PONE-D-24-13222Epidemiology of Canine Ehrlichiosis and Molecular Characterization of Erhlichia Canis in Bangladeshi Pet DogsPLOS ONE

Dear Dr. Ehsan,

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:

• It is suitable for re-review after major comments.  

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

Please submit your revised manuscript by Jul 11 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Faham Khamesipour, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

I want you to respond to the comments and revise the manuscript. Given the growing dog population in Bangladesh and the significant zoonotic implications of the disease, we may find merit in favorably considering this manuscript.

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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: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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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: No

Reviewer #2: No

Reviewer #3: No

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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: No

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: The authors comprehensively explored the epidemiology of canine Ehrlichiosis (Ehrlichia canis), bolstering their insights with meticulous molecular and microscopic analyses. Their discoveries represent a significant advancement for the study region, shedding light on a topic often overlooked despite its zoonotic importance. I commend the authors for undertaking this vital research. Their manuscript is skillfully crafted and logically presented.

While the manuscript is commendable, there are some areas that could benefit from refinement:

1. Ensuring consistency in the presentation of scientific names, adhering to journal guidelines for formatting.

2. Correcting a typo in the methodology section regarding the abbreviation "EDTA" (line 132)

3. Standardizing the presentation of primer names and codon hyphenation (line 145).

4. Removing redundant terminology, such as "status," from line 165.

5. Adding punctuation for clarity in line 186 after mentioning the Rajshahi district's prevalence.

6. Inserting a comma after "Positive control" in line 192.

7. Streamlining Section 3.2 by summarizing significant findings rather than duplicating data from tables.

8. Providing high-resolution versions of figures for enhanced clarity, particularly Figure 1.

9. Clarifying the source of the positive control used in Figure 2, considering it's the first report of E. canis in the study area.

10. Enhancing Figure 4 by incorporating the 18 partial sequences obtained in the study to offer a comprehensive view of diversity.

11. Providing the questionnaire used in this study as a supplementary file

Reviewer #2: The article in question suffers from several methodological and conceptual flaws that compromise its suitability for publication. First, the lack of rigor in standardizing the writing of scientific terms, the lack of adequate references for key statements, the inadequate sample size, the non-random sampling, and the lack of justification for the choice of expected prevalence in the sample analysis are significant flaws that undermine the study's credibility.

Other problematic issues include a lack of clarity in the definition of the target population of dogs studied, as well as a sample collection interval that is too long for a cross-sectional study. In addition, there are conceptual inaccuracies, such as the misuse of the term "risk factor" in a cross-sectional study and statements about the sensitivity of diagnostic techniques that lack substantiation. Finally, the quality of the presented visual evidence is questionable, featuring poor-quality images and subjective interpretations. All considerations regarding the manuscript are listed below. Therefore, I recommend rejecting the article as submitted for publication in Plos One.

Major Revision

Line 112. To calculate the sample size, justify the use of an expected prevalence of 20%. It would be correct to use 50% of the expected prevalence. This is especially true in areas where there are no molecular epidemiologic studies to determine the prevalence of E. canis infection in dogs. With an expected prevalence of 50%, the authors would achieve the highest possible sample size with a precision of 5%.

Line 108. The criteria for determining the target population of dogs is not entirely clear. Did the study include both sick and healthy dogs, or did it only include sick dogs? Regardless of their living area, dogs exhibiting clinical signs are more likely to contract E. canis.

Line 124. Contrary to what the authors claim, the sampling was not random because the authors used the criterion of animals attending veterinary clinics to select the animals. This is an important selection bias since the dogs that visit the veterinary clinics do not adequately represent the same characteristics as the population of dogs that live in the rural and urban areas of the different districts of Bangladesh. Therefore, we cannot directly extrapolate the results of the analysis of the sample dogs to the dog population in Bangladesh.

Line 165. The text uses the term "risk factor" inappropriately throughout. It is not possible to analyze risk factors in cross-sectional epidemiologic studies because it is not possible in this type of study to determine the temporality between cause and effect, i.e., it is not possible to establish a precise time between exposure to associated factors and PCR analysis of E. canis in dogs.

Lines 168-169. The authors cannot state the mortality rate with absolute accuracy. It is not possible to guarantee that all dog owners have reported their dog's death.

Lines 167-168. The PCR is more sensitive than the blood smear test. Using this case definition significantly increases the number of false negative results. This introduces a measurement bias into the study, which will impact the analysis of factors associated with E. canis infection in dogs.

Lines 188-189. Ehrlichia canis is usually found in monocytes but rarely in neutrophils. The presence of E. canis morulae in lymphocytes is unlikely. The authors present an image (Figure 3) that lacks credibility and is of poor quality. The arrows indicate the presence of artifacts within the erythrocytes.

Minor Revision

Line 57: Write E. canis in full. Write scientific names in full at the beginning of a sentence.

Line 60. Write E. canis in full.

Lines 62-64. The etiologic agent (E. canis) is responsible for the disease. Provide a reference for this sentence.

Lines 64-66. The clinical manifestations of CME are not directly comparable to those of HME. HME typically manifests as a mild disease in the vast majority of cases, whereas immunocompromised individuals experience the severe form of the disease.

Lines 65-66. Add a citation for this sentence.

Line 67: Replace the term subclinical with subacute.

Line 71. E. canis in full

Lines 131-132. This sampling period is relatively long for a cross-sectional epidemiologic study. The authors should justify the sample collection interval and its implications for cross-sectional studies.

Line 132 EDTA

Lines 136-139. Explain how each variable was categorized.

Line 147: Ehrlichia in italics.

Reviewer #3: PONE-D-24-13222

The data presented in this manuscript are very relevant addressing a vector-borne disease in dogs prevalent in several countries, but some changes are recommended.

Title: Epidemiology of Canine Ehrlichiosis and Molecular Characterization of Erhlichia canis in Bangladeshi Pet Dogs

Change to lower case in the species.

• Introduction

“Ehrlichia canis (E. canis) is the most critical and deadly species among different members of the genus Ehrlichia, causing canine monocytic ehrlichiosis (CME), also known as tropical canine pancytopenia”.

Although E. canis can be fatal, especially in the chronic phase of the disease. I recommend changing this phrase, as it may convey the erroneous idea that this agent is more pathogenic than others of the same genus for different hosts.

• Material and Methods

When calculating the mortality rate, was the population at risk considered?

“Mortality: The estimated total number of deaths in a population of a given sex and/or age, divided by the total number of this population, expressed per 100,000 population, for a given year, in a given country, territory, or geographic area.”

(https://www.who.int/data/gho/indicator-metadata-registry/imr-details/1157)

• Discussion

“Notably, half of the dogs that contracted ehrlichiosis succumbed to the disease”.

Review the discussion.

As the sample was collected from veterinary clinics, and not through household surveys, there is a sampling bias. There is a tendency for dog owners to seek veterinary assistance when their dogs are sick, therefore, dogs that have mild signs or those that are in a subclinical phase may not have had the infection investigated. Furthermore, the mortality rate must consider the entire population at risk, not just those cared for in clinics.

• References

Standardize formatting

Improve the quality of Figure 3.

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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: Jayedul Hassan

Reviewer #2: No

Reviewer #3: 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.]

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PONE-D-24-13222R1Epidemiology of Canine Ehrlichiosis and Molecular Characterization of Erhlichia Canis in Bangladeshi Pet DogsPLOS ONE

Dear Dr. Ehsan,

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:

• Minor revisions are needed. I want you to respond to the comments and revise the manuscript. 

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

Please submit your revised manuscript by Nov 29 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,

Faham Khamesipour, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

Minor revisions are needed. I want you to respond to the comments and revise the manuscript.

[Note: HTML markup is below. Please do not edit.]

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 #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: No

**********

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 #1: Yes

Reviewer #3: Yes

**********

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 #1: 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 #1: I appreciate your response and the thoughtful way the comments were addressed. However, the supplementary materials were not cited in the main text. I recommend that the authors cite the supplementary files "Supplementary 1.docx" and "Erlichia Supplementary.xlsx" in the appropriate sections of the main text. Additionally, please remove "Supporting Information Fig. 2 raw.tif.

Reviewer #3: Line 54 - "E. canis", scientific genus name at the beginning of a sentence should be written in full.

Line 93 – correct “… in south a sian countries…”

Item 2.8 Statistical analysis also contains mortality

“…The mortality and case fatality was calculated based on the follow-up communication with the dog owners. Z-tests for proportions were used to compare prevalence, mortality, and case fatality among districts [24, 25].

Format tables according to the journal's standards. For example, Table 2 has rows without data

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7. 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: Yes: Jayedul Hassan

Reviewer #3: 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.

PONE-D-24-13222R2Epidemiology of Canine Ehrlichiosis and Molecular Characterization of Erhlichia Canis in Bangladeshi Pet DogsPLOS ONE

Dear Dr. Ehsan,

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:

• Minor revisions are needed. The authors have made the suggested changes, but should carefully review the entire text, as it still contains information about mortality. I want you to respond to the comments and revise the manuscript.

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

Please submit your revised manuscript by Dec 19 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,

Faham Khamesipour, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comment:

Minor revisions are needed. The authors have made the suggested changes, but should carefully review the entire text, as it still contains information about mortality. I want you to respond to the comments and revise the manuscript.

[Note: HTML markup is below. Please do not edit.]

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 #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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 #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: 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 #1: Yes

Reviewer #3: Yes

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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 #1: Yes

Reviewer #3: Yes

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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 #1: The authors are highly appreciated for making modifications as suggested. I believe this study will contribute combating the deadliest disease in the study area and pave the way to further research.

Reviewer #3: The authors have made the suggested changes, but should carefully review the entire text, as it still contains information about mortality. For example: line 252 "The present study's overall mortality and case fatality were 3.3 % and 47.1%, respectively"

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Reviewer #1: Yes: Jayedul Hassan

Reviewer #3: No

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Epidemiology of Canine Ehrlichiosis and Molecular Characterization of Erhlichia Canis in Bangladeshi Pet Dogs

PONE-D-24-13222R3

Dear Dr. Ehsan,

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

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 #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #1: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

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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 #1: Yes

Reviewer #3: Yes

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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 #1: 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 #1: (No Response)

Reviewer #3: (No Response)

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7. 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: Yes: Jayedul Hassan

Reviewer #3: No

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