140 High-resolution characterization of the gut microbiota of neonatal dairy calves using full-length 16S rRNA gene amplicon sequencing

Abstract

Abstract The gut microbiota has a crucial role in preventing pathogen colonization and training the immune system. An imbalanced gut microbiota tends to induce calf diarrhea, which can lead to dehydration and metabolic acidosis. Therefore, it is urgent to identify critical factors that influence the establishment of gut microbiota in the early stages of development in dairy calves. Most previous research relied on short-read-based partial 16S rRNA gene amplicon sequencing to study calf microbiota, which has limited taxonomic resolution. The aim of our study is to obtain a species-level characterization of calf gut microbiota using long-read based full-length 16S rRNA gene amplicon sequencing and investigate the factors that affect the gut microbiota composition in the early stages of development in dairy calves. We collected fecal samples from the recto-anal junction of 25 Holstein-Angus crossed calves at d 4 and d 14 after birth. The calves were raised in individual outdoor hutches from birth and fed milk replacer twice daily. The gut microbiota was analyzed using full-length 16S rRNA gene amplicon sequencing on an Oxford Nanopore sequencer. The results revealed that the gut microbial diversity measured by Chao 1 (P < 0.001) and Shannon index (P < 0.001) significantly increased from d 4 to d 14. We identified 15 and 36 core bacterial species (prevalence ≥ 80%) in d 4 and d 14 gut microbiota, respectively, and both stages share 7 bacterial species: Bacteroides fragilis, Butyricicoccus pulicaecorum, Enterocloster bolteae, Escherichia coli, Lachnoclostridium sp YL32, Rumiococcus gnavus and Streptococcus equinus. The calf gut microbiota was dominated by Bacteroides fragilis (18%), Escherichia coli (13%), Ruminococcus gnavus (10%) and Butyricicoccus pulicaecorum (6%) species on d 4, and Megamonas rupellensis (18%) and Fusobacterium varium (6%) species (relative abundance > 5%) on d 14. Notably, we found a significant impact of hutch location on the composition of gut microbiota, particularly on d 4, but the impact was reduced on d 14. Moreover, there were 4 dystocia calves among the 25 calves, which had a significantly lower Shannon index (P = 0.046) compared with 4 paired normal calves of similar age and sex, and location balanced. In conclusion, this study provides a species-level resolution of gut microbiota for neonatal dairy calves and indicates a potential impact of dystocia on early microbiota development. Additionally, calf microbiota composition is influenced by environmental factors, likely the soil microbiota where hutches were located, with a more substantial effect on the microbiota at an earlier age.