Characterization of the Fecal Bacterial Microbiota of Healthy and Diarrheic Dairy Calves

Gomez, D.E.; Arroyo, L.G.; Costa, M.C.; Viel, L.; Weese, J.S.

Incidence of diarrhea and mortality rates attributed to diarrhea, as well as treatment rates, were compared by a Fisher's exact test. The Mothur software package g was used for the bioinformatic analysis. 16 Paired-end reads were merged to fully overlapping reads and then aligned to the SILVA 16S rRNA reference database. 17 Sequences that were misaligned with the target region were removed. Irregular sequences including those with contiguous sequence lengths >245 bp or <239 bp and ambiguous base calls also were removed, as were those with runs of homopolymers >8 base pairs. Uchime was utilized to identify chimeras, 18 which then were removed. Sequences belonging to nonbacterial domains (chloroplasts, mitochondria, Archaea, and eukaryotes) also were removed. The remaining sequences were assigned into operational taxonomic units (OTUs) by an open OTU-picking approach, with a distance limit of 0.03 (97% similarity). The OTUs were classified by the Ribosomal Database Project classifier. h Relative abundances of the main phyla, classes, orders, and families (median relative abundance >0.1%) and the main genera (median relative abundance >0.05%) were calculated. The Shapiro-Wilk test was used to evaluate normality of the datasets. The majority of datasets did not meet the assumptions of normal distribution. Therefore, comparison of the relative abundances between groups (healthy calves between farms, and healthy and diarrheic calves within farms) was performed by the nonparametric Mann-Whitney U-test. P-values were adjusted for multiple 19 by a statistical software i to generate q-values. A q < 0.05 was considered statistically significant.