PONE-D-23-42435Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.PLOS ONE

Dear Dr. Kimemia,

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ACADEMIC EDITOR: The manuscript needs a revision according to the reviewers' notes and suggestions.

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“We thank Francis Ngere, Nicholas Odemba, David Oullo, Charles Waga, Richard Ochieng, Daniel Ngonga and Vitalice Opondo for their expert contribution in ticks sampling and identification.

This work was funded by the Armed Forces Health Surveillance Branch (AFHSB) and its Global Emerging Infections Surveillance (GEIS) Section, FY2022 ProMIS ID: P0116_22_KY and FY2023 ProMIS ID P0094_23_KY. The material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the true views of the Department of the Army or the Department of Defence.”

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

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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: Ticks were collected from cattle, sheep, goats, and camels, therefore, these ticks must have sucked blood. The microorganisms should be affected by the blood from different hosts. When ticks were grouped, the host they parasitized should be considered. For example, Rickettsia prowazekii are commonly asociated with fleas and body lice, and it identified in ticks may be from blood. Therefore, these limitation should be added in the disscussion.

When detecting the microorganisms, the ticks were pooled in groups of 1 to 8 individuals. Therefore, it is unreasonable to calculate the prevalence of microorganisms, and even the prevalence compared to previous studies was also unreasonable.

Table 4 was not shown in the text.

1. The MS should be reviewed by a native english speaker.

2. Species names: with full genus name at first mention, later with abbreviated genus name throughout the text (exception: at the beginning of a sentence). Species names and genus names should be italicized. Please check throughout the text.

3. "while the second was to confirm the morphological tick species identification", hence, can the COI gene of microorganisms also be amplified with the primers provided in the MS?

4. "COI" and "p" value should be italicized.

5. "Spotted Fever Group Rickettsias", replaced by "Spotted Fever Group of Rickettsiae".

6. table 4 was not shown in text, how to deternine the prevalence?

7. line 135, add as before per.

8. line 290, Pseudomonadales 410, please show IQR.

9. line 329, add "," after Rickettsia slovaca.

10. the section "Confirmation of Vector Identity using Cytochrome Oxidase Subunit I Sequences" should be shown in section "Tick Collection".

11. line 367, the endosymbiotic bacteria was not identified in this study, how to "explain why the overall bacteriome of different tick species collected from the two different regions is similar to a greater extent as indicated by the alpha diversity indices"?

Reviewer #2: The study highlights the occurrence of pathogenic bacteria of public health importance in tick species. The findings show the need for continuous surveillance efforts to create effective control and preventive measures.

I have a few suggestions to improve the manuscript.

Comments

L112-113: Did the authors consider pooling the ticks based on the animal host? If yes, please include it in the “Sampling and Pooling” section.

L139-140: The word “were” was omitted. The sentence should read…. reverse primer pair were used whereas…

L145: Remove the “min” after the “denaturation for 30 sec”.

L148: Remove the “min” after the “95ºC denaturation for 30 sec”.

Results:

For the tick collection section, the authors should consider first talking about the tick collection at the various sampled areas, the tick species identified and then zoom in to the pools.

Which of the livestock sampled were mostly infested by tick species? The authors can consider including the number of ticks collected from each livestock (cattle, sheep, goat, camel) in the results.

L202-206: The authors should consider revising the statements made for clarity.

L249-252: Kindly read the sentence and make the necessary corrections.

Table 2 captioned “Distribution of OTUs that were classified to the bacterial species level per Tick Genera” and Table 3 “The numbers of Operational Taxonomic Units (OTUs) contributed by each tick genera that were classified (≥60% confidence) to the bacterial species level” are practically the same. Remove the Table 2.

L402: The authors mentioned Table 4, but no Table 4 has been presented in the results.

There are two different captions of Figure 1 in the manuscript. I believe the second Figure 1 mentioned in the discussion (L432-434) is Figure 5. Please confirm and make the change.

Reviewer #3: Line 71: correct to “….reported spotted fever group Rickettsia in the United……..”

Line 97: “The study was also submitted for ……………..

Line 108: 15 mL

Line 121: 1 mL

Line 127: 4 m/s

Line 128: 1000 µL, also in line 130, correct to 200 µL

Line 136: 16S rRNA

Line 150: is 0.8% agarose gel not too low?

Line 201-203: The sentence is confusing. The way it began, it sounds like the total number collected from Isiolo was 2918 which is not so. 2918 was the total number collected from both Isiolo and Kwale. The sentence needs to be revised for clarity by stating explicitly, the total overall number collected and the total collected each from Isiolo and Kwale.

Line 205: it must be exactly and not approximately.

Line 207: The authors have mentioned that the collected tick’s falls into 10 species in total but in line 205, it was mentioned that the 372 tick pools represented 8 ticks. This is conflicting and needs to be clarified.

Line 215-217: this line of text is completely distractive considering its location. Preferably, this should come immediately after line 212.

Line 346-347: skewed in term of what? Numbers? If so, clearly and expressly state so and avoid ambiguous terms that are meaningless without been qualified.

Line 349-350”: The use of the word “respectively” in this sentence is meaningless as it has nothing to qualify here. Take a look and revise

Introduction

In the current form, the introduction is too generalist without focus and the background information is too scanty that attempts to provide good background. The authors need to rejig the introduction by making it more focused.

Methodology

They is need for the authors to provide details of the geography of the sampling locations including details of the average temperature, humidity and seasons. I understand that part of this information has been provided in the introduction; however, this can be removed and transferred to the methodology. Usually, the first part of the methodology deals with the description of the “study area”. In the study area, the authors should state specifically, the months and year when tick sampling was undertaken. In other words, the duration of sampling is necessary.

Discussion

Some of the results were embedded within the discussion section which is a misnormal and needs to be revised. For instance, table 2 and figure 1 was presented in between the text for the discussion. Take another look at this and address my concerns.

Results

Confirmation of the vector identity should be the first part of the results after stating the total number of ticks collected across all the study locations and the number of ticks. It is abnormal to state all the pathogens detected in the various tick species before providing details of how the tick vector identity was confirmed.

General comments

-Numerous grammatical and editorial errors that makes it difficult to grasp the meaning of some of the sentences.

- In the discussion, I will suggest that the authors devise a more robust way to discuss their findings. For instance, discuss the tick prevalence and distribution first, then tick borne alpha-proteobacteria that are potentially pathogenic, zoonotic and non cultivable before discussing the findings from gram negative bacteria that can be cultured on media.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: THANKGOD EMMANUEL ONYICHE

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PONE-D-23-42435R1Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.PLOS ONE

Dear Dr. Kimemia,

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.

==============================

ACADEMIC EDITOR: The authors should revise the manuscript according to reviewers comments. The comments of a reviewer are not replied to by the authors. 

==============================

Please submit your revised manuscript by Apr 26 2024 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,

Shawky M Aboelhadid, PhD

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: No

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

Reviewer #2: Yes

Reviewer #3: 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 #2: Yes

Reviewer #3: No

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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 #2: Yes

Reviewer #3: 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 #2: (No Response)

Reviewer #3: The author failed to address all my comments made in the earlier revision. I will recommend that they address my earlier comments or make satisfactory rebuttal to all my comments

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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 #2: No

Reviewer #3: Yes: ThankGod Emmanuel Onyiche

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Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

PONE-D-23-42435R2

Dear Dr. Kimemia,

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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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,

Shawky M Aboelhadid, PhD

Academic Editor

PLOS ONE

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