Dear Dr Boulangé,

Thank you very much for submitting your manuscript "Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

As specified by Reviewer 2, all primary data have to be provided in an appropriate format (e.g. a source data file) for transparence and documentation.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Philippe Büscher, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Michael Boshart

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The manuscript presents a comprehensive epidemiological study of three regions (Hauts-Bassins, Cascades and Sud-Ouest) located in the southwestern part of Burkina Faso, between April and May 2019, at the end of the dry season. The study included 1,898 domestic animals (cattle, sheep, goats and pigs). Various techniques were used to investigate the prevalence of infection of the trypanosomes responsible for AAT, transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei. The presence and species of tsetse flies were also assessed.

The diagnostic methods used in the survey were adequate: PCR with TRYP1(R & S) primers to amplify the ITS1 of the ribosomal DNA of most trypanosome species, which allows species discrimination based on size polymorphism. Species-specific PCR tests were then used on the positive samples: T. brucei s.l. 171 (TBR1/TBR2), T. congolense “Savannah” type (TCS1/TCS2) (20), T. congolense “Forest” 172 type (TCF1/TCF2), T. vivax (TVW1/TVW2) (21) and T.b. gambiense specific glycoprotein 173 TgsGP ”.

Serological diagnosis: whole soluble antigen of T. vivax, T. congolense or T.b. brucei and immune trypanolysis test.

The entomological survey used biconical traps for tsetse flies at various sites along the riverbanks in the three study areas for 48 hours each. This was followed by species identification. counting and determination of “the number of tsetse captured per trap per day”.

Data analysis and statistical tests were performed to compare: 1. prevalence of the different study areas, the animal species, and the sexes 2. prevalence between the different infections and the association of trypanosome infections with anemia and 3. to study the effects of parameters such as sex, age, PCV, animal species, and study areas on the molecular prevalence. Finally, a Ven diagram was constructed to analyze the levels of concordance of detection of the tests used between parasitological, serological, and molecular data.

Reviewer #2: The methods described in this study are clear and properly referenced. There is however one question that could be addressed by the authors and that is: why was Trypanosoma theileri not included in the study? While being considered mostly non-pathogenic, the presence f this infection can lead to to false positive results in a number of diagnostic approaches.

Minor comment: could the TRYP1 primers be added to the primer table?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results and Discussion:

1. Table 2 presents a summary of the sampled animals per region, species and sex and n=1898 animals. Table 3 includes the various animal species that were sampled, classified according to their physical condition and n=1813.

R. Why are the numbers of total samples and animal species in Tables 2 and 3 different?

In the following tables, which present summaries of BCT, PCR and ELISA results in the three study regions (Table 4) and the same results except for the classification by trypanosoma species (Table 5),

n= 1898.

R: Aside from the discrimination by trypanosome species, could the authors present the data about possible trypanosome co-infections based on their molecular diagnosis?

This seems relevant, since the authors state in the Discussion (lines 430-432): “In our study, animals infected with one species of trypanosome had lower mean hematocrit than those diagnosed as negative, and animal with multiple infections even lower.

See also reviewer’s next comment about seropositive animals for at least two species of trypanosomes.

2. Lines 314-317: Concerning the seroprevalence, the authors indicate:

“Overall, of the 934 positive cases, 527 were T. vivax infections, while 59 were T. brucei infections, 26 were T. congolense and 323 (34.6%) cases were positive for at least two species of trypanosomes (Table 5).

R. The values (total and %) on Table 5 (%) do not match those of the above mentioned text.

833 (43.9%) T. vivax, 325 (17.1%) T. brucei and 172 (9,1%) T. congolense.

In addition, there is no data in the table for co-infection with two trypanosome species.

Reviewer #2: While the paper is very clearly written, the result section is very cryptic and most true scientific results are not shown. Hence, the reader is forced to 'accept' the conclusion of the authors, without being given a chance to appreciate the scientific data. Tis is OK for a review, but not really acceptable for scientific 'data' publication.

Question 1: ELISA results in trypanosome research can be 'tricky' to interpret. The OD% explanation in the MM section makes one wonder what the real issue is the readout. This could be anywhere between 0.1 and 4.0 and while a % value will mask any inconveniences, it also makes the data very susceptible to individual interpretations. This reviewer understands that 10 plates of positive result is 'a lot' but the true results should be added as supplementary data. In particular for the 232 double PCR+ results. The reader should be given the chance to see 'what that means'.

The same holds true for the PCR data itself. While this reviewer understands that this is a lot of visual data, it is hard to accept that nothing is shown. For example: a reader could be interested in seeing how a PCR+/ELISA+ sample is scored n gel, versus a PCR+/ELISA- result. This data is important is the work is to be taken as a reference for others, and future studies.

A final questions: the results described in Lines 335- (the TL positive PCR positive T. brucei sample in pig and the T. b. gambiense positive sample in cattle: why are such important results mentioned without showing any true result. The authors must have the visual data in hand, so there is no reason not to show them (on not showing them, without mentioning 'data not shown' is truly not acceptable).

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes, the conclusions are supported by the data presented. The authors clearly state the limitations of the various tests performed, as well as the impact of other variables that may affect the trypanosome prevalence, such as the dry season, during which the sampling was carried out, the bias in the sampling towards animals in poor physical condition.

Yes, the authors addressed the importance of their study to better understand the dynamics of the epidemiology of AAT in Burkina Faso.

Reviewer #2: There are no major issues with the conclusions, other than that they cannot be challenged by the reader (or reviewer) because none of the actual data is shown. In fact, technically most of the 'result' section should be moved to the 'conclusion' section...leaving the actual scientific result/data section virtually empty.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Abstract:

Lines 31-32. Misspelling: It was therefore important to investigate the extend extent of the infection..

Author abstract:

Line 59: The low occurrence of tsetse fly

R: Tsetse flies?

Introduction:

Lines 81-82: The impact of trypanosomes on livestock productivity negatively affects millions of people in rural communities who depend on animal for their livelihood

R. Do the authors mean “animal products”?

Lines 83-84: “adding a public health component to an already pregnant economic issue”

R. Suggestion:” adding a public health component to an already complicated economic issue”

Lines 88-90: The potential benefits of better control of AAT on the continent, in terms of meat and milk productivity alone, are estimated to US$700 million per year (2).

R: are estimated up to US$700 million per year

Lines 448-449 and 450-452:

The concordance of the results obtained with BCT and PCR confirms the specificity of these tests for the diagnosis of TAA.

The difference in specificity between this serological diagnosis and the others is high and its significance could be questioned for the diagnosis of active TAA infections.

R: Please, clarify the meaning of TAA, do the authors mean AAT?

Reviewer #2: Data presentation?

See above...virtually no data is shown. Mostly conclusions are presented.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study provides a very good assessment of the epidemiology of AAT in Burkina Faso. The authors show excellent command of the strengths, weaknesses and limitations of the various diagnostic tools that were used. The combination and comparison of the various results as well as the statistical analyses performed to compare the data from three different regions, and various species of domestic animals are especially noteworthy and merit publication.

The manuscript requires minor revisions, as indicated above.

Reviewer #2: This is a paper with good potential. All this reviewer is asking for is to show the visual (PCR) and numerical (ELISA) data that has been used to draw the presented conclusions.

--------------------

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

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr Boulangé,

Thank you very much for submitting your manuscript "Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The original manuscript was revised according to the instructions of the reviewers and is almost ready for acceptance.

Only some minor corrections are suggested by reviewer 1. We trust that the authors will follow these suggestions and will submit a further revised manuscript soon.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Philippe Büscher, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Michael Boshart

Section Editor

PLOS Neglected Tropical Diseases

***********************

The original manuscript was revised according to the instructions of the reviewers and is almost ready for acceptance.

Only some minor corrections are suggested by reviewer 1. We trust that the authors will follow these suggestions and will submit a further revised manuscript soon.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The manuscript has been extensively revised by the authors and resubmitted. As far as this reviewer is concerned, all the previous comments and observations were addressed. The objectives of the study are clearly stated and justified: 1. to assess the prevalence of various trypanosome infections and 2. to carry out an entomological survey on the presence of tsetse flies in various regions in Burkina Faso. The design of the experiment, ethical aspects, methodology and statistical analysis are appropriate and sustain their conclusions.

Reviewer #2: All previously raised questions have been addressed

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Numerical data have been submitted to a data depository (Dataverse).

Tables 4,5 and 6 were modified in the revised manuscript, which makes it easier to analyze the data. Two tables, 7 and 8 were added. Table 7 presents the serological prevalence of various trypanosomes, measured through indirect ELISA with various soluble trypanosome antigens (T. vivax, T. congolense or T.b. brucei), while Table 8 presents the analysis of PCV values for various trypanosome species.

However, the following points must be revised/ addressed by the authors:

Lines 278-280: “The overall molecular prevalence assessed by PCR was 23.1% with a total of 438 positive samples. The Hauts-Bassins region showed the highest prevalence with 60% followed by Sud-Ouest 12% and Cascades 10.6% with 276, 80 and 82 positive animals respectively 280 (Table 4).

R: The authors must revise and correct either the text or the data in Table 4. If the values presented in Table 4 are correct, the text should read: “The Sud-Ouest region showed the highest prevalence with 60%, followed by Hauts-Bassins with 12% and Cascades with 10.6% with 276, 80 and 82 positive animals respectively (Table 4).

Line 360. Column heading for Table 8. PCV versus trypanosome species in cattle

R: The column heading “Nbre d’animaux” must be translated to English.

Lines 368-370: “In details, the Hauts-Bassins region presented the highest AD with 2.32 flies caught per trap per day followed by Sud-Ouest and Cascades regions with 0.42 and 1.23 respectively”.

R: The statement should be corrected to present the regions in decreasing order of AD:

“In details, the Hauts-Bassins region presented the highest AD with 2.32 flies caught per trap per day, followed by Cascades and Sud-Ouest regions with 1.23 and 0.42, respectively”.

Reviewer #2: All previously raised questions have been addressed

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are supported by the data and the authors clearly discuss the limitations of the study and their epidemiological data. They also highlight the importance of this study and the contribution to implement future control measures and a comprehensive path to control AAT in the region.

Reviewer #2: All previously raised questions have been addressed

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Introduction:

Lines 80-81: “In addition, the same tsetse flies also transmit the agents of Human African Trypanosomosis (HAT), also known as sleeping sickness (5, 6).”

R: Replace the term “Trypanosomosis” for the correct one for human disease: “Trypanosomiasis”.

“In addition, the same tsetse flies also transmit the agents of Human African Trypanosomiasis (HAT), also known as sleeping sickness (5, 6)”

Reviewer #2: All previously raised questions have been addressed

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study is novel and comprehensive and draws interesting conclusions as it compares various diagnostic methods and analyzes the prevalence of T. vivax, T. congolense and T. brucei sl, as well as the presence of tsetse flies. The manuscript is well written and greatly improved by the inclusion of numerical data, representative figures of the PCR results and additional tables that facilitate the interpretation of the results.

Reviewer #2: All previously raised questions have been addressed

--------------------

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

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

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.

Dear Dr Boulangé,

We are pleased to inform you that your manuscript 'Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Philippe Büscher, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Michael Boshart

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

This second revision of the original manuscript has now been adapted according to all reviewers' comments and is ready for acceptance.