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PONE-D-23-27370Molecular identification of Coxiella burnetii in raw milk samples collected from farm animals in districts Kasur and Lahore of Punjab, PakistanPLOS ONE

Dear Dr. Yasir Zahoor,

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https://www.hindawi.com/journals/ijmicro/2021/6632036/

http://erepository.uonbi.ac.ke/bitstream/handle/11295/101328/Thesis%20final%20.pdf?isAllowed=y&sequence=1

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0289944

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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

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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 describes the detection of Coxiella burnetii in milk samples collected from farms animals in districts Kasur and Lahore of Punjab, Pakistan. A PCR assay was performed. Unfortunately, too much information is lacking in the manuscript, particularly in Materials and Methods. My specific comments on the manuscript are shown in the attached file (highlighted in pink). Below are my other concerns:

1. For easier review, line numbers should be shown on the left side of each page. Also, please make sure to subject the manuscript to language review and editing.

2. In the introduction, please don't begin every sentence with C. burnetii, and the way punctuation marks are used confuse the reader

3. Tables in general, including the Supplementary ones are poorly organized and difficult to understand. I can't see the difference between table 1 and 2. And as a recomendation, when there are rows that add up to zero, it means that the sample size was insufficient, and that the analyzes carried out are not appropriate.

4. Phylogenetic analysis is insufficient. More isolates, both reported and those derived from their own study should be added.

5. The discussion is also poorly written. What was already mentioned in the summary, introduction and results is constantly repeated.

Reviewer #2: While the study addresses an important public health concern, there are several shortcomings and scientific mistakes that need attention.

Introduction:

The introduction is informative, but it lacks a clear statement of the research gap and a hypothesis. The authors could improve the introduction by providing more context on the global prevalence of C. burnetii in raw milk and its impact on public health. Furthermore, the reference citations in the introduction are somewhat outdated, and more recent literature should be incorporated to support the study's rationale.

Methodology:

The methodology section lacks details on the inclusion/exclusion criteria for farms and animals, potentially introducing selection bias. The use of a convenient sampling approach may not be representative of the overall population, and the authors should discuss the potential limitations associated with this sampling strategy. Additionally, the absence of information on the sensitivity and specificity of the PCR assay used raises concerns about the reliability of the results.

Discussion:

While the discussion compares the study findings with previous research, it fails to discuss the implications of the results for public health and the dairy industry in Punjab. Additionally, the discussion could benefit from a more critical analysis of the study limitations, such as the potential for contamination during sample collection and the use of a single gene target for detection.

Conclusion:

The conclusion provides a succinct summary, but it lacks specific recommendations for future research or practical implications for stakeholders. A more detailed discussion of the policy and public health implications of the study results would enhance the conclusion.

Overall, the manuscript should undergo thorough proofreading and editing for language and grammar issues. Additionally, the authors should consider updating references to include more recent publications in the field. Lastly, the manuscript would benefit from a more explicit statement on ethical considerations, especially regarding animal welfare and informed consent for sample collection.

Reviewer #3: General Comments:

The paper by Shujat et al. provided some interesting results of detection of Coxiella in milk samples of Pakistan. I understand this is the first study of its kind in Pakistan, and I think the results would be applicable to many other low-income countries with similar agriculture and dairy systems.

There were two aspects investigated: (1) molecular characterising and (2) prevalence estimation, investigating differences between species and geographic districts. The second part was a bit more restricted, in terms of sufficient number of animals across all species and district combinations, but nevertheless this is useful as a reference base for other future studies, and the authors provided a comprehensive analysis of the available data. One point that needs to be clarified is the overall objective due to a clash between the Introduction and Discussion. I think it really is both points above. In addition, some of the analyses need to be described in a bit more detail in the “Data Analysis” section to clarify some of the results being presented.

The following is a list of points I noted as I read through the manuscript. Most are minor editorial, but some may take some more time to work through. Please consider each point carefully, particularly the more substantial points, as this will result in a considerably improved manuscript.

Specific Comments:

Page 1: In the title page, I believe the affiliation for Wasim Shehzad should be ‘1’, not ‘2’.

Page 2, Abstract: (1) I think “convenience sampling” rather than “convenient”. (2) Also, be consistent in number of decimal places, suggest only one, so “7.4%” and “5.2%”. (3) Change “univariate” to “univariable” (The former indicates only one outcome variable. The latter indicates only one explanatory variable in the model). (4) Change “was differed” to “differed”.

�, Paragraph 3: Suggest “contaminated milk” rather than “contagious milk”.

Page 3, Paragraph 2: Change “Multicopy” to “multicopy”.

�, Paragraph 3: Change “about six” to “six” as that is what you have reported. However, please be more specific about “international databank”, e.g. a PubMed search? Make sure this list is current.

�, Paragraph 4: Change “convenient” to “convenience sampling”. Please provide a breakdown of the 304 samples by species.

�, Paragraph 5: Change “-20 °C temperature” to “-20 °C”.

Page 4, Paragraph 3: For the logistic regression, you need to mention the terms in the model, i.e. explanatory variable(s), not just the outcome variable. Also, where you used a chi-square analysis, is that what you labelled “univariate” (or “univariable” as mentioned above? Please clarify.

�, Paragraph 4: Perhaps write as “The University of Veterinary and Animal Sciences Advanced Studies and Research Board approved the study at its 50th meeting held on 8 February 2019” (suggesting explicit month name because of differing UK / US date format conventions – or was it “2 August 2019”?

Page 5, Figure 1: Change to “Lahore” in caption.

�, Paragraph 1: (1) As in Abstract, please round to 1 d.p., as sample sizes don’t lend to 2 d.p. accuracy. After “not statistically significant’, add P-value for the Kasur vs Lahore overall comparison (P = 0.63 I think). (2) Suggest change to “Coxiella burnetii prevalence in the four species of ruminants was”. (3) Suggest change to “although the differences between districts for each species were not statistically different (Table 2). I am not sure if the logistic regression model for looking at species × district interaction adds more, and it will be affected by the sample sizes, but will leave to authors if they decide to retain it. (4) Please add a bit more explanation about abortion as an outcome variable, I wasn’t sure how it was analysed giving an overall P-value of P = 0.03 (not described in Data Analysis section).

Page 6, Table 2: For the more detailed results in Table 2, comparing districts within each species, the Pearson chi-square procedure will fail due to small sample sizes. I would recommend using the Fisher exact test in that instance, the resultant P-values are cattle: 0.30, buffalo: 0.37, goat: 0.39, and sheep: 0.34. They don’t change much apart from goats.

Page 7, Figure 2: By “Labeled sequences”, do you mean the samples with the solid square symbol, i.e. your samples? Please clarify. Also, for publication, you may need higher quality image resolution than in this manuscript.

Page 9, Paragraph 1: Please clarify what the overall aim was: In the Introduction it was stated it was about prevalence estimation, but here it is about molecular characterisation. Also please avoid too much repetition of information already provided in the Introduction.

�, Paragraph 2: Suggest change “The previous study” to “A previous study”.

�, Paragraph 3: Suggest change “The distribution” to “The estimated prevalence”.

Page 10, Paragraph 1: The description of cattle and buffalo should be in a new paragraph, separate from your discussion of sheep. And then your overall recommendation in a final separate paragraph, which could be expanded with the specifics of recommendations.

References:

[2] Benenson & Tigertt: Missing volume number.

[13] Derrick: Missing page numbers.

[23] Tozer et al: Change to “Queensland”.

[26] Iqbal et al. This has now been published in Pak. J. Zool., please update reference.

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Reviewer #1: Yes: Ruth Cabrera

Reviewer #2: No

Reviewer #3: No

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Attachments

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Submitted filename: PONE-D-23-27370_reviewer.pdf

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Submitted filename: PONE review.docx

Molecular identification of Coxiella burnetii in raw milk samples collected from farm animals in districts Kasur and Lahore of Punjab, Pakistan

PONE-D-23-27370R1

Dear Dr. Muhammad Yasir 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.

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Kind regards,

Gianmarco Ferrara, PhD, MVD

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 #3: All comments have been addressed

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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 #3: (No Response)

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

Reviewer #1: Yes

Reviewer #3: (No Response)

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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: Yes

Reviewer #3: (No Response)

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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 #3: (No Response)

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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: After review, I consider the article suitable for publication. However, as a last comment, the figures still do not have the necessary resolution, and are difficult to view.

Reviewer #3: (No Response)

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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 #3: No

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