Analysis of the sheep (Ovis aries) vaginal microbiota preceding spontaneous abortion: a pilot study

  1. Lucille C. Jonas
  2. Stephan Schmitz-Esser
  3. Curtis R. Youngs

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Abstract

Little is known regarding the vaginal microbiota of sheep that undergo spontaneous abortions. The aim of this pilot study was to characterize, using 16S rRNA gene sequencing and shotgun metagenomics, the vaginal microbiota throughout gestation of two ewes (Ewe1 and Ewe2) that spontaneously aborted. Ewe1’s vaginal microbiota contained high abundances of Histophilus, Staphylococcus, and Unclassified Pasteurellaceae. Most notable was the high abundance of Campylobacter in Ewe1’s vaginal microbiota following the abortion. Ewe2’s vaginal microbiota was characterized by high abundances of Pasteurella throughout gestation. Shotgun metagenomic sequencing produced two high quality metagenome-assembled-genomes (MAGs), identified as Campylobacter jejuni and Histophilus somni. The C. jejuni MAG had 99.95% average nucleotide identity (ANI) to the most abundant sheep-abortive C. jejuni clone in the US. The H. somni MAG was most similar to a pathogenic H. somni strain, and contained genes that contribute to serum resistance and sialic acid utilization. The results presented here demonstrate the need for continued research into the vaginal microbiota, specifically to identify potential predictors of spontaneous abortion.

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  1. Access Microbiology

    I thank the authors for a nice paper presenting a small study which adds to the literature overall. From the three reviewer reports the main requirements for publication are; - Adding in additional methodology details - Adding in discussion on the switch in the vaginal microbiome of ewe 1 prior to abortion. Two reviewers have commented that adding a graph on functional analysis with your shotgun data would be a great addition to the paper. I would like to see this as I think it would strengthen the study and make the most of the small data set, but this is at the authors discretion and not mandatory for publication. I have issued a decision of "major revise" to allow time for this should the authors wish to add it. Please respond to each reviewer comment, point by point, and provide a track changes document with your resubmission.

  2. Access Microbiology

    Comments to Author

    Jonas et al. conducted an intriguing pilot study investigating the sheep vaginal microbiome before spontaneous abortion. This study is particularly significant in the field of vaginal microbiome research in sheep, as it fills a gap in existing knowledge. However, there are a few limitations to consider. Firstly, the study's sample size is relatively small. Secondly, a more detailed analysis of shotgun metagenomics would provide valuable insights and enhance the manuscript's overall quality. Here are my comments to the authors: 1. Line 78 - "to enrich microbial reads using BBmap" - This needs to be rephrased as this step is mainly performed to remove host reads (sheep) and there is no enrichment of microbial reads that takes place by doing this. 2. Line 82 - Can you mention the reference database used with this tool? Is it the standard reference database or any custom database used? If the webserver was used, please change it to JspeciesWS. 3. Line 83 - This needs to be a bit more detailed as Bacterial and Viral Bioinformatics Resource Center is hosting service, not a bioinformatics tool. Could you provide more details on which tools were used for annotation and proteome comparisons? 4. Line 107 - Why did you opt for a co-assembly approach with all the samples from two ewes together? Given the taxonomic composition of the samples is quite different at the timepoints sequenced, that might add a lot of noise for binning algorithms. Have you tried reconstructing MAGs by assembling samples individually? 5. Line 109 - The average length of the contigs (457 basepairs) is not ideal. The fragmented assemblies could be due to low sequencing depth in each sample. Could you include the average number of reads per sample after removing host-contamination reads? 6. It would be interesting to look into the functional potential with the shotgun metagenomics data using tools like Humann3 as there is a significant change in taxonomic composition in Ewe1 from 13 weeks to 15 weeks. 7. Line 132 - "characterization of the vaginal microbiota not only over time but also through metagenome shotgun sequencing." There is no taxonomic profile/ functional profile generated from shotgun metagenomic data. Did you find the same taxonomic composition in shotgun data as Figure 1? 8. With shotgun data, it would be possible to reconstruct the taxonomic composition at species-level resolution and allows you to strengthen the claims that (Campylobacter jejuni is linked with abortion in Ewe1) - Line 135 - "Ewe1's abortion appears to be connected to the presence of Campylobacter in her vaginal communities." And also "P. multocida can cause a variety of pathologies"- line 155. 9. Line 99 - In the figure legend, include that these plots are generated using 16S rRNA sequencing data.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  3. Access Microbiology

    Comments to Author

    The present work reports relevant preliminary findings on the microbiota observed in sheep that suffered a spontaneous abortion. The authors employed a novel approach, not commonly used in the study of small ruminant reproductive pathologies. As the first work focused on describing the microbiota of these spontaneous aborted ewes, the presented results are comparable to previous ovine studies where vaginal microbiota has been analyzed in cases of reproductive disorders. Based on these preliminary results, further studies should be developed to evaluate and describe the vaginal microbiota in abortive processes in sheep with a higher number of animals in order to performed statistical analysis. Key words: It may be beneficial to change the terms "vaginal microbiota" and "spontaneous abortion" in the key words, as they are mentioned in the title, in order to enhance the article's visibility on scientific platforms. Abstract: It may be beneficial to include briefly information about the study population, sampling times, pregnancy status at the sampling points, etc., to help the reader gain a better understanding of the article. Additionally, indicate the relative abundance of the most abundant genera in the abstract. Introduction: The main hypothesis of the study could be included to provide further context. Material and methods: Is the health status of the animals known? This includes vaccination program, history of infectious disease and veterinary check-ups. Line 48. Ovine articles that describe the microbiota in females with reporductive disorders could be included. Sample collection, Line 64: Add body condition score value Results: What was the level of subsampling for the 16rRNA? Did you have some dominant phyla or species results? Was the shotgun technique unable to detect viruses or fungi in the analyzed samples? Table 1. Add the bottom line of the table Table 1. Add at the table title the description of MAGs Discussion, Line 125. Add some small ruminant references Line 136: Some references indicated that Campilobacter was statistically more abundant in aborted females than in normal-delivery ewes and Histophilus somni had greater abundance in vaginal microbiota of aborted sheep (Toledo-Perona et al., 2025; (https://doi.org/10.1016/j.onehlt.2025.100996). Linea 159: In cattle, author's proposed the effect of gut-lung axis which revealed emerging insights into the interaction between microbiota from different anatomical locations (https://doi.org/ 10.1038/nrmicro.2016.142 and https://doi.org/10.3389/fcimb.2021.583600). These interactions could occurred between respiratory and vaginal microbiota through the ingestion of mucus and its excretion. Despite the cost of the technique, would you recommend the use of these molecular methods for abortion diagnosis in small ruminants? Author Contributions: In order to follow the author contributions using the CRediT taxonomy format, some contributor roles need to be add: Validation, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization, Resources, Software, Supervision, Visualization.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  4. Access Microbiology

    Comments to Author

    The subject of the paper is novel and of interest to the wider research community. I have no comments/changes relating to the methodology, representation of the key fundings and literature analysis. There are two minor suggestions below which I think would strengthen the paper by discussing which relate to discussion and presentation of the results. There is no discussion of the shift in the vaginal microbiome pre-abortion in ewe1 that is visible in Figure 1. I also thing that the shotgun metagenomics data could be used to generate a figure to better represent the results I have elaborated on these points below. Once these have been addressed the paper is ready for publication as a short communication in Access Microbiology. Line 67: Can the type of DNA extraction used and the region used for 16S amplicon sequencing be mentioned here even if it is included in the reference mentioned. Figure 1: Why is the barplot for ewe1 during week 5 and 6, along with that for ewe2 during week 8 so small? Are all the reads unknown at genus level or belong to genera that were not one of the 15 most abundant so were not visualised? Can an explanation of what these very small bars represent be added to the corresponding results paragraph. Week 8 seems to be around the time that the vaginal microbiome in ewe 1 shifts to being predominantly Staphylococcus, in the lead up to the ewe aborting (weeks 8 - 13). The sudden increase in Staphylococcus during this period is not mentioned in the results and discussion and I think it would be worth adding this. Could a graphic be added to demonstrate the results from the metagenomics sequencing? Is there a way of showing the abundance of the functional pathways mentioned? It would also be interesting to see the difference in abundance of the pathways between the two ewes and between the samples analysed.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  5. Version published to 10.1099/acmi.0.001005.v1 on Access Microbiology