained Th1 and mixed Th1/Th2 responses. Low-dose but not high-dose infection also results in Th1 imprints, guaranteeing a Th1 response upon high-dose challenge, and resulting in optimal bacterial clearance. Our observations on low-dose Th1 imprinting are intriguing in the context of the well-known madras trial. In this trial, the highest dose of BCG, which had insignificant side effects, was administered to over 250,000 human subjects. This high-dose vaccination resulted in insignificant protection against tuberculosis.