A window of opportunity : Modulation of the porcine gut microbiota and immune system by feed additives in early life

In early life and around weaning piglets are susceptible to infection due to abrupt dietary, social and environmental changes that may directly or indirectly impact gut health and animal performance. In the past, antibiotic growth promoters (AGPs) were intensively used in the livestock industry to prevent the development of gastro-intestinal disorders and to optimise animal performance. However, because of the worldwide concern on the emergence of multi-drug resistant bacteria, the use of in-feed AGPs has been banned in the European Union since 2006. As a result, sustainable alternatives to in-feed AGPs that promote animal health and performance received great interest. In the last few decades, researchers have been studying the effects of several promising microbial and non-microbial nutritional factors for pigs. Pre- and probiotics, for example, have been shown to influence the porcine gut microbiota, improve gut epithelial barrier function and modulate the immune system. It is evident that these complex systems are inherently linked to each other and are important determinants of health and disease. However, dietary interventions are mostly administered post-weaning. During this period, feed additives are less likely to exert strong effects because of the with age increasing resilience of the gut microbiota to (dietary) perturbations. In order to induce transient and/or long-lasting changes, we hypothesized that early life serves as a ‘window of opportunity’ to modulate the gut microbiota, the gut epithelial barrier, and the immune system with the aim to make pigs more resilient to infections during the weaning period and later in life.Dendritic cells (DCs) are important immune cells that link the innate and the adaptive immune system and are the first cells to encounter (dietary) antigens that pass the gut epithelial barrier. In this thesis we assessed the immunostimulating potential of feed additives in vitro, using fresh and cryopreserved bone marrow-derived dendritic cells (BMDCs). Prior to the in vitro study, we selected two promising feed additives that are commercially available; yeast-derived β-glucans (MacroGard®) and the Gram-negative probiotic strain E. coli Nissle 1917 (Mutaflor®/Ponsocol®). We showed that E. coli Nissle 1917 (EcN), but not yeast-derived β-glucans, induces a dose-dependent upregulation of the DC maturation marker CD80/86. In addition, we demonstrated that both feed additives promote a dose-dependent production of several cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10. Overall, EcN more significantly enhanced upregulation of CD80/86 and induced significantly higher levels of cytokines than yeast-derived β-glucans. These immunomodulatory effects were observed in fresh as well as cryopreserved in vitro cultured porcine BMDCs. Taken together, these results illustrated that both yeast-derived β-glucans and EcN are promising candidates to modulate the immune system, but they do so in a differential manner. A more mature DC phenotype may contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved BMDCs can be used as in vitro pre-screening tools that enable an evidence-based prediction of the potential immunomodulatory effects of feed additives.We also investigated the presence, abundance, and location of EcN in the porcine gastrointestinal tract after administration of different concentrations of EcN. In a pilot study, we sampled faeces at different time points, and at dissection we collected digesta from different gut segments. In addition, the pH of digesta were measured and the mesenteric lymph node (MLN) was removed to investigate if EcN is able to translocate to porcine gut-associated lymphoid tissues (GALT). Overall, no adverse health effects were observed that were attributed to EcN. EcN was detected by qPCR in faeces of all piglets that received either high, medium, or low concentrations of EcN (109, 108, or 107 CFU/mL, respectively). In addition, no major differences with regards to the relative abundance of EcN were observed between these piglets. Although at a low abundance, we also detected EcN in one of the control piglets, indicating cross-contamination. This underlines the importance of implementing adequate hygiene measures when designing in vivo studies that involve microbial products such as probiotics. Another interesting observation was the detection of EcN in the MLN of a single animal that received the highest concentration of EcN, which suggests that EcN is able to (spontaneously) translocate to GALT. Taken together, this pilot study shows that after peroral administration of EcN in early life, EcN can be detected in feaces, digesta, and MLN of pre-weaning piglets.After investigating the effects of feed additives in vitro study, we analyzed if oral supplementation of yeast-derived β-glucans (MacroGard®) and EcN in early life affects the porcine gut microbiota and immune system in vivo. In addition, this study also provided valuable insights into the temporal development of these complex systems. From day 2 after birth until day 44 post-weaning, piglets perorally received yeast-derived β-glucans, EcN or a control treatment (water) every other day. On days 21 and 45, all pigs were vaccinated against Salmonella enterica serovar Typhimurium (Salmoporc ®) to determine the effect of yeast-derived β-glucans and EcN on the vaccination response. To study the gut microbiota composition and immune function, faeces, digesta, blood, and tissue samples were collected at different time points during the study. Overall, yeast-derived β-glucans and EcN did not affect the vaccination response. In addition, (modest) effects were observed on faecal microbiota composition (e.g., reduced α-diversity) and immune parameters (e.g., enhanced IL-10 production by stimulated MLN cells). Furthermore, most effects were transient and only observed in the pre-weaning period, and no long-lasting effects were detected.Based on the in vitro and in vivo results presented in this thesis, I conclude that yeast-derived β-glucans and EcN are able to modulate the porcine gut microbiota and the immune system, but in a differential manner. In addition, the majority of these effects were observed pre-weaning, which is in line with our hypothesis that early life serves as a ‘window of opportunity’ to modulate the porcine gut microbiota and the immune system by feed additives. Effects observed post-weaning were modest and any (long-lasting) health effects induced by yeast-derived β-glucans and EcN remain to be elucidated