Lack of reproducibility compromises conclusions on the effects of three Bacillus species on Salmonella enterica Enteritidis colonisation in layer-type chickens.

1. Salmonella spp. are one of the most important foodborne zoonotic pathogens, often transmitted to humans through table eggs and fresh meat. Strategies for the prevention and reduction of Salmonella spp. in poultry include various approaches, such as biosecurity measures, vaccination and the use of feed additives, like probiotic bacteria.2. This study investigated the impact of a commercial Bacillus-based probiotic on Salmonella enterica serovar Enteritidis (SE) colonisation in layer-type chicks, a critical issue for food safety. Lohmann Selected Leghorn chicks were divided into two groups, where one received GalliPro® Fit (Chr. Hansen A/S, Hoersholm, Denmark; 1.6 × 10⁶ CFU/g feed) from day-of-hatch, while the control group did not. On d 8, all chicks were orally challenged with SE (7-8 × 10⁵ CFU/bird). Cloacal swabs were collected on d 8, 10, 11 and 12 to assess shedding, and caecal contents were analysed for SE counts after euthanasia on d 12. The study was repeated three times with 30 chicks per group in each trial under consistent housing, husbandry and feeding conditions, except for differing parental bird origins. Shedding analysis was not performed in the final trial.3. The results were inconsistent. In the first trial, probiotic-treated chicks showed significantly reduced SE shedding and caecal loading compared to the control group. However, in the second trial, shedding and caecal loads were significantly higher in the probiotic group. The third trial revealed no significant differences between the groups.4. These findings showed that the probiotic effect on SE colonisation was inconclusive, despite identical experimental conditions, apart from parental bird origin. This highlighted the potential influence of parental health on offspring immunocompetence and gut microbiota, underscoring the challenges in interpreting in vivo studies. This study emphasised the need for reproducibility and careful evaluation of factors affecting trial outcomes.