Long-lasting effects of early-life antibiotic treatment and routine animal handling on gut microbiota composition and immune system in pigs

Abstract

Background: In intensive pig husbandry systems, antibiotics are frequently administrated during early life stages to prevent respiratory and gastro-intestinal tract infections, often in combination with stressful handlings. The immediate effects of these treatments on microbial colonization and immune development have been described recently. Here we studied whether the early life administration of antibiotics has long-lasting effects on the pig's intestinal microbial community and on gut functionality.

Methodology/principal findings: To investigate the long-lasting effect of early-life treatment, piglets were divided into three different groups receiving the following treatments: 1) no antibiotics and no stress, 2) antibiotics and no stress, and 3) antibiotics and stress. All treatments were applied at day four after birth. Sampling of jejunal content for community scale microbiota analysis, and jejunal and ileal tissue for genome-wide transcription profiling, was performed at day 55 (~8 weeks) and day 176 (~25 weeks) after birth. Antibiotic treatment in combination with or without exposure to stress was found to have long-lasting effects on host intestinal gene expression involved in a multitude of processes, including immune related processes.

Conclusions/significance: The results obtained in this study indicate that early life (day 4 after birth) perturbations have long-lasting effects on the gut system, both in gene expression (day 55) as well as on microbiota composition (day 176). At day 55 high variance was observed in the microbiota data, but no significant differences between treatment groups, which is most probably due to the newly acquired microbiota during and right after weaning (day 28). Based on the observed difference in gene expression at day 55, it is hypothesized that due to the difference in immune programming during early life, the systems respond differently to the post-weaning newly acquired microbiota. As a consequence, the gut systems of the treatment groups develop into different homeostasis.

Fig 1. Schematic representation of the experimental design.

Fig 2. Diversity in microbiota in the three treatment groups on day 55 and 176.

The Shannon index (y-axis) was calculated for all three treatments (T1, T2, and T3) on both days (55 and 176) (x-axis) based on probe-level data from the PITChip.

Fig 3. Triplot for RDA analysis of jejunal microbiota composition on day 55 and day 176.

Nominal environmental variables T1, T2 and T3 are represented by red triangles (▲). Samples are grouped by treatment: T1 (red; ○), T2 (blue; □) and T3 (green; ◇), each symbol represents a pool of four pigs, and numbers represent pool identifiers. A) Top-panel shows the RDA analysis of jejunal microbiota composition on day 55. Microbial groups contributing at least 40% to the explanatory axes are represented as vectors. Both axes together explain 15% of the total variance in the dataset. B) Bottom-panel shows the RDA analysis of jejunal microbiota composition on day 55. Microbial groups contributing at least 52% to the explanatory axes are represented as vectors. Both axes together explain 27.8% of the total variance in the dataset.

Fig 4. Principal Component Analysis of jejunal and ileal tissue gene expression for three different treatments at day 55 and 176.

Each symbol represents all expressed genes (approximately 44k probes) of a particular sample. A) top-panel represents day 55 and B) bottom-panel represents day 176. Three different treatments are depicted, T1 (red), T2 (blue) and T3 (green) and two different tissues, jejunum (JEJ, triangles) and ileum (IL, squares).

Fig 5. Schematic representation of results.

Overview of the time-line, birth (day 0), administration of the treatments (day 4), measurements days 8, 55, and 176, as well as the hypothetical interpretation of all results from the whole experiment, results from the previous paper about day 8 are included too [16]. On the left we categorized the gut system in three different ‘blocks’, immune programming which occurs in early life, followed by an instable period which includes weaning and later in life a stable period (homeostasis). Note that the treatments, antibiotic and/or stress, were at day 4 during the immune programming period. Next to this the significant findings of microbiota or gene expression data between treatments per time-point are depicted by “+”, and no differences between treatments with a “-”. On the right a metaphorical landscape of the gut system in time is depicted, where the top is day 0 (birth) and bottom is day 176 (slaughter). Spheres depict the current state of the system for day 8, 55, and 176, and colours correspond to the different treatments (T1; red, T2; blue, and T3; green).