Vaginal microbiome and serum metabolite differences in late gestation commercial sows at risk for pelvic organ prolapse

Abstract

Sow mortality attributable to pelvic organ prolapse (POP) has increased in the U.S. swine industry and continues to worsen. Two main objectives of this study were, (1) to develop a perineal scoring system that can be correlated with POP risk, and (2) identify POP risk-associated biological factors. To assess POP risk during late gestation, sows (n = 213) were scored using a newly developed perineal scoring (PS) system. Sows scored as PS1 (low), PS2 (moderate), or PS3 (high) based on POP risk. Subsequently, 1.5, 0.8, and 23.1% of sows scored PS1, PS2, or PS3, respectively, experienced POP. To identify biomarkers, serum and vaginal swabs were collected from late gestation sows differing in PS. Using GC-MS, 82 serum metabolite differences between PS1 and PS3 animals (P < 0.05) were identified. Vaginal swabs were utilized for 16S rRNA gene sequencing and differences in vaginal microbiomes between PS1 and PS3 animals were detected on a community level (P < 0.01) along with differences in abundances of 89 operational taxonomic units (P < 0.05). Collectively, these data demonstrate that sows with greater POP risk have differential serum metabolites and vaginal microflora. Additionally, an initial and novel characterization of the sow vaginal microbiome was determined.

Figure 1

Perineal score in late gestation as an indicator of pelvic organ prolapse (POP) risk. Perineal scoring was conducted on sows (n = 213) during late gestation (days 105–117). Sows assigned a PS1 (n = 68), PS2 (n = 119) and PS3 (n = 26) were assumed low, medium or high risk, respectively, for POP. As predicted, 23.1% of PS3 scored sows subsequently died due to POP while 1.5 and 0.8% of sows scoring PS1 and PS2, died respectively. These data demonstrate the ability to distinguish differential risk of POP for animals during late gestation.

Figure 2

Most abundant microbial phyla (A) and genera (B) in vaginal swabs from sows with low and high risk for pelivc organ prolapse (POP). (A) Vaginal swabs were collected from sows with low (PS1) and high (PS3) risk for POP and microbial detection and abundance were determined through 16 s rRNA sequencing and analysis. Spirochaetes abundance between PS1 and PS3 sows was significantly different (P < 0.05). (B) Stacked bar charts comparing the ten most abundant vaginal microbial genera. Relative abundance of vaginal swab microbial communities on a genus level represent the mean across each perineal score. Animals assumed low risk for POP are considered PS1 and high risk animals for POP are considered PS3.

Figure 3

Vaginal swab community comparisons. (A) Canonical correspondence analysis showing maximum variation between samples in community due to perineal score (PS). Data for low risk animals (PS1) are depicted in light grey and high-risk animals (PS3) are in black. All points represent Bray Curtis dissimilarity measures for each sample. (B) Principal Coordinates Analysis showing differences in beta diversity of vaginal microbial communities from sows with different perineal scores (PS). All points represent Bray Curtis dissimilarity measures for each sample. Ellipses were based on a multivariate normal distribution of data points. There were statistical differences in overall microbial communities found using ANOSIM due to PS (P < 0.01).

Figure 4

Different OTUs in the vaginal microbiota of sows with different perineal scores (PS). Significantly different (P < 0.05) OTUs were identified with LEfSe. Only significant OTUs within the 100 most abundant are shown. Error bars represent the standard error of the mean. Out of all significantly different OTUs, 24 were considered significantly different among the 100 most abundant OTUs. Out of these 24 OTUs, 12 were more abundant in PS1 animals and 12 were more abundant in PS3 animals. See Table 1 for more details.

Figure 5

Serum metabolites in sows differing in perineal score (PS) and pelvic organ prolapse (POP) risk. This plot represents important features and demonstrates those which are different between sows with a PS3 (high risk for POP) compared to PS1 (low risk for POP). The pink circles represent features above the threshold (twofold change and P < 0.05). PS1 averages were set as the base level and PS3 values of individual metabolites are represented by points. The pink dots represent the individual metabolites that are considered significantly different between PS. Pink dots in the upper right are metabolites that are more abundant, on average, in PS3 animals, and pink dots in the upper left less abundant, on average, in PS3 animals. Note both fold changes and P-values are log transformed. The further the position from the (0,0), the greater level of statistical significance for that the feature.

Figure 6

Differences in serum metabolites between PS1 and PS3 sows. This heat map displaying the 75 molecular features with the highest statistical significance (lowest p-values), illustrates relative abundance of serum metabolites (represented by color) between low POP risk (PS1) and high POP risk (PS3) sows. PS3 scored sows (black) cluster to the left of the dendrogram separate from PS1 scored sows (grey).

Figure 7

Perineal scoring (PS) methodology as an indicator of pelvic organ prolapse (POP). The perineal region was visually evaluated for swelling, redness and protrusion to assign scores. (A) If a sow lacked swelling, redness and protrusion they were considered low risk for POP and assigned as PS1. (B) Sows with some characteristics such as moderate swelling, redness and protrusion of the perineal area were considered moderate risk for POP and assigned as PS2. (C) Sows demonstrating all of the characteristics of severe swelling, redness and protrusion of the perineal area were assigned to the PS3 category and considered high risk for POP.