Dear Dr. Rahman,

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.

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ACADEMIC EDITOR:

• The paper has now been reviewed by an expert in the field. While the reviewers acknowledge that your study addresses an important gap — namely the absence of data on Mycobacterium avium subsp. paratuberculosis (MAP) in Bangladeshi dairy herds — they have identified several issues that must be resolved before the manuscript can be considered further. After careful consideration of the reviewer’s comments and my own evaluation, I am returning the manuscript for major revision. The reviewer appreciates the novelty and potential value of your work, but notes that the current version overstates its representativeness and certainty. Specifically, the survey’s non-random sampling, limited geographical balance, and use of milk ELISA only restrict the generalizability of the conclusions. Furthermore, several methodological details and interpretations require clarification or revision. Please submit your revised manuscript. Along with your revision, include a point-by-point response detailing how you have addressed each reviewer comment and where changes were made in the text.

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Faham Khamesipour, Ph.D.

Academic Editor

PLOS ONE

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7. We are unable to open your Supporting Information file [Supplementary_file_3_R_code_herd_level_risk_factors.Rmd, Supplementary-file_4_R_code_cow_level_risk_factors.Rmd and Supplementary_file_5_R_code_herd_cow_level_true_prevalence.Rmd]. Please kindly revise as necessary and re-upload.

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Additional Editor Comments:

The paper has now been reviewed by an expert in the field. While the reviewers acknowledge that your study addresses an important gap — namely the absence of data on Mycobacterium avium subsp. paratuberculosis (MAP) in Bangladeshi dairy herds — they have identified several issues that must be resolved before the manuscript can be considered further. After careful consideration of the reviewer’s comments and my own evaluation, I am returning the manuscript for major revision. The reviewer appreciates the novelty and potential value of your work, but notes that the current version overstates its representativeness and certainty. Specifically, the survey’s non-random sampling, limited geographical balance, and use of milk ELISA only restrict the generalizability of the conclusions. Furthermore, several methodological details and interpretations require clarification or revision. Please submit your revised manuscript. Along with your revision, include a point-by-point response detailing how you have addressed each reviewer comment and where changes were made in the text.

[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: Partly

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

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Title and Abstract

The phrase “True prevalence” could mislead readers to assume diagnostic certainty; consider “Estimated true prevalence.”

The abstract contains excessive numerical detail i.e. confidence intervals and regression coefficients, that might be summarized more succinctly for readability.

The term “nationwide” might be overstated given sampling in only 14 districts.

Introduction

This section provides a comprehensive background on Mycobacterium avium subsp. paratuberculosis (MAP), its global distribution, and regional context and clearly identifies the knowledge gap and study rationale for Bangladesh.

Materials and Methods

The sampling strategy for herd selection is described as “convenient,” which introduces potential bias and limits national representativeness.

The criteria for selecting 14 districts and seropositive herds for individual sampling are not fully explained.

The diagnostic sensitivity and specificity values for the ELISA (herd-level Se = 9.58%) seem unusually low; justification or source verification is necessary.

Results

Results are systematically presented from descriptive statistics to modeling outcomes. Figures and tables are well structured and appropriately referenced. The clear distinction between apparent and true prevalence is valuable.

The jump from apparent to true herd-level prevalence (7.2 % → 71.2 %) seems implausibly large; discussion of possible model over-correction or low test sensitivity should appear here.

Map need better captions specifying sample numbers per district.

Discussion

This section is well written and compares results effectively with studies from South Asia and other countries. Also appropriately discusses diagnostic limitations and implications for disease control. Integrates productivity and reproductive performance findings logically.

Reviewer #2: General comments:

The manuscript describes the outcome of a survey to evaluate the prevalence of paratb in milk samples in Bangladesh. Although the manuscript contains useful information, as nothing was known about the prevalence in the county before, it has some weaknesses which should be addressed before it can be considered for publication. The main issues are, that the survey was not statistically balanced and therefore does not reflect the situation in the whole country. Furthermore, the authors tend to overinterpret their results and the manuscript lacks a critically discussion, addressing the limitations of the study. Specific comments are provided below which should be addressed during a thorough review of the manuscript.

Specific comments:

Line 22: Why seroprevalence? Serum was not tested throughout the study.

Line 32-35: There were some more significant risk factors, please list them all

Line 63: “…limited sensitivity in eraly stages of the infection, meaning…”

Line 66: “…the paratuberculosis status and prevalence…”

Line 70: “…PCR (polymerase chain reaction).”

Line 71: “…ELISA (enzyme linked immunosorbent assay) …”

Line 71: specify “other tests”

Line 71: Why approximately? Be precise

Line 72: Replace “although” by “and” or “while”

Line 73: Please specify large, how many animals?

Line 77: What kind of skin test?

Line 96: Please rephrase, as farms >25 lactating cows still are small and should not be addressed as large-scale herds, although this might be the case for the situation in Bangladesh.

Line: Why was the expected prevalence 50%? Above it is stated that it is around 30-40% in other Asian countries. Please justify.

Line 108: How were the farms selected for the convenient sampling?

Line 109: If bulk milk samples were tested, why “seropositive” herds? Were there serum samples taken also? Please check and change throughout the manuscript.

Line 110: Provide more details of the farms and cows enrolled in the study, move lines 200-213 to this part of the manuscript, as they are a description of the study population and not a result of your study.

Line 110: Provide manufacturer for the software

Line 113: Each cow of the farm or from selected cows? Was this a combined ample of all for quarters?

Line 115: Department for Medicine of which institution?

Line 117: What are pretested questionnaires? How were they pretested?

Line 119: Purchase/trading of animas and contact to other herds (pasture etc.) are well known risk factors for the transmission of MAP. Why were they mot included in the survey?

Line 137: (ODNC)

Line 150: Where were the cut off values taken from? Manufacturer?

Line 163-164: Are these the Sp and Se values for milk? Please provide a reference, the values could not be found on the home page of the producer of the ELISA.

Line 202: Delete .0 for the mean of 119

Line 209: How were the sampled cows distributed to the farms? In the end, only 4 farms were included concerning single animal testing in the whole survey, although you identified 10 positive farms.

Line 211: please check your numbers, this is not possible: If there was a cow with 0 calves included, the lactation period can not range from 1-11 months.

Line 212: How was the body weight determined?

Line 221: Seroprevalence/Milk?

Line 222: In line 107-109 it is stated, that 10 out of 138 sampled herds were positive = 13.8%, here a prevalence of 7.2% is calculated, how? And why seroprevalence again? Was serum sampled or only milk?

Line 228: Seroprevalence?

Line 230: Seroprevalence/Milk?

Fig. 3: What does the line in the middle of the figures stand for? Add key

Fig. 5: Add key (SP), each figure hast to be self-explaining without connection to the text of the manuscript.

Line 267-269: This paragraph should be moved to the beginning of the results, as these are quite general outcomes of the study

Table 1: The variable “semen source” is not explained at any point of the manuscript. What is source 1, 2 etc.?

Table 1 and 2: Add key and level of significance

Line 310: This gap was not reveled by the study, this is obviously the case when diagnostic tests have a low sensitivity, delete statement.

Line 314-320: Please be more modest when interpreting the results of your study. The survey was not statistically balanced for the whole country, milk samples were used only (which are known not to be the best medium for Paratb detection), the herds showed a high variety in size. All this should be considered and discussed critically. Fig. 1 for example shows, that the herd prevalence was 0% in one province and 100% in a neighbor province-this does most probably not reflect the real situation but is caused by the study design.

Line 321-326: Same as above, consider and discuss weaknesses of the study and interpret with caution.

Line 339: There are also some studies showing, that cows with a high milk yield more often are MAP-positive, this also should be discussed.

Line 344-349: This should be discussed to a further extend, only because there is an association between two findings it doesn’t necessary imply that they are related. There is much more literature available concerning risk factors for Paratb in dairy cows. Also, some well known risk factors (purchase, contact to other herd) were no included in this study. Also, some found strong associations like age are not discussed at all.

Line 325-355: Not really a result of the study, delete. On the other hand, the main results (prevalence on the herd and individual level) should be added to the conclusions.

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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: Yes: Muhammad Avais

Reviewer #2: No

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Estimated true prevalence and associated risk factors of bovine paratuberculosis antibodies in dairy herds in Bangladesh

PONE-D-25-53226R1

Dear Dr. Rahman,

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,

Faham Khamesipour, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: The authors performed a thorough and detailed revision of the manuscript, addressing all questions pointed out at the first revision, This markedly improved the quality of the manuscript!

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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: Yes: Muhammad Avais

Reviewer #2: No

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