Dear Dr. Iqbal,

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Abdelfattah Selim, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

[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?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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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

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: General Evaluation:

This manuscript presents a well-structured and scientifically sound study investigating the prevalence and genetic characterization of three major blood-borne pathogens Anaplasma spp., Anaplasma ovis, and Theileria ovis in sheep populations from six districts of Punjab, Pakistan. The topic is timely, relevant, and important for both veterinary health and livestock productivity, particularly in low-income rural regions where small ruminants play a key role in livelihoods and food security.

The authors have combined molecular techniques, including PCR amplification of 16S rRNA, msp4, and 18S rRNA genes, with phylogenetic analysis to assess pathogen diversity. This approach provides both prevalence data and evolutionary insights that are critical for understanding pathogen ecology and potential disease transmission dynamics in the region.

Strengths of the Manuscript:

Novelty and Regional Relevance:

The study fills an important knowledge gap regarding the epidemiology and molecular diversity of blood-borne pathogens in sheep from Pakistan, a country where such data are limited.

Comprehensive Sampling Strategy:

Sampling across six districts ensures geographic diversity and strengthens the generalizability of findings.

Robust Molecular and Phylogenetic Methods:

The use of highly conserved genetic markers for molecular identification and phylogenetic analysis is appropriate and yields informative results.

Relevant Risk Factor Analysis:

Associations between infection prevalence and factors such as herd size, breed, and geographical location are well-presented and contextualized with previous studies.

Well-Structured Discussion:

The discussion effectively compares local data with global findings, providing valuable insight into the pathogen's prevalence, diversity, and epidemiological trends.

Clear Conclusion and Implications:

The conclusion rightly emphasizes the need for continued surveillance, regional-scale phylogenetic studies, and the development of prevention strategies.

Minor Suggestions for Improvement:

Some grammatical and typographical corrections can improve readability. These are mostly minor and do not affect the scientific content. Figures and tables should be properly formatted according to journal guidelines. For clarity, brief information on the tick vectors present in the sampled regions could be added in future studies to strengthen the host-vector-pathogen relationship

Final Recommendation:

Accept with Minor Revisions

This manuscript makes a valuable contribution to veterinary parasitology and molecular epidemiology. The study is scientifically rigorous, the data are relevant, and the findings provide useful baseline information for future surveillance and control programs. I recommend acceptance after minor editorial revisions.

Reviewer #2: The manuscript reports molecular detection of four pathogens in sheep from six different regions of Pakistan. The authors collected the data and analyzed statistically for its significance regarding sex and geographic region of the sample collection. However, the manuscript suffers with several shortcomings:

1. The criteria for the selection of four pathogens and six geographic regions have not been stated. Why did authors ignore other pathogens and/or regions?

2. How did authors define ‘large herds’ versus ‘small herds?

3. What was the probability of the movement of herds from one region to other region?

4. How did authors calculated sample size? Why the number of samples differed in each district?

5. Method in 2.1 is not supported by the data. There wasn’t a structured epidemiological survey.

6. Method in 2.2 needs critical scrutiny. First, the authors did not use standard or type species as control. Also, the amplicon sizes are too small to assess the results. Have authors provided correct references for these primers? The reference with the doi 10.1016/j.parint.2017.09.002 led to a totally different paper. Other papers are not accessible.

7. How did authors confirm the information given in the lines 194-197.

8. On what basis authors decided to sequence some of the samples and not all? What was the criteria in deciding the number of samples to be sequenced?

9. Information in line 61 is contradictory to the information given in the abstract.

10. There are several language errors including spelling mistakes, formatting issues and redundancy.

Reviewer #3: The article titled "Molecular epidemiology and genetic diversity of Anaplasma and Theileria infections in sheep" by Shahzadi et al. and submitted by Dr. Furhan Iqbal is technically sound and contain important information related to anaplasmosis and theileriosis in sheep of Pakistan. Pakistan being agricultural country with over 70 percent population depends on livestock for their hands to mouth life, this study present important data about livestock parasitic diseases. I will recommend this article for acceptance based on the below mentioned minor edits.

1. Please remove extra information from abstract.

2. Please add more scholarly updated information about the tick borne diseases in Pakistan in the introduction section. This will help improve the rationale part of the introduction.

3. please also clearly mention the gap and need of this study in the last portion of introduction.

4. Please provide a clear map of the study using coordinates of the study area.

5. Results\are well interpreted and presented.

6. Please add some sentences in the discussion portion to make it more interested for the readers.

7. Please revise the conclusion portion based on the results of the study. Also tell the readers about what in future is possible regarding this topic.

Reviewer #4: Dear Authors,

I have reviewed the manuscript titled "Molecular epidemiology and genetic diversity of Anaplasma and Theileria infections in sheep" by Shahzadi et al. Below is my evaluation based on scientific content, language, and adherence to international standards.

Scientific Issues:

1. Title:

- The title is appropriate but could be more concise. Consider: "Molecular Epidemiology and Genetic Diversity of Anaplasma and Theileria spp. in Pakistani Sheep."

2. Abstract:

- The abstract lacks clarity in stating the novelty of the study. The significance of the findings should be emphasized.

- The phrase "phylogenetic evaluation of sp." is unclear and grammatically incorrect.

3. Keywords:

- "Molecular prevalence" is redundant; "Prevalence" suffices.

4. Introduction:

- The economic impact of these infections in Pakistan should be better justified with recent data.

- Some statements lack citations (e.g., "Pakistan is third in the World...").

5. Objectives:

- The aims are clear but could be more explicitly linked to gaps in existing literature.

6. Materials and Methods:

- The sampling strategy (e.g., random vs. convenience sampling) needs clarification.

- PCR conditions (annealing temperatures, cycle numbers) are missing.

- Statistical analysis: Clarify why Fischer’s exact test was used over chi-square for some comparisons.

7. Results:

- Table 1: The co-infection data is useful but should be presented more clearly.

8. Discussion:

- The discussion compares findings well but overstates some conclusions (e.g., genetic similarities with global strains without deeper analysis).

- The absence of Theileria lestoquardi should be discussed in the context of regional vector ecology.

Suggested Addition to the Discussion Section:

Add the two below citations to the reference list and the discussion.

https://doi.org/10.1016/j.smallrumres.2022.106617

https://doi.org/10.1007/s11250-021-03007-4

Two recent works by Noaman and colleagues provides valuable insights into the epidemiology of tick-borne pathogens in small ruminants.

Overall Assessment:

The manuscript presents valuable data on tick-borne pathogens in Pakistani sheep but requires minor revisions for clarity, grammatical accuracy, and methodological details. The results support the discussion and references need verification.

Recommendation: Accept after minor revisions.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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Molecular epidemiology and genetic diversity of Anaplasma and Theileria infections in sheep

PONE-D-25-28992R1

Dear Dr. Iqbal,

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,

Abdelfattah Selim, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have thoroughly addressed all reviewer comments and significantly improved the manuscript. I recommend acceptance in its current form.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #1: Thank you for your thorough and thoughtful revision of the manuscript. I have carefully reviewed the revised version and the authors’ responses to the previous comments. I am pleased to note that all the minor concerns raised in the initial review have been fully addressed.

The revised manuscript demonstrates improved clarity, coherence, and scientific rigor. The additional data and explanations provided have significantly strengthened the overall quality and impact of the work. The methods are now clearly described, the figures and tables are appropriate, and the conclusions are well-supported by the results.

Based on the comprehensive revisions and the satisfactory responses to all reviewer comments, I recommend the manuscript be accepted for publication in its current form.

Reviewer #3: All my comments on the previous version is well addressed. Hence, i recommend acceptance of this article.

Reviewer #4: Corrections are acceptable. The authors made all the proofreading and suggestions. Now, the manuscript can be considered for publication in the PLOS ONE as a Research Article.

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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

Reviewer #3: No

Reviewer #4: No

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