The composition of the perinatal intestinal microbiota in cattle

Abstract

Recent research suggests that the microbial colonization of the mammalian intestine may begin before birth, but the observations are controversial due to challenges in the reliable sampling and analysis of low-abundance microbiota. We studied the perinatal microbiota of calves by sampling them immediately at birth and during the first postnatal week. The large size of the bovine newborns allows sampling directly from rectum using contamination-shielded swabs. Our 16S rDNA data, purged of potential contaminant sequences shared with negative controls, indicates the existence of a diverse low-abundance microbiota in the newborn rectal meconium and mucosa. The newborn rectal microbiota was composed of Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The microbial profile resembled dam oral rather than fecal or vaginal vestibular microbiota, but included typical intestinal taxa. During the first postnatal day, the rectum was invaded by Escherichia/Shigella and Clostridia, and the diversity collapsed. By 7 days, diversity was again increasing. In terms of relative abundance, Proteobacteria were replaced by Firmicutes, Bacteroidetes and Actinobacteria, including Faecalibacterium, Bacteroides, Lactobacillus, Butyricicoccus and Bifidobacterium. Our observations suggest that mammals are seeded before birth with a diverse microbiota, but the microbiota changes rapidly in the early postnatal life.

Figure 1

16S rDNA copy numbers per sample. Blue = negative controls, green = calf rectal sampling swabs. Values represent 16S rDNA copy numbers per sampling swab or the corresponding amount of extraction reagent (extraction control) or ultra pure H₂O (no-template control). 24 h and 7 d samples were diluted more before amplification, in order to fit in the qPCR dynamic range (dashed line). The boxes represent the interquartile ranges (IQR) containing the middle 50% of cases. The horizontal line in a box indicates the median. Whiskers show maxima and minima within 1.5× IQR. Circles indicate outliers between 1.5–3× IQR.

Figure 2

Effect of data decontamination on the animal microbiota samples. Each bar represents all sequence reads obtained from one sample. Green color shows the proportion of genotypes (de-noised 16S rDNA sequences) found only in the actual samples. Yellow shows genotypes which were shared between samples and negative controls, but were accepted due to more than four-fold relative abundance in samples. Red shows sequences which were removed due to their abundance in the controls.

Figure 3

Microbiota composition and microbial diversity in calves (n = 21) and adults (n = 10). (a) Median microbiota compositions (scaled to totals of 100%) and sequence numbers. Within phyla, the relative abundances of individual genera are shown with different shades. The lightest shades in each phylum show the combined abundance of the least abundant genera (<0.5% of total). For sequencing, control and newborn samples were preamplified by 21 PCR cycles, 7-day and adult feces by 15, and others by 18 to compensate for different quantities of 16S rDNA copies in the samples. (b) Shannon diversity. Boxplot as in Fig. 1.

Figure 4

(a) Principal coordinates analysis (PCoA) of newborn rectal microbiota and dam fecal, oral and vaginal vestibular microbiota. The sample type explained 53% of the variance across all samples. n = 21 calves and 10 cows. (b) Similarity of microbiota in newborns and their own dams, expressed as Spearman correlations (ρ). Boxplot as in Fig. 3; n = 10 calf-dam pairs. (c) Similarity of microbiota in newborns and the same calves at 24 h and 7 d (ρ). n = 21 calves.

Figure 5

Shared genus-level bacterial taxa. (a) Taxa shared between newborns and various dam samples (total: 136 taxa). (b) Taxa shared between newborns and older calves (total: 73 taxa). For each sample group, all taxa with a median abundance >0 were included. Note that a larger number of taxa were shared at lower abundance levels (see text).

Figure 6

Co-existence of genus-level bacterial taxa in newborns and their own dams. The heatmap shows the occurrence of each taxon in the feces, vaginal vestibule and mouth of the cow, if detected in the cow’s own calf (cutoff: ≥20 hits/taxon/animal), and the occurrence of each taxon in the calves. Only the taxa present in >50% of the newborns are shown.

Figure 7

Rectal sampling swab. The sterile equine uterine sampling swab is protected by a double sheath, which prevents contamination at the anus. The sampling head was only exposed within the rectum and then in the laminar flow cabinet upon DNA extraction.