PONE-D-23-03478Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, PakistanPLOS ONE

Dear Dr. Zahoor,

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 Apr 27 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,

Gizat Almaw

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 

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

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. 

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

a) The name of the colleague or the details of the professional service that edited your manuscript.

b) A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file).

c) A clean copy of the edited manuscript (uploaded as the new *manuscript* file).

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. Thank you for stating the following financial disclosure: 

"The authors received no specific funding for this work."

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. 

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[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: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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: Yes

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: Overall, the present article is pertinent since it explored the frequency of C. burnetii and its molecular characterization in raw meat from ruminant sources in Pakistan. However, some items need attention and adjustment:

-Please, all paper needs a complete revision of references.

For example, references 1 and 4 are de same (duplicated)

Reference 26 is incomplete.

Line 39: About following affirmation and references: "It infects over 40 tick species, which is an important transmission vector in ruminants (5). It replicates in ticks; thus sufficient 41 amounts of this pathogen are eliminated in their faeces and deposited on the skin of animal hosts during feeding" It is recommended to review and address this issue more assertively and support it with recent references. The role of ticks as vectors of this bacterium is still controversial...

Line 71. The following phrase, "PCR was used to detect the IS1111 element of the C. burnetii genome" could be deleted from here.

Line 73: Is there any reference supporting the DNA extraction protocol from the meat sample? Please include it.

Line 87: To explain the PCR condition, please include the following:

-The final concentrations of the primers

-Reference of the type of taq used

- How were possible PCR amplification inhibitors in the sample evaluated?

Line 98 For the Phylogenetic analysis, please clarify with more detail. It needs to be improved:

-Was the Nucleotide substitution model (model test) verified and adjusted for the data before the ML method?

-Did Bootstrap? Please present it.

-Outgroup? Please include and present it.

Line 118: It is unclear in the methodology; how was it possible to verify the age of the animals from which the meat sample came?

In the discussion, the authors should address the issue of whether or not good meat cooking could inactivate this bacterium. Furthermore, how much could molecular detection be expected to ensure viable transmission of this species?

Reviewer #2: The manuscript entitled “Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan” investigates 200 meat specimens collected in slaughter houses in two districts of Punjab, Pakistan. Samples obtained from 4 species (goat, sheep,cattle and buffalo) were tested for the presence of specific C.burnetii sequence by PCR, also phylogenetic analysis was performed. The manuscript is easy to follow and provides valuable information on presence of C.burnetii DNA in raw meat samples – a subject that has been poorly known so far.

Major concerns:

1. The data about tested meat samples are too general. There is no information whether skeletal muscles or offal samples were collected. It might be an important issue, taking into consideration that one of the studies showed significant difference between prevalence of pathogen DNA in skeletal muscle meat and offal. Moreover there is lack of information if the same type of meat was collected from all animal species.

2. The data about age of tested animals are not present in the manuscript, but they are included as one of variables in chi square analysis.

3. Lack of essential information in phylogenetic tree i.e. scale bar and bootstap values of the nodes makes the figure non-informative. Once completed, the data should be analysed and discussed.

4. The discussion is a little bit one-sided, possible limitation of the research are not adressed. Moreover, the conclusion that „C. burnetii prevalence, especially in meat samples, pose a severe risk of Q fever” seems to be far-reaching. The presence of C. burnetii DNA was confirmed but the viability of bacteria in specimens was not evaluated and the risk level is unknown, so far. In adition, it would be advisable to refer to findings about milk and dairy products and importance of transmission via alimentary route.

Minor comments:

line 17: I suggest “in raw ruminant meat”

line 22: Please replace “ruminant sources” with “ruminant species”

line 26: I suggest “strains are genetically diverse (…)” rather than “strains are not genetically identical”

line 38: reservoirs of

line 46: Flu is a contagious respiratory illness caused by influenza viruses. Please correct

49: Delete dot after word France

line 52: Please correct as follows: to detect Coxiella burnetii in samples

lines 62-63: Please precise the aim of this study (add regions where prevalance was estimated)

line 63: Please correct “used for human consumption” to “intended for human consumption”

Methodology

It would be interested for readers to have more information about tested meat. Could you add information about type of tested meat? Were it skeletal muscle meat samples or offal? Did you collect the same type of meat from all animals? Is it possible to present some details i.e. types of offal or meat cuts that were incuded in the study?

line 68: between 2021 and 2022/in 2021 and 2022- please correct

line 69: I suggest: This study included 200 meat samples(…)

line 73: Please delete word temperature, it is unnecessary

line 75 and 76: Please add full names of the reagents SDS and PCI

line 77: Then it was centrifuged under the same conditions as mentioned above – Please complete the information about centrifugation conditions as none were mentioned in this section.

line 81: I suggest “remained pellet“

line 83: “(…) evaporate ethanol and act as a PCR inhibitor”- it is uncelar, please rephrase

line 83: I suggest: “pellet was resuspended”

Section molecular assay and sequence analysis

Please add information about controls that were used in DNA extraction and PCR.

line 87: Assay (not assays), please correct

lines 93-94: I suggest rephrasing the sentence e.g. The reaction conditions were as follows (…)

line 94: delete final

line 97: by Macrogen

line 100: Please add information about bootstap value.

lines 115-116: What about sheep vs. goat meat?

lines 118-120: The results of chi square analysis on animals age as one of variables were described, but the data about age of tested animals are not present in the manuscript. Please complete this information!

line 124: the sequences were clustered because their genetic similarity not geographical proximity, please rephrase to clarify.

lines 122-126: A map showing all district of the Punjab province would make easier for the readers to follow the analysis presented in this paragraph.

line 126: I suggest: genetically diverse.

Table 1: Please redesign the table and add information about age of animals (see comment for lines 118-120)

Figure 1: Phylogenetic tree has not got a scale bar and bootstap values of the nodes, please add them. Moreover, the tree would be more informative if you add the data about host orgin of the sequences and year of isolation.

line 142: I suggest: for human consumption

line 147: - the sentence is misleading. It was mentioned that in other study the prevalence of C.burnetii DNA was determined for blood samples collected from ruminants in Lahore and Kasur. Nextly you referred to publication that describes prevalence only in Kasur. The publication 19 ( also do not analyse the prevalence of C.burnetii DNA in animals from Lahore. Please correct the sentence or add appropriate publication.

line 152: Please correct the reference number 23 as the publication describes first isolation of pathogen from cheese in Brasil…

lines 154-156: It would be worthwhile to familiarize readers with culinary habits in Pakistan. What is the most consumed meat type in Pakistan? Do Pakistanis often consume raw meat and thus what might be the zoonotic risk?

lines 165-166: What was the prevalence for buffalo samples in each district (Kasur and Lahore)?

line 167: Please correct the reference number

References need to be adjusted to the requirements of the journal.

**********

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.]

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.

PONE-D-23-03478R1Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, PakistanPLOS ONE

Dear Dr. Zahoor,

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 Aug 07 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,

Gizat Almaw

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 needs to strictly follow the 'Style and Format' of PLOS ONE. Their manuscript is a mix of Arial and Times New Roman. The authors need to revise and submit by strictly following the 'authors guideline'. eg. tables, figures.

[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.

Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan

PONE-D-23-03478R2

Dear Dr. Zahoor,

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,

Gizat Almaw

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: