-->PONE-D-25-57063-->-->Peripheral leukocyte transcriptomic changes in preweaned Holstein heifer calves with varying stages of Bovine Respiratory Disease-->-->PLOS One

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Reviewers' comments:

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-->Comments to the Author

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

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Yes

Reviewer #2: Yes

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: Yes

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: Bovine Respiratory Disease is a persistent and damaging disease in cattle and efforts to understand the disease in greater detail and potentially identify targets for improving diagnosis and treatment are welcome. The methods are comprehensive and very well written. The manuscript makes a new contribution to our understanding of the disease, it is well written, and the Figures are of high quality.

I have the following comments for the authors:

1. Abstract: More detail needs to be included on the FDR cut off and numbers of DEG for some important contrasts.

2. Table 1 should be included as supplementary data

3. My major issue with this manuscript is the selection of the most appropriate data for inclusion in the Figures. Figure 1 should graphically represent the numbers of DEG in each contrast. Subsequent Figures should show volcan plots for time point comparisons. There is no need for 15 Figures showing functional enrichment visualization of GO terms – the most important ones should be included in the main manuscript, and the others should appear in supplementary data. The cellular component Figures are not as valuable in the context of the aims of the paper. What about a Figure showing the predictive classification of the samples using the RFA conducted? What genes most effectively correctly classified resolvers from non-resolvers?

Reviewer #2: The manuscript by Makratzakis et al. is a very interesting study that aims to identify many leukocyte transcriptomic aspects for the resolution of BRD.

In my opinion, the article is very interesting, but in some aspects it is confusing in its presentation of tables and data. Unfortunately, it is very long and hard to read and the information may appear confusing when reading the manuscript.

I believe that the authors should summarise and reorganise the presentation of the data and their results.

I believe that there is a bias in the study in terms of the number of animals sampled and the detection of active or chronic lesions by ultrasound.

If possible, I would suggest that the authors avoid this division into years and consider only the disease, perhaps including time and stables as variables.

Questions and suggestions:

1) The relevant question that I have is related to Ultrasound scans and how many animals had active or chronic pneumonie per group.

In recent years, many variations have been made to the TUS score (Ollivett and Buczinski, 2016). Recent studies suggest that consolidation can be viewed and interpreted in different ways. Consolidation refers to the functional loss of lung aeration, which is shown in ultrasound in different ways: disappearance of A lines, presence of fluid and B lines (inflammatory phlogosis, probable bacteria? active pneumonia?), lesions and pleural effusions, liver-like lesions (viral lesions or chronic lesions?), or lesions with mixed aspects between these (liver-like associated with B lines). All this is used to identify if the lesion is active or not. (I recommend reading the score by Fiore et al 2022 and modified by Lisuzzo et al., 2024; other authors are Feitoza et al 2024 and 2025.) I am writing you that because I expect a better leukocyte and transcriptome response, especially in active lesions.

- Are the authors able to identify if the lesion is active in the study? Did you have B lines (active)?

- Is it possible to define the differences in leukocyte transcriptomics in active and chronic pneumonia on the basis of these changes?

2) the representation of table 1a, 1b, 1c have to be resumed and clarify for readers

3) I cannot read the tables and I cannot vision figures where you describe relations with genes and other variables. The tables appear cut to the right beyond the ‘Gene Name’ column. Maybe I have techical problem or author had submitted tables in the text in a not correct way.

4) You describe more than 20 tables and figures probably related to the same significance. I ask you to simplify and summarise the results and discussions section.

Reviewer #3: The manuscript presented the molecular mechanisms underlying the Bovine Respiratory Disease in different stage using RNA seq approach although the manuscript provided the interesting findings following concern should be addressed by the authors

1- the introduction and discussion must be flourished by the relevant published papers. for example i found that recently the integration of systems biology approach with machine learning for BRD in cattle"Weighted gene co-expression network analysis identifies functional modules related to bovine respiratory disease" It may better authors discuss and compare their findings with prior reports,

2- The material and method must be provided by details, for example the applied script for machine learning modeling must be provided in supplementary files.

3- for modeling the expression profiles, it may be better author perform the modeling with different ML approach to compare the model with higher accuracy rate

4- authors although mentioned that they perfomed the network construction using PPI, but I have not found any constructed network

5- The must popular approach for ranking and prioritizing the functional genes in network analysis is the hub genes analysis, I dint find such analysis. It is essential that authors perfom and provides the hubs for functional prioritization the candidate genes.

6- Authors must be mentioned that the functional enrichment performed in what level of Gene ontology?

7- It must be perfomed KEGG enrichment analysis for the more dissection of functional enrichment of overlapped genes of DEG and Machine learning candidate genes

Minor revision

1- figure quality is very low

2- it may be appropriate that several Tables transfer to the supplmentary fils, and authors perfom and provides the ven diagram for the coverage analysis of candidate genes beween DEG and ML approach

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Reviewer #1:  Yes: Kieran Meade

Reviewer #2: No

Reviewer #3: No

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Peripheral leukocyte transcriptomic changes in preweaned Holstein heifer calves with varying stages of Bovine Respiratory Disease

PONE-D-25-57063R1

Dear Dr. Makratzakis,

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,

Tofazzal Md Rakib, PhD

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

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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

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

Reviewer #1: 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

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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

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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: My comments have been addressed and the manuscript has been improved. One final comment is that volcano plots are much more informative if the top number of DEG are labelled in the diagram.

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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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