Characterization of pig saliva as the major natural habitat of Streptococcus suis by analyzing oral, fecal, vaginal, and environmental microbiota

Abstract

It is generally difficult to specify the sources of infection by which domestic animals may acquire pathogens. Through 16S rRNA gene amplicon sequencing, we compared the composition of microbiota in the saliva, vaginal mucus, and feces of pigs, and in swabs of feeder troughs and water dispensers collected from pig farms in Vietnam. The composition of the microbiota differed between samples in each sample group. Streptococcus, Actinobacillus, Moraxella, and Rothia were the most abundant genera and significantly discriminative in saliva samples, regardless of the plasticity and changeability of the composition of microbiota in saliva. Moreover, species assignment of the genus Streptococcus revealed that Streptococcus suis was exceptional in the salivary microbiota, due to being most abundant among the streptococcal species and sharing estimated proportions of 5.7%-9.4% of the total bacteria in saliva. Thus, pig oral microbiota showed unique characteristics in which the major species was the pig pathogen. On the other hand, β-diversity analysis showed that the microbiota in saliva was distinct from those in the others. From the above results, pig saliva was shown to be the major natural habitat of S. suis, and is suggested to be the most probable source of S. suis infection.

Fig 1. Comparison of OTUs between the five sample groups.

The Venn diagram presents the common and unique OTUs among the five sample groups. The number of common OTUs is indicated in parenthesis under each sample name.

Fig 2. Genera distribution of the microbiota in the biological and environmental samples from pig farms.

Only the bacterial genera share more than 2% relative abundance, which is indicated by different colors. The genera that share less than 2% are collected and then indicated by gray bars. Prefixes before the bacterial names indicate the following ranks: “f” for family, and “g” for genus.

Fig 3. Common, unique and top three genera.

(a) The Venn diagram presents the common and unique genera among the five sample groups. The number of common genera that were present in all samples in each group is indicated in parenthesis under each sample’s name. (b) The top three genera identified in the five sample groups.

Fig 4. Results of the LEfSe analysis show significantly discriminative genera among the five sample groups.

Genera with an LDA score >4.0 (p<0.01) are depicted, based on their scores. Prefixes of the bacterial names indicate the following ranks: “o” for order, “f” for family, and “g” for genus.

Fig 5. Comparison of species distribution in the genus Streptococcus between different growth stages of the pigs.

Twenty different species were identified. The top 10 streptococcal species are depicted in different colors. The remaining 10 species are indicated as “others.” The other OTUs identified as Streptococcus were not matched to the sequences of the original database; these are indicated as “unknown”.

Fig 6. Comparison of microbiota among different sample groups by using β diversity.

(a) PCoA measured using unweighted UniFrac distance matrices. The colored ovals show 95% confidence intervals calculated by R. The inset at the lower right shows the symbols and the corresponding samples. (b) The relationship among the microbiota of samples is presented by hierarchical cluster analysis. Symbols at the bottom of the chart are described in the inset in image (a).