Vaginal Microbiome Characterization of Nellore Cattle Using Metagenomic Analysis

Abstract

Understanding of microbial communities inhabiting cattle vaginal tract may lead to a better comprehension of bovine physiology and reproductive health being of great economic interest. Up to date, studies involving cattle microbiota are focused on the gastrointestinal tract, and little is known about the vaginal microbiota. This study aimed to investigate the vaginal microbiome in Nellore cattle, heifers and cows, pregnant and non-pregnant, using a culture independent approach. The main bacterial phyla found were Firmicutes (~40-50%), Bacteroidetes (~15-25%) and Proteobacteria (~5-25%), in addition to ~10-20% of non-classified bacteria. 45-55% of the samples were represented by only ten OTUs: Aeribacillus, Bacteroides, Clostridium, Ruminococcus, Rikenella, Alistipes, Bacillus, Eubacterium, Prevotella and non-classified bacteria. Interestingly, microbiota from all 20 animals could be grouped according to the respiratory metabolism of the main OTUs found, creating three groups of vaginal microbiota in cattle. Archaeal samples were dominated by the Methanobrevibacter genus (Euryarchaeota, ~55-70%). Ascomycota was the main fungal phylum (~80-95%) and Mycosphaerella the most abundant genus (~70-85%). Hormonal influence was not clear, but a tendency for the reduction of bacterial and increase of archaeal populations in pregnant animals was observed. Eukaryotes did not vary significantly between pregnant and non-pregnant animals, but tended to be more abundant on cows than on heifers. The present work describes a great microbial variability in the vaginal community among the evaluated animals and groups (heifers and cows, pregnant and non-pregnant), which is significantly different from the findings previously reported using culture dependent methods, pointing out the need for further studies on this issue. The microbiome found also indicates that the vaginal colonization appears to be influenced by the gastrointestinal community.

Fig 1. Average alpha diversity in the analyzed groups, expressed as antilog of Shannon’s index.

NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 2. Domains abundance in each group.

NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 3. Bacterial phyla found in the vaginal tract.

Relative abundance comparisons within each group are shown in graphics a to d, and phyla comparison between groups are shown in graphic e. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 4. Major bacterial OTUs at the genus-level in each group.

Average relative abundance is presented for the ten most abundant bacterial OTUs in the vaginal tract. (a-d) Comparison within each group. (e) Comparison between groups. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 5. Bacterial phylogenetic tree, including all bacterial families encountered in the samples.

The seven major families, comprising nine of the ten most abundant OTUs at the genus level are indicated by the red dots. Red circles indicate three important orders, namely Clostridiales (a), Bacillales (b) and Bacteroidales (c).

Fig 6. Relative abundance of the 10 major OTUs found at the genus-level in vaginal microbiota types “A” (a), “B” (b) and “C” (c).

The blue bars refer to aerobes and facultative anaerobes, red bars to obligate anaerobes and white bars to unspecified bacterial metabolisms. Results are shown as the mean and standard deviation of each group, and different symbols represent statistical difference (p < 0.05).

Fig 7. Major archaeal OTUs at the genus-level in each group.

Average relative abundance is presented. (a-d) Comparison within each group. (e) Comparison between groups. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 8. Major eukaryotic phyla found in the vaginal tract.

Relative abundance comparisons within each group are shown in graphics a to d, and phyla comparison between groups are shown in graphic e. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).

Fig 9. Major eukaryotic OTUs at the genus-level in each group.

Average relative abundance is presented. (a-d) Comparison within each group. (e) Comparison between groups. NPH—Non-pregnant Heifers, PH—Pregnant Heifers, NPC—Non-pregnant Cows, PC—Pregnant Cows. Different symbols represent statistical difference (p < 0.05).