-->PONE-D-25-67529-->-->A simulation-based approach to strengthen chronic wasting disease surveillance in captive cervid populations-->-->PLOS One

Dear Dr. Belsare,

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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PLOS One

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Additional Editor Comments (if provided):

After careful evaluation by three independent experts in the field, I am pleased to inform you that your manuscript has been recognized as a potentially valuable contribution to chronic wasting disease (CWD) surveillance policy. The reviewers found the study to be well written and the overall approach promising.

However, several substantial concerns have been raised that must be adequately addressed before the manuscript can be considered for publication. Please submit a revised manuscript that addresses all of the points raised, along with a detailed, point-by-point response to the reviewers’ comments.

We look forward to receiving your revised 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: Partly

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

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: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: Wakefield et al. “A simulation-based approach to strengthen chronic wasting disease surveillance in captive cervid populations”

The authors have taken historical data from the US state of Texas and performed simulations to determine the likelihood of having detected CWD in negative farms. This tool can be useful for identifying facilities that should increase testing so that they can be confident that negative results in a subset of animals are more likely to be reflective of a CWD negative facility. It remains unclear, however, whether this information could be gleaned from simply looking at the proportion of animals tested in each facility.

Major points:

The requirements of the USDA herd certification program should be clearly stated. It is my understanding that while there is no requirement that a percentage of the heard be tested in a given year, in addition to animals that die at an age greater than 12 months, any animal greater than 12 months old slaughtered on or off site must also be tested. On a farm, one would expect this to be a large number of the heard, or at the very least a relatively consistent percentage from harvest to harvest. This leads into my second point: it would benefit the reader to have a summary of the real-life data associated with each facility in Tables 1 and 2. How many animals were in these facilities in each year, and how many/what proportion were tested? What tests were used? What was the sensitivity of these tests? Without this information, it is difficult to assess the benefit of this modeling. Are these detection probabilities simply a reflection of the proportion of animals tested?

It is unclear from the methodology exactly how animal movement from one facility to another is taken into account. Movement would generally be assumed to entail additional risk to the facility, is this incorporated into the model? Or is this Texas specific, where animals that move must first be tested?

Figure 1: What do the individual data points represent?

The statement on line 85: "Even with the requirement for anti-mortem testing of all cervids prior to movement from a facility, sample sizes are often insufficient to confidently detect CWD in low prevalence scenarios". This requirement, presumably, refers to the additional mandates from the state of Texas. However, if all animals are tested, how could the sample size be insufficient? Please clarify this statement.

Editorial points:

Line 36: “misfolded isoforms” should be changed, as prions are not isoforms of the prion protein. Suggestion of “misfolded forms” or “misfolding of the normal..”

Line 39: CWD has also been detected in Europe and Asia

Line 47: suggest “the US state of South Dakota”

Line 48: suggest “the Canadian province of Saskatchewan”

Line 51: “fully impermeable” suggest “impermeable”

Line 54: “prevalence levels” suggest “prevalence”

Line 57: “infectious prions” suggest “prions”

Line 59: “infection” suggest “infectivity”

Line 61: suggest reference for the environmental persistence of prions

-Johnson, Christopher J., et al. "Prions adhere to soil minerals and remain infectious." PLoS pathogens 2.4 (2006): e32.

Line 289: “infectious prions” suggest “prions”

Reviewer #2: There are two major limitations to this approach. Firstly, cervids do not exhibit positivity in peripheral tissues like the tonsil until 6-18 months post exposure (see Mathiason et al. 2009 and others). This sensitivity issue is compounded when only IHC is used instead of sensitive amplification assays (e.g. real-time quaking induced conversion). This delay to positivity was not accounted for in the simulation, and further, the authors claim a 99% negative predictive value. This is simply impossible with a disease like chronic wasting disease that can take years to manifest. Secondly, the percentage of the herd tested needs to be accounted for in the model. The authors mention the importance of this consideration, yet it was unclear if the percentage of herd tested was accounted for in the model.

Minor comments include the following: authors should consider biological sex as a variable for chronic wasting disease modeling, and figure legend specifies green when the color in the figure appears blue.

Reviewer #3: The manuscript by Wakefield, L. et al (PONE-D-25-67529) entitled, “A simulation-based approach to strengthen chronic wasting disease surveillance in captive cervid populations,” explains the authors’ simulation-based method to analyze CWD surveillance results with the purpose to support monitoring of cervid herds. The study is straightforward and the manuscript is well written. Overall the approach may prove to be useful for CWD surveillance policy. While enthusiasm for the manuscript is high, there are a few concerns the authors should address.

Lines 240-243: As the authors note here, the approach is retrospective in nature. Such a point should be highlighted and discussed throughout the manuscript, beginning in the abstract. The abstract does not clearly state how the approach should be used, but rather summarizes that the approach addresses the issue with negative CWD results. Instead the authors should state early on that this is a retrospective approach that can be applied to historical CWD testing data and be used to identify at-risk herds. Throughout the manuscript, especially the management implementation section, the authors should highlight the application of the approach to identify at risk herds. This is a valuable application of the approach and should be a focus of the authors descript of the use of the application.

Lines 85-87: The authors write, “…sample sizes are often insufficient to confidently detect CWD in low-prevalence scenarios,” but can the authors provide a literature citation to statistical data that support this statement? Have there been any studies that performed power analyses, false discovery rate (q-value), or other statistical analyses to determine the sample size needed for confident determination of CWD presence or absence for a given testing method?

The introduction/background section would benefit from a brief discussion of the CWD testing methods and their sensitivity, as mentioned in the section of Lines 122-141. For example, the RT-QulC test on deer urine other biological samples, like blood or cerebrospinal fluid. Such a discussion would help put the shortcomings of testing in context for the manuscript.

Lines 50-51: Can the authors state this in the positive? For example, “There is a persistent risk of reciprocal CWD transmission between captive and wild cervids, because fences are permeable.”

Lines 142-161: A salient point that the authors should explicitly state is whether testing scenarios re-tested previously tested deer and what proportion of the annual sample test size (e.g. was 1 out of 10 deer previously tested?). Additionally, the authors state that 1,000 iterations were tested. How was each iteration varied between one another? Was a new iteration a random selection, semi-random selection, or non-random selection of tested deer? Finally, how were sampling sizes determined? Is a percentage of the heard tested? It would be helpful if there was some statistical analysis to help determine the testing sample size given the population size (and possibly including the variable of pen size- like 1 sampled deer per 10 deer per acre). If such parameters are already accounted for in CapOvCWD-then please reference these points in this section.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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-->PONE-D-25-67529R1-->-->A simulation-based approach to strengthen chronic wasting disease surveillance in captive cervid populations-->-->PLOS One

Dear Dr. Belsare,

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.

Please submit your revised manuscript by Jul 01 2026 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 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Jung-Yong Yeh

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

The reviewers identified several areas requiring clarification, further development, and correction. I therefore invite the authors to revise the manuscript in accordance with the reviewers’ comments and to submit a revised version together with a detailed point-by-point response addressing each issue raised.

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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 #2: (No Response)

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 #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

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 #2: 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 #2: 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: The authors have responded adequately to my initial critiques. My suggestion that “infectious prions” be changed to “prions” is rooted in the fact that prions are, by definition, infectious, making the statement “infectious prions” redundant. I will not belabour this point further, however.

The inclusion of the new table 1, which provides the proportion of animals tested in each year, reiterates a point made in the first round of review. At first glance, it appears that the model is simply assigning confidence to years in which a very large proportion of animals were tested. For example, in 2023 TxCF1-4 had the following proportions of animals tested: 208/260, 237/265, 423/477, 41/6, 227/288. Looking at these numbers alone, I would conclude that it is unlikely that CWD was present in these facilities, and it is unclear to me what additional insight is provided by this modeling in these scenarios. In the case of TxCF18-19, there is high confidence that fawns in 2020 did not have CWD despite no fawns being tested. In other instances in 2023, the confidence is low using multi-year data despite relatively high testing percentages (TxCF5, 11, 13). I feel the manuscript would be improved if this, and potentially other examples, are explicitly discussed in this light to further highlight the usefulness of this modeling.

Reviewer #2: The authors did not address limitations pointed out previously. Concise recommendations are provided below.

As the authors point out, a 99% negative predictive value can be achieved from low prevalence scenarios. However, the current verbiage is "Across this sensitivity range, the NPV exceeded 99%, indicating a high likelihood that deer with CWD not detected results using ante-mortem methods were free of CWD, supporting the inclusion of CWD not detected ante-mortem tests as reliable indicators of CWD-negative status in the model.” Change to “Across this sensitivity range and prevalence <1%, the NPV exceeded 99%, indicating a high likelihood that deer with CWD not detected results using ante-mortem methods were free of CWD, supporting the inclusion of CWD not detected ante-mortem tests as reliable indicators of CWD-negative status in the model. However, NPV will decrease as prevalence increases, and as such, the 99% NPV value is only applicable for low prevalence scenarios” or something similar. This should be abundantly clear in the manuscript.

At a minimum, authors should mention the unique aspects of CWD pathogenesis that lead to delays in positivity, even for antemortem samples. Their simulation relies on gold standard diagnostic tests that require months post exposure to show positivity. This must be mentioned in the manuscript as a limitation to data interpretation. Specifically, a negative test result could be interpreted as the animal is truly CWD-free or the animal has been exposed but has not reached the threshold to positivity. For example, "... a CWD not detected test result indicates that the deer was not infected in any previous year" is simply not accurate.

In the response to reviewer comments, the authors mention “…the approach is designed to evaluate the confidence that can be placed in existing surveillance rather than to declare disease freedom.” As written, the manuscript does not reflect this. For example, "... a high likelihood that deer with CWD not detected results using ante-mortem methods were free of CWD, supporting the inclusion of CWD not detected ante-mortem test results as reliable indicators of CWD-negative status in the model." Further, "..CapOvCWD described here provides a mechanism for assessing the comprehensive risk of undetected CWD in captive cervid facilities."

Lastly, as presented, it is still unclear whether the percentage of herd tested was explicitly included in the simulation. The authors refer to two separate lines that point out these variables, but it would be helpful to make this more concise.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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A simulation-based approach to strengthen chronic wasting disease surveillance in captive cervid populations

PONE-D-25-67529R2

Dear Dr. Belsare,

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jung-Yong Yeh

Academic Editor

PLOS One

Additional Editor Comments (optional):

The authors have addressed all previous comments and improved the manuscript accordingly. I recommend acceptance for publication.

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 #2: 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 #2: Yes

**********

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

Reviewer #2: N/A

**********

-->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 #2: 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 #2: 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 #2: All concerns were addressed in the most recent round of edits. Hopefully the CWD antemortem tests improve in the future!

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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.

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Reviewer #2: No

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