Dear Dr. TALUKDER,

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.

• 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'.
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We look forward to receiving your revised manuscript.

Kind regards,

Shahin Tajeri, D.V.M. Ph.D.

Academic Editor

PLOS ONE

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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

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“The authors acknowledge the financial support (Principal investigator, M. H. T.; research and innovation subproject; project code: (RP-C-03-24) from the Livestock and Dairy Development Project (LDDP), jointly funded by the World Bank and Department of Livestock Services (DLS), Ministry of Fisheries and Livestock, Govt. of Bangladesh.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Additional Editor Comments:

When revising your manuscript please consider below comments plus those of the #reviewer 1:

• Lines 35-36: Please change the sentence to: “In contrast, T. orientalis does induce lymphoproliferation.”
• Lines 36-37: Please provide a reference for the tick vectors. I do not believe all tick species listed act as vectors.
• Please remove the abbreviation “Md.” from the author names.
• Please delete the Acknowledgments  section, as it is not relevant.
• Please ensure that the bibliography follows PLOS ONE  formatting guidelines.
• Since the study focuses on T. orientalis , which causes a disease distinct from other Theileria  species, please be specific throughout the manuscript. Avoid using the general term “theileriosis” for T. orientalis  infection (e.g., line 236).

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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?

Reviewer #1: Yes

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

Reviewer #1: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: Thank you for the opportunity to review your manuscript. This is an original and well-structured study reporting the molecular detection and genetic diversity of Theileria orientalis in cattle across Bangladesh. The large sample size, national coverage, and sequence confirmation make this work a valuable contribution to regional epidemiology and tick-borne disease surveillance. Overall, the study is technically sound, conclusions are supported by the data, and the English is clear. Only minor revisions are needed to enhance clarity and reporting consistency.

1. Sampling description

The random sampling strategy is appropriate and ensures diversity of age, breed, and sex. To enhance transparency, please briefly describe how randomization was implemented within farms, herds, or markets (e.g., random selection on site).

2. PCR methodology

It is clear that positives were confirmed by sequencing, which supports assay specificity. Please indicate explicitly whether no-template or extraction controls were included.

Although homologous MPSP-like genes exist in T. parva and T. annulata, the primer pair used here targets a region unique to the T. orientalis complex (as validated in Kamau et al., 2011). Mentioning this would clarify assay specificity given the absence of local DNA from other Theileria species in Bangladesh.

3. Statistical reporting

The use of Pearson’s chi-square and Fisher’s exact tests (SPSS v27) is appropriate for the descriptive objective, and this information is already clearly stated in the Methods. Reporting 95 % confidence intervals for prevalence estimates would enhance interpretability, but no further analysis is required.

4. Sequencing subset

A total of 510 bovines' blood were found positive with T. orientalis. Could you please state briefly how the 32 sequenced isolates were selected (randomly or by gel quality/representation). Clarify that genotype diversity reflects this subset and may not capture all variants nationwide.

5. Genotype classification

The study identifies T. orientalis MPSP genotypes 5 and 7, both of which have been reported previously in Asia. While these genotypes are not novel, documenting their concurrent circulation across multiple Bangladeshi divisions adds valuable epidemiological context. The authors could emphasize this contribution more clearly in the Discussion.

6. Figures and presentation

Figure 1 (Map): Please cite the map source/shapefile and ensure boundary lines are clearly visible in print.

Figure 3 (Haplotype network): Please just verify label legibility (≥ 8 pt).

7. Discussion

The Discussion is well written and adequately contextualizes the results within regional and global T. orientalis epidemiology. The comparative analysis with previous studies is informative. However, the section could be strengthened by briefly explaining the ecological or management factors influencing regional differences, and by adding a concise concluding paragraph linking the findings to surveillance or control strategies. Overall, this is a solid and publishable discussion after minor revisions.

The manuscript is written in standard English. Minor typographical and taxonomic corrections (e.g., Amblyomma testudinarium, italicised species names, consistent “Chattogram”) can be made at proof stage.

This manuscript provides useful baseline data on T. orientalis distribution and diversity in Bangladesh. With the small clarifications above, it will fully meet PLOS ONE’s publication criteria

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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

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[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 ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Insights into the genetic diversity and population structure of prevalent Theileria orientalis in Bangladesh

PONE-D-25-52299R1

Dear Dr. TALUKDER,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Shahin Tajeri, D.V.M. Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Dr. Talukder,

Please consider correcting line 36 to 'does not induce' when finally checking the accepted proof. Please excuse me as this was a typing error of mine when writing my review.

Regards,

Shahin Tajeri

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: Thank you for the opportunity to review your manuscript. This is an original and well-structured study reporting the molecular detection and genetic diversity of Theileria orientalis in cattle across Bangladesh. The large sample size, national coverage, and sequence confirmation make this work a valuable contribution to regional epidemiology and tick-borne disease surveillance. Overall, the study is technically sound, conclusions are supported by the data, and the English is clear.

**********

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

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