PONE-D-21-31323Prevalence of tick infestation and Molecular characterization of spotted fever Rickettsia massiliae in Rhipicephalus species parasitizing domestic small ruminants in North-Central NigeriaPLOS ONE

Dear Dr. Ola-Fadunsin,

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 all of the points raised during the review process.

Please submit your revised manuscript by Dec 29 2021 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,

Brian Stevenson, Ph.D.

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. Thank you for including the following ethics statement on the submission details page:

'University of Ilorin, Nigeria.'

Please amend your current ethics statement to confirm that your named institutional review board or ethics

committee specifically approved this study.

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. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.  

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

5. 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.

[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: No

Reviewer #3: Partly

Reviewer #4: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: No

Reviewer #4: No

**********

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

Reviewer #3: Yes

Reviewer #4: 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: It will be good to see other statistical analysis on the manuscript. The authors need to use scientific words. There must also be consistency throughout the manuscript (structure, language used). It would be good to also get result on the other ticks species that were collected, even though in low numbers but they must also be tested for the pathogen. For interest sake were there any other pathogens tested or any co infection observed.

Reviewer #2: Nusirat Elelu., et al, reported in the present manuscript entitled: “Prevalence of tick infestation and Molecular characterization of spotted fever Rickettsia massiliae in Rhipicephalus species parasitizing domestic small ruminants in North Central Nigeria” a survey on tick infesting sheep and goats in the Ipata municipal. The authors have collected the tick from the animals at the local slaughterhouse. The authors report that ticks belonging the Rhipicephalus genre were the most collected from the small ruminants. PCR amplification targeting the 350 bp DNA of the Rickettsia 23S-5S intergenic spacer followed by sequencing have revealed the presence of R. massiliae in two Rhipicephalus turanicus ticks. The data collected and presented in the manuscript lack originality. All the results have been already reported in Nigeria regarding the presence of the different tick species identified in the study. In addition, R. massiliae were also reported in Nigeria. Albeit, the authors claim that the manuscript is the first report of the amplification of the DNA of R. massiliae from tick infesting small animals. Even if the primers used in the study targeting the 23S-5S region of Rickettsia were reported able to amplify the DNA of R. massiliae, the shortness of the amplicon (350 bp DNA) is a limitless to compare between related species of Rickettsia. Using other genes like gltA and rpoB were suitable to clarify the genetic position of the species amplified in these study compared to other closet species. Finally, it was suitable that the authors investigate the presence the presence of Rickettsia species in the other tick species rather than Rhipicephalus ticks. Authors can also consider a molecular survey of other related Rickettsiaceae species like Anaplasmataceae to add more valuable data.

Minor revision

Add tick identification methodology to the material and method section.

Reviewer #3: Major comments

- Why was DNA extracted only in ticks that were identified as Rhipicephalus? Number of ticks is not a scientific reason. Hyalomma ticks can also be infected with Rm.

- The bootstrap values in the Rickettsia massieliae grouping is really poor. Only 40, which in most cases would not be reported. This may be due to using different lengths of sequences. Were the sequences trimmed to represent the same 350 bp piece amplified in the PCR? Also, how do the authors explain that their potential strain is closer to USA strains than strains in Tunisia.

- The authors should show the alignment and blast results. The authors do not report performing any BLAST analysis, however, this should be the first step before moving into the ClustalW alignment and MEGA analysis.

- Likewise, the sequences obtained after Sanger sequencing should be deposited into NCBI and the Genbank numbers should be made available to the scientific community.

- The role of sheep as “reservoir” host is a stretch (see minor comments). Unless, several animals are found infected. The significance of two infected ticks in whether an animal may serve as a “reservoir” is unknown. That statement should be deleted.

- Although the reviewer believes that studies in the epidemiology of diseases are very beneficial. It would be better to start by determining the actual incidence of the pathogen among ticks in the area since only two ticks were found.

Minor comments

- Although the issues in grammar and English are not substantial, the reviewer recommends that the authors consult a colleague for editing.

- Lane 75 – rather than regarded, the Rickettsia belong to the SFG or are classified.

- Be consistent, sometimes the authors use “μl” and sometimes “ul”.

- Ticks tend to be seasonal. May the differences reported in this study differ from other studies due to the seasons when they were carried out?

- Lane 261. Given that only two Rht were collected from sheep and the two were infected with Rm that is a 100% infection rate. By comparing to the rest of the Rhipicephalus sp in the study, the authors are underestimating the infection rates. If fact this study has much higher infection rates that the cited work. Are these sheep from a particular farm? What information is available. Were the two ticks collected from the same animal? And what other ticks were collected from this particular animal? Was it only these two ticks? If so, was the animal infected?

Reviewer #4: This manuscript determined the prevalence of tick infestation among small ruminants and molecularly described a clinically significant spotted fever Rickettsia massiliae from Rhipicephalus ticks collected from North-Central, Nigeria, which means regional study of a pathogen and its host.

Title and keywords title must use different words.

Are the sheep where the ticks were found hairless? or not?

Discuss regarding the life cycle of these ticks if it is from one or more hosts?

Concerning the 23S-5S IGS Rickettsia molecular taxonomy it will be important to provide a database on the sequences found.

as in the reference in line 19. García-García JC .......,

review all references

**********

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

Reviewer #3: No

Reviewer #4: 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.

Prevalence of tick infestation and Molecular characterization of spotted fever Rickettsia massiliae in Rhipicephalus species parasitizing domestic small ruminants in North-Central Nigeria

PONE-D-21-31323R1

Dear Dr. Ola-Fadunsin,

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,

Brian Stevenson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: