Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk

Amira Dewair; Mohamed Bessat

doi:10.1371/journal.pone.0233453

Peer Review History

Original SubmissionDecember 12, 2019
25 Mar 2020 Decision Letter - Jacopo Guccione, Editor PONE-D-19-34351 Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk PLOS ONE Dear Dr. Mohamed Bessat 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. We would appreciate receiving your revised manuscript by May 09 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Jacopo Guccione Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. Thank you for stating in your Funding Statement: "The study was in-part supported financially by the Vmerge FP7 grant (613996) from the EU. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. " Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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). 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 Reviewer #2: 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: No Reviewer #2: 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: The manuscript PONE-D-19-34351 reports the results of a study aimed at detecting the experimental and natural infection with Toxocara vitulorum larvae in the bovine milk applying two techniques, i.e. the classical microscopy-based centrifuge-sedimentation method and a molecular-based PCR assay. The experimental set-up permitted to recover larvae of T. vitulorum from milk samples both after experimental contamination and from colostrum samples of naturally infected animals. I recommend the publication of the paper in Parasites and Vectors after major revisions. The questions posed by the authors are well defined, the methods appropriate and the results, discussion and conclusions well balanced and adequately supported by the data. However, there some issue which should be addressed by the Authors. • Introduction is confusing and with too many paragraphs on Toxocara canis and T. cati (e.g. lines 62 to 72) • In the introduction, I would suggest to provide prevalence of T. vitulorum in different geographical areas • Figure 1 is not necessary to the MS. I would suggest to remove it. • Line 190. I think that “Mancianti et al., 2013” is not an appropriate reference for Kappa coefficient ranking. I suggest the authors to use a statistical reference. • In the discussion the authors mention microscopy and molecular methods for detection of larvae in milk samples, as well as antibody-based serological methods. What about antigen-based milk ELISA methods? And what about copromicroscopic methods? • Furthermore, do the author have any clue regarding the costs of each diagnostic method? Reviewer #2: Ref: PONE-D-19-34351 Title: Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk The objective of this study was to detect the experimental and natural infection with Toxocara vitulorum larvae in the bovine milk applying two techniques, the classical microscopy-based centrifuge-sedimentation method and the more advanced molecular-based PCR assay. The manuscript is well written, and the results are very interesting. The paper is accepted after minor revision. However, see comments below: Abstract Line 11: replace “T. vitulorum” with “Toxocara vitulorum” Introduction Line 41: Please, put T. vitulorum between two brackets and remove the commas. Material and Methods Lines 96-106: Insert the bibliographic reference of the method used. Lines 106-119: Insert the bibliographic reference of the method used. Lines 119-129: Insert the bibliographic reference of the method used. Line 151: Explain if the 50 animals have undergone copromicroscopic examination to know their positivity to T. vitulorum. Lines 232-234: add for each stage the time (days) of development of the larvae in the egg. Line 234: In the legend of Fig. 2, remove "Scale bar is 50 μm" and add "50 μm" on the white bar in the photos. Results Line 284: Add a photo of larvae identified with the optical microscope. Discussion Lines 297-298: The bibliographic references cited are very outdated, use or add more updated ones. Line 358: cursive writing in vitro. General comments: The overall paper is well written, but I think you should add more bibliographic references regarding the methods used in laboratory tests. Moreover, bibliographic references of the introduction and discussion should be more up to date. ****** 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 Reviewer #2: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Review_PONE-D-19-34351.docx https://doi.org/10.1371/journal.pone.0233453.r001
Revision 1
6 Apr 2020 Author Response Reviewer #1: The manuscript PONE-D-19-34351 reports the results of a study aimed at detecting the experimental and natural infection with Toxocara vitulorum larvae in the bovine milk applying two techniques, i.e. the classical microscopy-based centrifuge-sedimentation method and a molecular-based PCR assay. The experimental set-up permitted to recover larvae of T. vitulorum from milk samples both after experimental contamination and from colostrum samples of naturally infected animals. I recommend the publication of the paper after major revisions. The questions posed by the authors are well defined, the methods appropriate and the results, discussion and conclusions well balanced and adequately supported by the data. However, there some issue which should be addressed by the Authors. • Introduction is confusing and with too many paragraphs on Toxocara canis and T. cati (e.g. lines 62 to 72) Response: This is due to the matter of fact that the dog and cat Toxocara species are the most studied, and to pin down the fact that T. vitulorum did received the least attention despite its economic and medical importance. Thus it highlights the importance of our study, and why it was initiated. • In the introduction, I would suggest to provide prevalence of T. vitulorum in different geographical areas Response: A paragraph describes the prevalence of T. vitulorum in different geographical areas has since been added to the “Introduction” section (Lines: 48-55). • Figure 1 is not necessary to the MS. I would suggest removing it. Response: Figure 1 has since been removed, as per our agreement with the reviewer’s recommendation. • Line 190. I think that “Mancianti et al., 2013” is not an appropriate reference for Kappa coefficient ranking. I suggest the authors to use a statistical reference. Response: A more specialized statistical reference has been applied to cite the statistical method of “Kappa coefficient”. I would thank the reviewer for pinpointing the correction. • In the discussion the authors mention microscopy and molecular methods for detection of larvae in milk samples, as well as antibody-based serological methods. What about antigen-based milk ELISA methods? And what about copromicroscopic methods? Response: Unfortunately and after deep and rigorous mining of all publications on T. vitulorum, only antibody-based serological methods had been used to monitor excretion of parasite in the colstrum/milk samples, with no data on the antigen-based milk ELISA. Also since our study was only focused on the milk excretion with no interest of faecal excretion data, the copromicroscopic methods become of less significance to be mentioned in the “Discussion” part. But again I would appreciate the reviewer for raising the point here. • Furthermore, do the author have any clue regarding the costs of each diagnostic method? Response: In short, the PCR-based molecular method would be more expensive than the microscopic examination after the concentration-sedimentation technique (would approximate 3000 Egyptian pound/180 USD per 50 samples versus ~ 800 EGP/50 USD, respectively). Albeit, the molecular method still to give more sensitive detection levels when compared with the microscopy method when applied in the experimental part of the study. Thus, and again as mentioned in the “Discussion” part, the molecular technique would fit more the research purposes, while the microscopic examination would constitute a golden standard for surveying the milking animals. I shall thank the reviewer for recommending these constructive corrections, which with no doubt have added much to the manuscript, leading to much improvement in its quality and readability. Reviewer #2 Title: Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk. The objective of this study was to detect the experimental and natural infection with Toxocara vitulorum larvae in the bovine milk applying two techniques, the classical microscopy-based centrifuge-sedimentation method and the more advanced molecular-based PCR assay. The manuscript is well written, and the results are very interesting. The paper is accepted after minor revision. However, see comments below: o Abstract: • Line 15: replace “T. vitulorum” with “Toxocara vitulorum” Response: It has been corrected o Introduction: • Line 43: Please, put T. vitulorum between two brackets and remove the commas. Response: It has been corrected o Material and Methods • Lines 96-106: Insert the bibliographic reference of the method used. Response: A suitable bibliographic reference of the method used has been inserted. • Lines 106-119: Insert the bibliographic reference of the method used. Response: The method used was based solely on our trials and observations during the pilot and preliminary experiments in the Lab. It has been written in enough details so giving the fellow researchers the opportunity of copying it into their Labs. • Lines 119-129: Insert the bibliographic reference of the method used. Response: The method used was based solely on our trials and observations during the pilot and preliminary experiments in the Lab. It has been written in enough details so giving the fellow researchers the opportunity of copying it into their Labs. • Line 151: Explain if the 50 animals have undergone copromicroscopic examination to know their positivity to T. vitulorum. Response: No, Milk/Colostrum samples were collected blindly from 50 animals, without performing any copromicroscopic examination. This is because the main specific aim was to compare the same two methods blindly on the same milk samples, as it is not necessarily that the same copro-positive animal to excrete the parasite in its milk. • Lines 232-234: add for each stage the time (days) of development of the larvae in the egg. Response: Information about developmental periods (in days) has been added. • Line 234: In the legend of Fig. 2, remove "Scale bar is 50 μm" and add "50 μm" on the white bar in the photos. Response: It has been corrected on the photos. o Results • Line 284: Add a photo of larvae identified with the optical microscope. Response: Supporting files (Supporting video and figure) have been added to show the viable larvae inside the fully-embryonated eggs, as well as larvae being extracted from mechanically-broken eggs. o Discussion • Lines 297-298: The bibliographic references cited are very outdated, use or add more updated ones. Response: More updated references have been added accordingly, many thanks for the reviewer for his recommendation. • Line 358: cursive writing in vitro. Response: It has since been corrected. o General comments: The overall paper is well written, but I think you should add more bibliographic references regarding the methods used in laboratory tests. Moreover, bibliographic references of the introduction and discussion should be more up to date. Response: As it can be easily figured out that the parasite of T. vitulorum is considered as a neglected when compared to the heavily-researched counterparts of dog and cat ascarids, and thus just barely over 20 papers had been published throughout the last decade. Nevertheless, more updated references have been added to the introduction and discussion sections, to reflect upon the reviewer’s recommendations. We finally would greatly appreciate the reviewer for his comments which led to much improvement in the quality of the manuscript. In all, we consider the paper much improved after the suggested revisions, and would like to thank you and the reviewers for your kind help. We do hope that the paper is now deemed acceptable for publication in PLoS One. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0233453.r002
6 May 2020 Decision Letter - Jacopo Guccione, Editor Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk PONE-D-19-34351R1 Dear Dr. M Basset, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Jacopo Guccione Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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: No Reviewer #2: Yes ****** 5. 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 Reviewer #2: Yes ****** 6. 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: The authors have addressed the issues required by the reviewers. The manuscript can be accepted for publication. Reviewer #2: Dear Authors, you have performed all required comments and congratulations on your paper. ****** 7. 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: Laura Rinaldi Reviewer #2: No https://doi.org/10.1371/journal.pone.0233453.r003
Formally Accepted
11 May 2020 Acceptance Letter - Jacopo Guccione, Editor PONE-D-19-34351R1 Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk Dear Dr. Bessat: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jacopo Guccione Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233453.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .