Influence of medium-chain fatty acids and short-chain organic acids on jejunal morphology and intra-epithelial immune cells in weaned piglets.

Medium-chain fatty acids (MCFA) and short-chain organic acids (SOA) are often used as feed additives in piglet diets. There are limited studies in pigs describing the impact of MCFA or SOA on gut morphology and the local immune system. The aim of this study was to investigate whether the supplementation of SOA (0.41% fumaric acid and 0.32% lactic acid), or the combination of SOA with MCFA (0.15% caprylic and capric acid) would have effects on gut morphology and intestinal immune cells in weaned piglets. A total number of 72 weaned piglets were randomly allocated into three experimental groups. Tissue samples of six animals per group were used to investigate the potential impact of the feed additives on villus length and crypt depth of the jejunum and to quantify intra-epithelial lymphocytes (IEL). CD3-positive IEL were determined via immunohistochemistry (IHC) and flow cytometry (FC), whereas CD2-, CD5-, CD8ß-, CD16- and γδ TCR-positive IEL were only analysed by FC. The supplementation of MCFA and SOA did not significantly affect morphometric data. The FC data indicated that SOA significantly increased the quantity of CD2- CD8- γδ T cells in the jejunum epithelium. Both IHC and FC analyses of pig jejunum confirmed that the majority of IEL expressed the surface marker CD3 and could be classified as cytotoxic T lymphocytes. In conclusion, the data indicated that SOA increased the proportion of CD2- CD8- γδ T cells in the jejunal epithelium. Thus, SOA might enable a beneficial effect on the local immunity by increasing the constitutive number of potential effector cells to defeat infectious diseases.

Bovine milk-based formula leads to early maturation-like morphological, immunological, and functional changes in the jejunum of neonatal piglets.

Artificial rearing and formula feeding is coming more into the focus due to increasing litter sizes and limited nursing capacity of sows. The formula composition is important to effectively support the development of the gut and prevent intestinal dysfunction in neonatal piglets. In this study, newborn piglets ( = 8 per group) were fed a bovine milk-based formula (FO), containing skimmed milk and whey as the sole protein and carbohydrate sources, or were suckled by the sow (sow milk [SM]). After 2 wk, tissue from the jejunum was analyzed for structural (i.e., morphometry) and functional (i.e., disaccharidase activity, glucose transport, permeability toward macromolecules, and immune cell presence) changes and concomitant expression of related genes. Formula-fed piglets had more liquid feces ( < 0.05) over the entire experimental period. Although FO contained twice as much lactose (46% on a DM basis) as SM (21%) and no maltose or starch, the lactase activity was lower ( < 0.05) and glucose transport capacity was higher ( < 0.05) in FO-fed pigs. The relative proportion of intraepithelial natural killer cells and proinflammatory cytokine gene expression (, , and ) was higher in FO-fed pigs ( < 0.05). Piglets fed FO had deeper crypts, larger villus area, and higher expression of caspase 3 and proliferating cell nuclear antigen ( < 0.05). Epithelial permeability toward fluorescein isothiocyanate-dextran was higher and expression of claudin-4 was lower in FO-fed piglets ( < 0.05). The data suggest an early response to bovine milk-based compounds in the FO accompanied with early onset of functional maturation and impaired barrier function. Whether lactose, absence of species-specific protective factors, or antigenicity of foreign proteins lead to to the observed intestinal reactions requires further clarification.

Effect of dietary zinc oxide on jejunal morphological and immunological characteristics in weaned piglets.

The aim of this study was to evaluate age-related changes and the effect of dietary Zn concentration on morphological and immunological characteristics in the gastrointestinal tract of piglets. A total of 96 purebred Landrace piglets were weaned at the age of 26 ± 1 d, and randomly allocated into 3 treatment groups fed with low (57 mg Zn/kg), medium (164 mg Zn/kg), and high (2425 mg Zn/kg) dietary Zn (ZnO). Piglets (4 males and 4 females per treatment group) were killed at 33 ± 1, 40 ± 1, 47 ± 1, and 54 ± 1 d of age. In the jejunum, villus height, crypt depth, and the number of goblet cells producing neutral, acidic, sulfated, and sialylated mucins were measured. Intraepithelial lymphocytes were characterized by flow cytometry and the gene expression of mucin 2 (MUC2), mucin 20 (MUC20), ß-defensin 3, and trefoil factor 3 (TFF3) was determined by reverse transcription quantitative PCR. Villus height and crypt depth in the jejunum showed age related differences (P < 0.01), whereas the dietary concentrations of Zn had no effect. The mucin types were modified mainly by age, and dietary Zn had no effect in the proximal jejunum. In the distal jejunum, age and Zn had effects on the mucin types. The abundance of sulfomucins decreased (P < 0.001) and sialomucins increased with age (P < 0.001), while high dietary ZnO reduced the sulfomucins (P < 0.001) and increased the sialomucins (P < 0.05) in the crypts. The phenotypes of lymphocytes in the epithelium of the proximal jejunum showed relatively constant percentages of T-cells, as well as natural killer cells. High dietary Zn treatment led to a reduced abundance of CD8(+) γδ T-cells (P < 0.05). The apportionment of different cytotoxic T-cell was age dependent. Although the percentage of CD4(-)CD8ß(+) increased (P < 0.01), the relative amount of CD4(+)CD8ß(+) decreased with age (P < 0.05). The expression of MUC2 and MUC20 was not influenced by age or dietary Zn concentration. High Zn intakes resulted in a reduced gene expression of ß-defensin 3 (P < 0.05), but did not affect the expression of TFF3. It is concluded that Zn in the form of ZnO appears to have specific effects on the innate and adaptive gut associated immune system of piglets. These might contribute to the positive effects of Zn on the prevention of postweaning diarrhea.

A novel lineage transcription factor based analysis reveals differences in T helper cell subpopulation development in infected and intrauterine growth restricted (IUGR) piglets.

Research in mouse and human clearly identified subsets of T helper (Th) cells based on nuclear expression of specific lineage transcription factors. In swine, however, transcription factor based detection of functional subpopulations of porcine Th cells by flow cytometry is so far limited to regulatory T cells via Foxp3. T-bet and GATA-3 are the transcription factors that regulate commitment to Th1 or Th2 cells, respectively. In this study we prove GATA-3 and T-bet expression in porcine CD4(+) cells polarized in vitro. Importantly, GATA-3 and T-bet expressing cells were detectable in pigs infected with pathogens associated with Th2 and Th1 immune responses. Increased frequencies of GATA-3 positive CD4(+) cells are found in vivo in pigs experimentally infected with the nematode Trichuris suis, whereas porcine reproductive and respiratory syndrome virus (PRRSV) infection elicited T-bet positive CD4(+) T cells. Analysing the immune status of pre-weaning piglets with intrauterine growth restriction (IUGR) we found an increased expression of Foxp3, T-bet and GATA-3 in CD4(+) and CD4(+)CD8(+) double-positive T cells in systemic and intestinal compartments of IUGR piglets. Hence, we established the detection of porcine Th1 and Th2 cells via T-bet and GATA-3 and show that the porcine lineage transcription factors are differentially regulated very early in life depending on the developmental status.

Cross-talk Between Host, Microbiome and Probiotics: A Systems Biology Approach for Analyzing the Effects of Probiotic Enterococcus faecium NCIMB 10415 in Piglets.

A comprehensive data-set from a multidisciplinary feeding experiment with the probiotic Enterococcus faecium was analyzed to elucidate effects of the probiotic on growing piglets. Sixty-two piglets were randomly assigned to a control (no probiotic treatment) and a treatment group (E. faecium supplementation). Piglets were weaned at 26 d. Age-matched piglets were sacrificed for the collection of tissue samples at 12, 26, 34 and 54 d. In addition to zootechnical data, the composition and activity of intestinal microbiota, immune cell types, and intestinal responses were determined. Our systems analysis revealed clear effects on several measured variables in 26 and 34 days old animals, while response patterns varied between piglets from different age groups. Correlation analyses identified reduced associations between intestinal microbial communities and immune system reactions in the probiotic group. In conclusion, the developed model is useful for comparative analyses to unravel systems effects of dietary components and their time resolution. The model identified that effects of E. faecium supplementation most prominently affected the interplay between intestinal microbiota and the intestinal immune system. These effects, as well as effects in other subsystems, clustered around weaning, which is the age where piglets are most prone to diarrhea.

Bacillus cereus var. Toyoi modulates the immune reaction and reduces the occurrence of diarrhea in piglets challenged with Salmonella Typhimurium DT104.

A feeding trial with sows and their piglets was performed with the probiotic feed additive Bacillus cereus var. Toyoi in two consecutive experimental periods. Sows (n = 8) were allocated into treatment (Bc) and control (CO) groups. Sows of Bc group (n = 4) were fed 3.14 × 10(5) cfu/g Bacillus cereus var. Toyoi with the diet from d 87 of pregnancy on. Their piglets received Bacillus cereus var. Toyoi supplemented feed (8.7 × 10(5) cfu/g) starting on d 14 of life and further on after weaning (6.5 × 10(5) cfu/g), whereas sows and piglets of the CO group remained untreated. One day after weaning, piglets from both groups (n = 24 each) were challenged orally with Salmonella Typhimurium DT104 (3 × 10(9) viable bacteria). Health status, shedding of B. cereus in the feces, and performance of the piglets were monitored. At 24 h, 72 h, 6 d, and 28 d postinfection (PI), six piglets from each group were euthanized and cell counts of Salmonellae were determined in the colon contents, mesenteric lymph nodes, and tonsils. Peripheral blood mononuclear cells and jejunal intraepithelial lymphocytes (IEL) were analyzed by flow cytometry. The incidence of scours was lower in the Bc group than in the CO group (P = 0.004). In addition, the fecal shedding of Salmonella was significantly lower in the Bc group at 25 d PI (P = 0.004). Shortly after infection, the γδ T cells were significantly less frequent in the blood of Bc piglets. For both CD8-positive γδ T cells (P = 0.033) and CD8-negative γδ T cells (P = 0.028), significant differences were observed. Furthermore, 28 d PI piglets from the treated group showed lower numbers of γδ T cells in the jejunal epithelium (P = 0.036). To investigate the role of intestinal γδ T cells during the infection with S. Typhimurium, IEL were gained from six healthy 40-d-old piglets and infected in vitro with S. Typhimurium. CD8ß cells and γδ T cells were detected by flow cytometry and the infection rates of both populations in the cell suspensions were compared. The infection rate (IR) of γδ T cells was higher in all six cell suspensions than the IR of CD8ß expressing T cells (P = 0.002). In conclusion, B. cereus var. Toyoi supplementation of sows and their piglets had a positive impact on the health status of the piglets after a challenge with Salmonella, likely due to an altered immune response marked by reduced frequencies of CD8+ γδ T cells in the peripheral blood and the jejunal epithelium.

Effects of dietary combinations of organic acids and medium chain fatty acids on the gastrointestinal microbial ecology and bacterial metabolites in the digestive tract of weaning piglets.

Organic short and medium chain fatty acids are used in diets for piglets because they have an impact on the digestive processes and the intestinal microbiota. In this study, 48 pens (2 piglets/pen) were assigned randomly to 4 diets, without additive (control), with organic acids (OA; 0.416% fumaric and 0.328% lactic acid), with medium chain fatty acids (MCFA; 0.15% caprylic and capric acid), and a combination of OA and MCFA, to assess changes in the gastrointestinal microbiota with 12 pens per diet. Eight to nine piglets from each group were euthanized after 4 wk. Organic acids, MCFA, and pH in the digesta were determined and the intestinal microbiota was quantified by real-time PCR. The different diets had no effect on the growth performance. Concentration of added fumaric acid was below the detection limit in the upper small intestine whereas the concentration of lactic acid in the digesta was not affected by the treatments. The added MCFA was recovered in the MCFA treated groups in the stomach, but the concentrations declined in the upper small intestine. Concentration of short chain fatty acids was reduced in the colon digesta in piglets fed diets with OA compared with those fed unsupplemented diets (P = 0.029). The MCFA resulted in a pH reduction of the digesta, likely because of the effect on bacterial acid production. The addition of OA increased cell counts of Bacteroides-Porphyromonas-Prevotella group and clostridial clusters XIVa, I, and IV in the stomach, the clostridial cluster XIVa in the jejunum, and Bacteroides-Porphyromonas-Prevotella in the ileum and reduced counts of Streptococcus spp. in the colon (P < 0.05). The MCFA induced only minor changes in the gastrointestinal microbiota but increased cell counts for the Escherichia-Hafnia-Shigella group in the jejunum and the clostridial cluster XIVa in the colon digesta (P < 0.05). In the colon of piglets fed diets with organic OA, reduced mean cell counts of STb (est-II) positive Escherichia coli were found. In conclusion, OA and MCFA had effects on the intestinal microecology in piglets. The decrease of the intestinal pH and the reduction of E. coli virulence genes by OA could make the combination of short chain fatty acids and MCFA as interesting gut flora modifiers, which can eventually prevent postweaning diarrhea.

Proliferation capacity of T-lymphocytes is affected transiently after a long-term weight gain in Beagle dogs.

Across species obesity is associated with several disorders but in companion animals little information is available on the impact of chronic obesity on immune competence. The aim of the present study was to investigate whether weight gain and stable obese bodyweight affects the immune cell response. Obesity was induced in eight adult healthy beagle dogs (weight gain group; WGG) by a weight gain period (WGP) of 47 weeks, which was immediately followed by a period (stable period: SP) of stable obesity of 26 weeks. Eight adult healthy beagle dogs were included as a control group (CG) and remained at their ideal bodyweight throughout the entire study. Body composition was measured at five intervening time-points. Concentration of serum leptin and inflammatory cytokines, functionality of lymphocytes and phagocytic activity of neutrophils and monocytes were evaluated at ten intervening time-points. Serum leptin concentration was rising during the WGP in the WGG but went to lower concentrations during the SP. At the end of long-term weight gain, a decreased mitogen-induced proliferation of T-lymphocytes was noted but this alteration seemed to be transient after stabilization of bodyweight. This finding may imply an altered immune response for dogs with different energy balances. However, no systemic low grade inflammation or alteration in other immune cell functions was observed. Consequently it is suggested that the change in energy balance during the onset of obesity (becoming obese versus being obese), evokes an additional obesity-related disorder in dogs, i.e. impaired T-lymphocyte immune function.