PSI-17 A 16S rDNA reference database of the deep-nasopharynx in steers for bacterial community analysis of cattle with bovine respiratory disease complex

Bovine respiratory disease complex (BRDC) is caused by a variety of complex factors that involves host elements, environmental elements, inadequate management and housing, as well as viral and bacterial pathogens, such as Mannheimia haemolytica and Mycoplasma bovis. The objective of the study was to develop and evaluate a deep-nasopharyngeal bacterial community database to provide greater assignment of taxa within the bovine nasopharynx and as a site-specific tool for examining microbiome dysbioses involved in BRDC. A total of 180 cattle with an overall BW ranging from 164-269 kg were obtained from Athens, Tennessee (n = 60), Maryville, Missouri (n = 60), and Richmond, Kentucky (n = 60). Cattle were transported to a feeding facility in Kansas and housed in open air, dirt floor, group housing pens. At the first diagnosis of BRD, deep nasopharyngeal samples were collected. DNA library preparation of the bacterial 16S rRNA V1–V8 hypervariable regions was performed and sequenced on the Pacific Biosciences RSII instrument. Database construction and all sequencing data were curated using mothur (v1.39.5). For validation analyses using, a subset of cattle was sampled as described above, and three samples sequenced using the V1–V3 hypervariable 16SrRNA regions and the Illumina MiSeq. In the database, Mycoplasma accounted for over 57% of the reads and Mannheimia was present at 0.7%. When validating the database using short-reads, unassigned taxa in the Greengenes analysis was 4.22% contrasted to the 0.91% of the closed reference database analysis. The two taxonomic compositions were not correlated and the correlation was statistically significant (r = -0127; P = 0.001). All alpha diversity indices between the two analysis methods differed (P < 0.05). Development of this reference database provides researchers a tool with considerable improvements in performance for the analysis of bacterial communities in the nasopharynx relating to cattle diagnosed with BRDC.