Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysentery

Jéssica A. Barbosa; Juan C. P. Aguirre; Roman Nosach; John C. S. Harding; Vinícius S. Cantarelli; Matheus de O. Costa

doi:10.1371/journal.pone.0294273

Peer Review History

Original SubmissionJuly 1, 2023
15 Aug 2023 Decision Letter - Gianmarco Ferrara, Editor PONE-D-23-20538 Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysentery PLOS ONE Dear Dr. Costa, 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. Please submit your revised manuscript by Sep 29 2023 11:59PM. 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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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: Partly Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: Yes ****** 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). 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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: The manuscript is designed to characterize the intestinal microbiota of piglets prior to inoculum with the bacterium that causes swine dysentery. , and contrast the findings with the profiles of the microbiota of pigs exposed 1 or 2 days before the presence of hemorrhagic diarrhea . I want to understand that the purpose is to define if there is a bacterial profile in these pigs that is permissive to develop hemorrhagic dysenteria. However, the purpose is not explicitly clear because the methodology does not mention a study design as such, on the other hand , although the hypothesis of the authors is mentioned . The methodology section is mixed with what would be part of the results and in the section where the description of the results should be, the figures are described as the figure captions were. Thus, the manuscript does not conform to the standards of the journal. Reading the introduction section, I was struck by the fact that the authors mention that there is evidence on this same model, with results comparable to those presented in this manuscript. In my opinion, the manuscript should be structured in a consistent way, making it clear: 1- what is the purpose and the novelty of this research strategy with respect to what has already been published; 2- the methodology section describe the methodology or, where appropriate, 3- gather methodology and results in a single section and 4- discuss the results and contrast them with those obtained by other authors, mention the limitations of the model and highlight the most important findings in a conclusion section Reviewer #2: General comments: This is very well written manuscript that has a clear study design. The main rationale for the study was to understand the change in the fecal microbiota few days before the development of mucohemorrhagic diarrhea due to swine dysentery in pigs. For the study, they used a seeder exposure method that it is interesting to mimic natural infection. Despite the difference in the days until the animals started with mucohemorrhagic diarrhea, fecal samples were collected daily, demonstrating the complexity of the study design. Chao1 Index between d-1SD and MHD compared to D0 was demonstrated as the only change in alpha diversity. Alistipes dispar and Parabacterioides gordonii increased in MHD sound as an interesting finding that may help to elucidate B. hyodysenteriae pathogenesis. It would be interesting to see some discussion about why P. gordonii has been observed in health pigs but had demonstrated a 3-fold increase in diseased animals (MHD). Overall, this is an interesting study that will improve the knowledge about modifications observed in swine dysentery diseased pigs. Specific comments: Line 88: As diet is considered an important variable to the development of swine dysentery, it will be interesting to add supplementary table with the diet composition including ingredients and protein, energy, macro and microelements. Reference 31 is not cited in the text. Please include this citation or delete it from the list of references. Line 244: SCFAs is cited for the first time as an abbreviation. What does it mean? Line 272: “….findings, Burrough et al. (11) found…” Reviewer #3: The manuscript deals with a relevant and interesting theme. This topic will be of interest to the readership of PLOSONE. Generally, the structure of each section of the paper is very clear and well defined. The methods used are sound and properly described. The results are interesting and well discussed. The limits of the study are reported within the text with a sufficient discussion. The English is correct and fluent for the readers. Not all the images included in the paper are sharp enough. Authors should submit better quality figures. I did not find the captions of figures, authors should provide them. Lines 263 and 266: please modify “actinobacteria” in “Actinobacteria” Line 287: ante-mortem should be italicized ****** 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: Yes: CECILIA XIMENEZ MD PhD Reviewer #2: No Reviewer #3: Yes: Francesca Romana Massacci ******** [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. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0294273.r001
Revision 1
5 Oct 2023 Author Response We appreciate the reviewer’s unpaid time and efforts in reviewing this manuscript. Thank you! Reviewer #1: The manuscript is designed to characterize the intestinal microbiota of piglets prior to inoculum with the bacterium that causes swine dysentery. , and contrast the findings with the profiles of the microbiota of pigs exposed 1 or 2 days before the presence of hemorrhagic diarrhea. 1- I want to understand that the purpose is to define if there is a bacterial profile in these pigs that is permissive to develop hemorrhagic dysenteria. However, the purpose is not explicitly clear because the methodology does not mention a study design as such, on the other hand , although the hypothesis of the authors is mentioned. A: The purpose of the study is to characterize changes in the fecal microbiome immediately prior to disease. Previous studies have only characterized the changes in the microbiome of clinically affected pigs versus healthy controls or samples collected prior to inoculation (See change in L54). As stated in L74 (where a hypothesis and goal are described), we aimed to profile the changes in fecal microbiome composition immediately prior to the development of SD. 2- The methodology section is mixed with what would be part of the results and in the section where the description of the results should be, the figures are described as the figure captions were. Thus, the manuscript does not conform to the standards of the journal. A: It is unclear to us as what the reviewer perceived as “methodology section is mixed with results”. Unfortunately, the Reviewer did not provide specifics, so we are unable to address this issue. That being said, we believe that they might refer to Table 1, which is truly part of the methods rather than results. Sampling (and the “result” from inoculation) is a key aspect of the methodology used in this study since we required a prior knowledge of clinical outcomes in order to analyze the correct samples (1 and 2 days prior to SD). PLOS One guidelines to authors states that “Each figure caption should appear directly after the paragraph in which they are first cited.”. The Reviewer can familiarize themselves with these guidelines by clicking here: https://journals.plos.org/plosone/s/submission-guidelines 3- Reading the introduction section, I was struck by the fact that the authors mention that there is evidence on this same model, with results comparable to those presented in this manuscript. In my opinion, the manuscript should be structured in a consistent way, making it clear: 1- what is the purpose and the novelty of this research strategy with respect to what has already been published; A: We regret that the introduction was not clearly written, leading to some confusion by the reviewer. We have modified the text (L54 and L73) to hopefully clarify it. Our results differ from other published work from the perspective that those studies did NOT sample pigs immediately prior to the development of disease. Pigs were sampled prior to inoculation, and not naturally infect but inoculated directly with an overload of pathogen. In our case, we analyzed samples from pigs that were naturally infected with Brachyspira, but were still in the incubation period (text modified in L77). 2- the methodology section describe the methodology or, where appropriate, 3- gather methodology and results in a single section. A: We hope to have clarified this in the comments above. 4- discuss the results and contrast them with those obtained by other authors, mention the limitations of the model and highlight the most important findings in a conclusion section. A: Thank you, this can be found in L223-300. Reviewer #2: General comments: This is very well written manuscript that has a clear study design. The main rationale for the study was to understand the change in the fecal microbiota few days before the development of mucohemorrhagic diarrhea due to swine dysentery in pigs. For the study, they used a seeder exposure method that it is interesting to mimic natural infection. Despite the difference in the days until the animals started with mucohemorrhagic diarrhea, fecal samples were collected daily, demonstrating the complexity of the study design. Chao1 Index between d-1SD and MHD compared to D0 was demonstrated as the only change in alpha diversity. Alistipes dispar and Parabacterioides gordonii increased in MHD sound as an interesting finding that may help to elucidate B. hyodysenteriae pathogenesis. It would be interesting to see some discussion about why P. gordonii has been observed in health pigs but had demonstrated a 3-fold increase in diseased animals (MHD). A: Acknowledged. We have added more info on P. gordonii in L230-240. Unfortunately, there’s nothing very clear regarding how this bacterium could be contributing to the disease, or if it is simply an opportunistic. Overall, this is an interesting study that will improve the knowledge about modifications observed in swine dysentery diseased pigs. Specific comments: Line 88: As diet is considered an important variable to the development of swine dysentery, it will be interesting to add supplementary table with the diet composition including ingredients and protein, energy, macro and microelements. A: We have added the reference for the diet used in line 89 and the diet composition in a supplementary table 1 (S1_File). Reference 31 is not cited in the text. Please include this citation or delete it from the list of references. A: Acknowledged. Reference 31 (updated for 32) included (Line 151). Line 244: SCFAs is cited for the first time as an abbreviation. What does it mean? A: Thank you, we have addressed this in lines 254 and 255. Line 272: “….findings, Burrough et al. (11) found…” A: Acknowledged. Line 283. Reviewer #3: The manuscript deals with a relevant and interesting theme. This topic will be of interest to the readership of PLOSONE. Generally, the structure of each section of the paper is very clear and well defined. The methods used are sound and properly described. The results are interesting and well discussed. The limits of the study are reported within the text with a sufficient discussion. The English is correct and fluent for the readers. Not all the images included in the paper are sharp enough. Authors should submit better quality figures. A: Acknowledged. Figures have been updated to publication quality. I did not find the captions of figures, authors should provide them. A: PLOS formatting guidelines is different from most journals. Captions are included in the text as figures are cited, instead of at the end of the manuscript. Figure 1 is in L167, Figure 2 L180, Figure 3 L186, and Figure 4 in L196. Lines 263 and 266: please modify “actinobacteria” in “Actinobacteria” A: Acknowledged (L274 and L277). Line 287: ante-mortem should be italicized A: Acknowledged (L298). Attachments Attachment Submitted filename: Responses to reviewer comments.docx https://doi.org/10.1371/journal.pone.0294273.r002
18 Oct 2023 Decision Letter - Gianmarco Ferrara, Editor PONE-D-23-20538R1Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysenteryPLOS ONE Dear Dr. Costa, 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. Please submit your revised manuscript by Dec 02 2023 11:59PM. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Gianmarco Ferrara, PhD, MVD Academic Editor PLOS ONE Journal Requirements: 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. Additional Editor Comments: The authors addressed the reviewers' comments satisfactorily. Below are some of my minor comments preceding acceptance. Abstract: I’m not sure that readers can understand what the authors intend for “seeder pig” and “contact pigs” reading only abstract section. The abstract can’t finish with this statement “Future investigations to verify the specific role of these taxa on the pathogenesis of SD is warranted” but requires the implications and the limitations of this study in details. Introduction: Line 40: Delete “initial” Lines 43-49: This list of studies should be preceded by an introductory sentence such as "Numerous studies in the literature have demonstrated the role etc." Line 56: italics? Line 63: I advise the authors to reverse "colonic microbiome" and "incidence of SD". Line 73-75: Change this sentence as follows: We assessed any disturbances in the fecal microbiome immediately before clinical SD was observed. Authors should specify what they consider “immediately prior to the clinical SD”. Was it during incubation? How many days? Discussion: Line 225: Please change “stepping-stone” in something else. What are the implications of this study? Understanding the microbiome in order to intervene in some way? The limitation of the few samples analyzed was discussed, not the fact that many parameters would not seem to differ between infected and non-infected animals. [Note: HTML markup is below. Please do not edit.] [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. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0294273.r003
Revision 2
25 Oct 2023 Author Response The authors addressed the reviewers' comments satisfactorily. Below are some of my minor comments preceding acceptance. Abstract: I’m not sure that readers can understand what the authors intend for “seeder pig” and “contact pigs” reading only abstract section. A: Addressed. The abstract can’t finish with this statement “Future investigations to verify the specific role of these taxa on the pathogenesis of SD is warranted” but requires the implications and the limitations of this study in details. A: Updated. Introduction: Line 40: Delete “initial” A: Deleted. Lines 43-49: This list of studies should be preceded by an introductory sentence such as "Numerous studies in the literature have demonstrated the role etc." A: Updated. Line 56: italics? A: Phyla names do not need to be itacilized. Line 63: I advise the authors to reverse "colonic microbiome" and "incidence of SD". A: Updated. Line 73-75: Change this sentence as follows: We assessed any disturbances in the fecal microbiome immediately before clinical SD was observed. Authors should specify what they consider “immediately prior to the clinical SD”. Was it during incubation? How many days? A: Addressed (L75). Discussion: Line 225: Please change “stepping-stone” in something else. A: Changed to “intermediary step” What are the implications of this study? Understanding the microbiome in order to intervene in some way? The limitation of the few samples analyzed was discussed, not the fact that many parameters would not seem to differ between infected and non-infected animals. A: We refrained from overinterpreting the data. Microbiome-host-pathogen interactions are extremely complex and cannot be fully dissected with a single study looking at the bacteriome. We have addressed this issue in lines 306-307. It is unclear what the editor means by “many parameters”? If this is related to alpha/beta diversity, that is expected given the short amount of time between sample collection points. All studies published to date looking at microbiome changes due to SD sampled animals at peak clinical signs vs before inoculation, when lesions and disease are well underway (which takes 7-14 days). In the case of our study, the very short period between d-2 and clinical disease (MHD, 48h) likely contributed to the lack of differences between parameters – which was expected. In addition, the editor may want to revise the methodology employed here: there is no infected vs non-infected comparison in this study. Attachments Attachment Submitted filename: response.docx https://doi.org/10.1371/journal.pone.0294273.r004
30 Oct 2023 Decision Letter - Gianmarco Ferrara, Editor Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysentery PONE-D-23-20538R2 Dear Dr. Matheus O. Costa, 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, Gianmarco Ferrara, PhD, MVD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294273.r005
Formally Accepted
2 Nov 2023 Acceptance Letter - Gianmarco Ferrara, Editor PONE-D-23-20538R2 Characterization of the bacterial fecal microbiota composition of pigs preceding the clinical signs of swine dysentery Dear Dr. Costa: 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. Gianmarco Ferrara Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294273.r006

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