SUMMARY While poultry microbiome discoveries are increasing due to their potential impact on poultry performance, studies examining the poultry respiratory microbiome are challenging because of the low microbial biomass and uniqueness of avian respiratory tract, which make it difficult to sample enough material for microbial analysis. Invasive sampling techniques requiring euthanasia are currently used to increase microbial mass for the analysis, thus making it impossible to sample individual birds longitudinally. In this study, we compared invasive (nasal wash, upper tracheal wash, lower tracheal wash, and lower respiratory lavage) and non-invasive (tracheal and choanal swabs) respiratory sampling techniques in two independent experiments using four-week-old chickens. We first established the experimental baseline of respiratory microbiota using invasive techniques to enable reasonable comparisons between sampling methods and between experiments. Although non-invasive sampling (live-bird swabs) resulted in lower 16S rRNA gene copy numbers compared to invasive sampling, live swabs were able to detect the dominant microbes captured by invasive techniques. Nevertheless, swabs from euthanized birds were more reflective of the microbiota captured through invasive methods than live swab. Furthermore, from two separate experiments, we also demonstrated that respiratory microbiota sampling is highly reproducible especially in the trachea and lower respiratory tract. Our study provides new insights and perspectives on decision-making when sampling and studying the poultry respiratory microbiota.

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