Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs

Abstract

Background: Immunoregulatory probiotics (immunobiotics) have been proposed to improve piglets' immune system to avoid intestinal infections and reduce unproductive inflammation after weaning. Previously, it was demonstrated that Lactobacillus jensenii TL2937 (LjTL2937) attenuated the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial (PIE) cells and antigen presenting cells (APCs) from porcine Peyer's patches (PP).

Objective: In view of the critical importance of PIE-APCs interactions in the regulation of intestinal immune responses, we aimed to examine the effect of LjTL2937 on activation patterns of APCs from swine PPs in co-cultures with PIE cells. In addition, we investigated whether LjTL2937 was able to beneficially modulate intestinal immunity of piglets after weaning to improve immune-health status.

Results: Stimulation of PIE-APCs co-cultures with LjTL2937 increased the expression of MHC-II, CD80/86, IL-10, and Bcl-3 in CD172a+CD11R1- and CD172a+CD11R1high APCs. In addition, the TL2937 strain caused the upregulation of three negative regulators of TLR4 in PIE cells: MKP-1, Bcl-3 and A20. These changes significantly reduced the inflammatory response triggered by TLR4 activation in PIE-APCs co-cultures. The in vivo experiments using castrated male piglets (crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D))of 3 weeks of age demonstrated that feeding with LjTL2937 significantly reduced blood complement activity and C reactive protein concentrations while no changes were observed in blood leukocytes, ratio of granulocytes to lymphocyte numbers, macrophages' activity and antibody levels. In addition, treatment with LjTL2937 significantly improved growth performance and productivity, and increased carcass quality.

Conclusions: We demonstrated that the use of immunobiotics strains like LjTL2937, as supplemental additives for piglets feedings, could be used as a strategy to maintain and improve intestinal homeostasis; that is important for the development of the pig and for health and performance throughout the productive life of the animal.

Figure 1

Effect of Lactobacillus jensenii TL2937 on porcine intestinal epithelial (PIE) cells co-cultured with adherent population from swine Peyer’s Patches (PPs). Antigen-presenting cells (macrophages and dendritic cells) from PPs were obtained after their adherence to glass. PIE-Adherent cells co-cultures were treated with L. jensenii TL2937 or L. plantarum TL2966 for 48 h. Untreated PIE-adherent cells co-cultures were used as controls. (A) Expression of IL-1β, IL-8, IL-6, MCP-I, and TGF-β mRNAs was examined in PIE cells using RT-qPCR. (B) Expression of IL-1β, IL-6, IL-10, IFN-γ, and TGF-β mRNAs was examined in adherent cells using RT-qPCR. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly. The results represent data from three independent experiments using ileal PPs from at least three different swine.

Figure 2

Effect of Lactobacillus jensenii TL2937 on porcine intestinal epithelial (PIE) cells co-cultured with adherent population from swine Peyer’s Patches (PPs). Antigen-presenting cells (macrophages and dendritic cells) from PPs were obtained by taking advantage of their capacity to adhere to glass. PIE-Adherent cells co-cultures were treated with L. jensenii TL2937 or L. plantarum TL2966 for 48 h. Untreated PIE-adherent cells co-cultures were used as controls. Expression of MHC-II, CD80/86, IL-10, and IFN-γ was studied in CD172a⁺CD11R1^-, CD172a⁺CD11R1^high, and CD172a^-CD11R1^low adherent cells by flow cytometric analysis. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly. The results represent data from three independent experiments using ileal PPs from at least three different swine.

Figure 3

Effect of Lactobacillus jensenii TL2937 on porcine intestinal epithelial (PIE) cells co-cultured with adherent population from swine Peyer’s Patches (PPs) under inflammatory conditions. PIE-Adherent cells co-cultures were treated with L. jensenii TL2937 or L. plantarum TL2966 for 48 h. Untreated PIE-adherent cells co-cultures were used as controls. After lactobacilli stimulation, treated cells and untreated controls were challenge with ETEC (5 × 10⁷ cells/ml). (A) Expression of IL-1β, IL-8, IL-6, MCP-I, and TGF-β mRNAs was examined in PIE cells using RT-qPCR. (B) Expression of IL-1β, IL-6, IL-10, IFN-γ, and TGF-β mRNAs was examined in adherent cells using RT-qPCR. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly. The results represent data from three independent experiments using ileal PPs from at least three different swine.

Figure 4

Effect of Lactobacillus jensenii TL2937 on porcine intestinal epithelial (PIE) cells co-cultured with adherent population from swine Peyer’s Patches (PPs) under inflammatory conditions. PIE-Adherent cells co-cultures were treated with L. jensenii TL2937 or L. plantarum TL2966 for 48 h. Untreated PIE-adherent cells co-cultures were used as controls. After lactobacilli stimulation, treated cells and untreated controls were challenge with ETEC (5 × 10⁷ cells/ml). Expression of MHC-II, CD80/86, IL-10, and IFN-γ was studied in CD172a⁺CD11R1^-, CD172a⁺CD11R1^high, and CD172a^-CD11R1^low adherent cells by flow cytometric analysis. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly. The results represent data from three independent experiments using ileal PPs from at least three different swine.

Figure 5

Effect of Lactobacillus jensenii TL2937 on negative regulators of the TLR signaling pathway in porcine intestinal epithelial (PIE) cells co-cultured with adherent population from swine Peyer’s Patches (PPs). PIE-Adherent cells co-cultures were treated with L. jensenii TL2937 or L. plantarum TL2966 for 48 h. Untreated PIE-adherent cells co-cultures were used as controls. Expression of SIGIRR, A20, Bcl-3, MKP-1, and IRAK-M mRNAs was measured in (A) PIE cells and (B) adherent cells using RT-qPCR. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly. The results represent data from three independent experiments using ileal PPs from at least three different swine.

Figure 6

Effect of Lactobacillus jensenii TL2937 on piglets’ growth and immune health. Pigs were grown from 3 weeks of age until week 24. Five pigs were used for each experimental group. The Control group was fed only the balanced conventional diet without antimicrobials ad libitum. The Medium, TL2937 and TL2766 groups were fed balanced conventional diet with supplemental bacteria medium only (200 g/day), L. jensenni TL2937 (3 × 10⁸ cfu/g) or L. plantanum TL2766 (3 × 10⁸ cfu/g) respectively, from 3 to 17 weeks of age. Body weight measurement, detection of enterotoxigenic Escherichia coli in feces, and plasma levels of alternative complement activity (ACA) and C reactive protein (CRP) were determined every 2 weeks. The line in body weight indicates the suitable body weight for shipping in Japan. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly.

Figure 7

Effect of Lactobacillus jensenii TL2937 on carcass quality. Pigs were grown from 3 weeks of age until week 24. Five pigs were used for each experimental group. The Control group was fed only the balanced conventional diet without antimicrobials ad libitum. The Medium, TL2937 and TL2766 groups were fed balanced conventional diet with supplemental bacteria medium only (200 g/day), L. jensenni TL2937 (3 × 10⁸ cfu/g) or L. plantanum TL2766 (3 × 10⁸ cfu/g) respectively, from 3 to 17 weeks of age. After sacrifice of pigs, carcass weight, oil-back fat thickness and unsaturated fatty acids were evaluated. Carcass grading evaluation was performed based on the standards of Japanese Meat Grading Association. Carcass meats were judged by high, middle or mediocre classes and out of standards. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly.

Figure 8

Effect of Lactobacillus jensenii TL2937 on meat quality. Pigs were grown from 3 weeks of age until week 24. Five pigs were used for each experimental group. The Control group was fed only the balanced conventional diet without antimicrobials ad libitum. The Medium, TL2937 and TL2766 groups were fed balanced conventional diet with supplemental bacteria medium only (200 g/day), L. jensenni TL2937 (3 × 10⁸ cfu/g) or L. plantanum TL2766 (3 × 10⁸ cfu/g) respectively, from 3 to 17 weeks of age. Visual impression of pork meat and evaluation of tenderness, juicy and overall palatability was performed by a panel of untrained persons. Pork from the different experimental groups was cooked with the same recipe and process. Panelists complete a questionnaire evaluating juicy, tenderness and overall palatability of pork. After tasting, all the dishes, the panelists were requested to grade taste based on three categories: distasteful, acceptable and extremely delicious. Values for bars with different letters were significantly different (P < 0.05). Values for bars with shared letters do not differ significantly.

Figure 9

Proposed mechanism for the immunomodulatory effect of Lactobacillus jensenii TL2937 on porcine intestinal mucosa. B-cell lymphoma 3-encoded protein (Bcl-3), Enterotoxigenic Escherichia coli (ETEC), Interferon (IFN)-γ, Interleukin (IL), Interleukin-1 receptor-associated kinase M (IRAK-M), Mitogen- activated protein kinase (MAPK), Mitogen-activated protein kinase 1 (MPK-1), Monocyte chemotactic protein-1 (MCP-1), Nuclear factor κB (NF-κB), Single immunoglobulin IL-1-related receptor (SIGIRR), Toll-like receptor (TLR)-4, Transforming growth factor (TGF)-β, Tumor necrosis factor (TNF), Ubiquitin-editing enzyme A20 (A20).