Comparison of the fecal bacterial communities in diarrheic and healthy dairy calves from multiple farms in Southeastern PA. Calf Metagenomics

Diarrhea is a major cause of illness and death in pre-weaned calves and causes significant economic losses to producers. A better understanding of the fecal microbiota in diarrheic and healthy calves has the potential to lead to the development of improved treatment and prevention strategies. The purpose of this study was to compare the microbiota of diarrheic and healthy calves to improve our understanding of what constitutes a healthy microbiome in pre-weaned calves. At each of 7 farms, fecal samples were obtained from 1-3 diarrheic calves (ages: 2-17 days old at sampling time) and age-matched (within 5 days) healthy controls for a total of 20 samples. Calves were fed either acidified bulk milk, pasteurized or unpasteurized waste milk, or milk replacer depending on farm. Fecal samples were extracted for genomic DNA, PCR-amplified, sequenced for the V1-V2 region of the 16S rRNA bacterial gene on the Illumina MiSeq platform, and analyzed using QIIME2. A significant difference between farms but not between groups (diarrheic versus healthy) was seen on unweighted UniFrac analysis; no difference was found on weighted UniFrac or in measures of alpha diversity between groups or farms. Firmicutes and Bacteroidetes were the most prevalent phyla in both groups; Fusobacteria was more prevalent in the sick group whereas Proteobacteria and Actinobacteria were more prevalent in the healthy group. At the genus level, Bacteroides was the most prevalent genus in both groups and was higher in the healthy group. Generalized linear mixed model analysis revealed that Ruminococcus and unclassified Ruminococcaceae were significantly influenced by farm, group, and their interaction; Bacteroides was significantly influenced by farm; and Lachnospiraceae was significantly influenced by the interaction between farm and group. Genera that were more prevalent in diarrheic calves included Fusobacterium, Sutterella, and Streptococcus, while genera that were more prevalent in healthy calves included Faecalibacterium and Ruminococcaceae. There was considerable difference at the genus level between farms, indicating that environmental and management factors play a large role in determining the microbiome of developing calves. Our results indicate that healthy commensal bacteria required in the neonatal period were replaced with opportunistic bacteria in diarrheic calves, which may have contributed to the incidence of diarrhea either directly or indirectly.