Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers

Ellen Ruth A. Morris; Ashley G. Boyle; Miia Riihimäki; Anna Aspán; Eman Anis; Andrew E. Hillhouse; Ivan Ivanov; Angela I. Bordin; John Pringle; Noah D. Cohen

doi:10.1371/journal.pone.0252804

Peer Review History

Original SubmissionMarch 8, 2021
7 May 2021 Decision Letter - Yung-Fu Chang, Editor PONE-D-21-07625 Differences in the Genome, Methylome, and Transcriptome Do Not Differentiate Isolates of Streptococcus equi subsp. equi from Horses with Acute Clinical Signs from Isolates of Inapparent Carriers PLOS ONE Dear Dr. Cohen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Your manuscript has been reviewed by an expert in your field. The reviewer did have two questions: Line 116: were any or all of the carriers examined by endoscopy to determine whether guttural pouch pathology was present? If so, it would be helpful to provide further information and to identify which carriers had endoscopically visible guttural pouch pathology. This point is picked up in the discussion (carrier isolates being obtained from animals with and without gross pouch abnormalities) and it would be useful to have more details provided. Line 258: This is an interesting observation – could the authors speculate on possible reasons for this in the discussion? For example, whether this is potentially due to the Swedish isolates all being obtained from a single outbreak and over a shorter time frame? Please answer these questions and make the necessary revision. Please submit your revised manuscript by two weeks. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 3. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is a well written, well designed and interesting paper that addresses an important question about SEE that has been debated for some years: to what extent is the clinical phenotype switch from acute disease to chronic, asymptomatic due to bacterial factors. Previous studies have found some genetic differences between strains isolated from acute cases and those from carriers, leading to the suggestion that pathogen-associated genetic changes represent a form of immune evasion, or result in other pathogen phenotypic changes, that enable the carrier state. This study design is intelligent and compares isolates from acute and carrier cases from an outbreak in Sweden with isolates from acute and carrier cases from outbreaks in the USA (Pennsylvania) for differences at DNA, methylated DNA and RNA levels. The matching of acute and carrier isolates from the same region addresses the geographical variation in SeM and ST that has been previously reported. The abstract accurately reflects the content of the paper and its conclusions. The introduction is complete and provides a clear justification and rationale for the study. The methods are clearly described. The results are well and clearly presented and the tables and figures provided are useful. The discussion is balanced and addresses the points of interest in the results, including The section discussing limitations is well constructed and addresses the intrinsic difficulties and limitations in defining carriers, the difference in duration of carriage between the Swedish and USA isolates, the limitations of the USA collection of isolates. The authors also make some useful comments about what can be inferred from these limitations, which is helpful to readers. The authors also acknowledge the inherent challenges of performing transcriptomics on isolates grown in vitro due to the alterations in gene expression compared with the in vivo state. The conclusions are supported by the data presented in the paper. Overall, this is useful and important study which adds significant new information to the field by addressing a question that is central to SEE pathogenesis. Specific questions: Line 116: were any or all of the carriers examined by endoscopy to determine whether guttural pouch pathology was present? If so, it would be helpful to provide further information and to identify which carriers had endoscopically visible guttural pouch pathology. This point is picked up in the discussion (carrier isolates being obtained from animals with and without gross pouch abnormalities) and it would be useful to have more details provided. Line 258: This is an interesting observation – could the authors speculate on possible reasons for this in the discussion? For example, whether this is potentially due to the Swedish isolates all being obtained from a single outbreak and over a shorter time frame? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
19 May 2021 Author Response May 19, 2021 Prof. Yung-Fu Chang Academic Editor PLoS One Subject: Revision of manuscript entitled, “Differences in the Genome, Methylome, and Transcriptome Do Not Distinguish Isolates of Streptococcus equi subsp. equi from Horses with Acute Clinical Signs from Isolates of Inapparent Carriers” Dear Dr. Chang: Thank you for your electronic message dated May 7, 2021 about the above-referenced manuscript. I also thank the reviewers who carefully considered our report. My coauthors and I have revised the report on the basis of the reviewers’ comments. Below, we detail how each point raised by the reviewers was addressed in the revised report. The revised report with marked changes and an unmarked manuscript will be uploaded to the PLoS One website along with this response letter. Comments from Reviewer 1: Line 116: were any or all of the carriers examined by endoscopy to determine whether guttural pouch pathology was present? If so, it would be helpful to provide further information and to identify which carriers had endoscopically visible guttural pouch pathology. This point is picked up in the discussion (carrier isolates being obtained from animals with and without gross pouch abnormalities) and it would be useful to have more details provided. AUTHORS’ RESPONSE: We thank the reviewer for raising this important point. All of the carrier horses were examined endoscopically. Unfortunately, follow-up information with details was not determined for 6 of the 11 horses from Pennsylvania. We have revised the report to include a table summarizing these findings (new Supplemental Table 1), and also included some discussion of these findings. Line 258: This is an interesting observation – could the authors speculate on possible reasons for this in the discussion? For example, whether this is potentially due to the Swedish isolates all being obtained from a single outbreak and over a shorter time frame? AUTHORS’ RESPONSE: We thank the reviewer for raising another great point. We think that the Swedish isolates had more similar (fewer) AGE because they were from a single outbreak. Interestingly, they were actually collected over a period of > 1 year, which demonstrates persistence of a single strain in horses without clinical signs for over 1 year (albeit with some variation including truncation of SeM). These isolates also had fewer contigs which might have contributed to more consistent results for isolates from the same outbreak. We have revised the report to address these points. Again, we appreciate your efforts and those of the reviewers. Please let us know if you have any questions or concerns regarding our revised report. Attachments Attachment Submitted filename: Response Letter PLoS One May 19 2021.pdf https://doi.org/10.1371/journal.pone.0252804.r002
24 May 2021 Decision Letter - Yung-Fu Chang, Editor Differences in the Genome, Methylome, and Transcriptome Do Not Differentiate Isolates of Streptococcus equi subsp. equi from Horses with Acute Clinical Signs from Isolates of Inapparent Carriers PONE-D-21-07625R1 Dear Dr. Cohen, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Yung-Fu Chang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0252804.r003
Formally Accepted
4 Jun 2021 Acceptance Letter - Yung-Fu Chang, Editor PONE-D-21-07625R1 Differences in the Genome, Methylome, and Transcriptome Do Not Differentiate Isolates of Streptococcus equi subsp. equi from Horses with Acute Clinical Signs from Isolates of Inapparent Carriers Dear Dr. Cohen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yung-Fu Chang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252804.r004

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