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This study investigated the effect of four different nutritional interventions, applied during the first week of life, on immune competence parameters of broiler chickens. The four (dietary) interventions were antibiotics in the drinking water, 25% inclusion of rye in the feed, beta-glucans from Saccharomyces cerevisiae, and coated butyrate. Broiler chickens (between 1 and 2 weeks of age) were subjected to a Necrotic Enteritis (NE) challenge. A positive and negative control treatment (challenged vs. non-challenged chickens, respectively) was included to estimate the effect of the NE challenge. It was hypothesized that applying a nutritional intervention in the first week of life would affect the microbiota colonization, immune system programming, and consequently the quantitative disease phenotype (lesions scores as a result of the NE challenge). The NE challenge did affect the overall performance during the first 2 weeks post-challenge. The antibiotics group showed significant effects on different biological levels: (temporary) increased performance, deviating microbiota composition, increased gene expression in barrier function processes, whereas decreased gene expression in immune related processes, and a higher villi to crypt ratio compared to the (un)challenged control treatment. In the other treatments only significant differences in bacterial genera were observed. In conclusion, this study has shown that it is probable to disturb the gut system development, whereby giving antibiotics (amoxicillin) gives an effect on multiple biological levels. In addition, the other dietary perturbations, i.e. change in feed composition (25% inclusion of rye), feed additives (beta-glucans or coated butyrate) only affected the microbiota composition to some extent.