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Sequencing technologies have recently facilitated the characterisation of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system.Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age 20 months) on three occasions (day 0, one month and three months). To further explore the spatial variability of the microbiota, more extensive brushings (n=16) and a throat swab were taken from a separate sheep. The V2-V3 hypervariable regions of the bacterial 16S rRNA gene were amplified and sequenced via Illumina MiSeq. DNA sequences were analysed using the MOTHUR software package. Quantitative PCR was performed to quantify total bacterial DNA.Some sheep lungs contained dramatically different bacterial communities at different sampling sites whereas in others airway microbiota appeared similar across the lung. In our spatial variability study, clustering was observed related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis progressing in a caudal direction. We conclude that both host influence and local factors have an impact on the composition of the sheep lung microbiota.
IMPORTANCE: Until recently, it was assumed that the lungs were a sterile environment which were only colonised by microbes during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the 'lung microbiota'. In this study we characterise the variability of the lung microbiota of healthy sheep. Sheep are not only economically important animals but are also often used as large animal models of human respiratory disease. We conclude that, whilst host influence does play a role in dictating the types of microbes which colonise the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of the lung microbiota.