PONE-D-22-25378Sores of boreal moose reveal a novel species of parasitic nematode within the genus OnchocercaPLOS ONE

Dear Dr. Benedict,

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.

I appreciate the effort of the authors in compiling this effort on identifying a novel Onchocerca sp. in moose. While I understand why the stages of molt are important in adult moose, I am not sure that level of detail is necessary in text or supplemental figures. Certainly not in moose calves considering none were detected in moose calves. I would suggest the authors spend considerably more text and effort in addressing comments from Reviewer 2 in expanding the methods and results to improve clarity on methods of scoring and whether that is as valuable as the histology and genetic analysis. As indicated by the reviewers, what is the basis for your molt scoring and is it a published scoring system or developed by the authors? If the authors are proposing a molt scoring system then that must be an objective and presented as such throughout the manuscript instead of focusing on adults vs. calves, captive vs. wild, etc.

All of this could be set up much better in the final paragraph in the introduction by explicitly stating your objectives. It appears the authors have 3 objectives but Objective 2 (calves) is simply molt progression which could be removed entirely? The very vague objectives could then focus less on molt patterns (unless it is being proposed here) and more on the findings and identification of the novel Onchocerca sp. as suggested by Reviewer 2. Please submit your revised manuscript by 21 November 2022. 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,

W. David Walter, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

Dates and formatting of dates throughout the manuscript are not consistent as written and very confusing.

Captive versus wild moose were described in the same paragraph of the Material and Methods but should not be treated as if all methods were done on both captive and wild. Why not organize Materials and Methods by the objective? Clearly, molt pattern and thermal imagery was not done on wild moose and cutting out the sore was not done in captive moose. The authors then go back and forth on Dates of molting and collecting roadkilled moose.

Lines 236-239: Again, it is not clear what Methods and Results of captive calf molt progression adds to this manuscript?

Figure caption text should be in consistent formatting. For example, panels are referenced with (A) and B. in Figure 2, A. in Figure 3, and A: in Figure 5.

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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: Partly

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

Reviewer #1: N/A

Reviewer #2: I Don't Know

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

The PLOS Data policy 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

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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

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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: This paper is a detailed account of a novel type of Onchocerca spp. retrieved from sores on moose legs. The methods were well-documented and the accompanying photographs and figures added a lot of value to the manuscript. There were a few places where I think clarification is warranted. Please see below. Overall, a terrific effort!

Abstract: please conclude with a statement about the significance of your findings...similar to what is stated in lines 58-370.

Line 67: suggested rephrasing: "Despite a superficial resemblance suggesting a similar cause, these sores were not cutaneous habronemiasis."

Line 74: please provide taxonomic information for "leeches"

Also, could leeches perhaps be responsible for sores if they were feeding on moose when moose were foraging in wetlands? The leeches may not be observable if the moose is standing in belly-deep water.

Line 80: suggest "whereas" in place of "while"

Line 85: is the degree of inflammation being evaluated via the thermal imagery? This brings up a general concern about how the thermal imagery is being used and interpreted.

Line 98: please provide taxonomic information for "black spruce"

Line 126: The entirety of the hind leg thermal imagery section is in passive voice. I would suggest rephrasing here (and elsewhere in the manuscript) that more active voice be incorporated.

Also, please say here why the thermal imagery is being used.

Line 194: This section is also very passive. Please think about rephrasing. Even if the work was not directly conducted by the authors, consider stating things like, "Technicians used nuclease-free water as a negative control..."

Using active voice would also help clarify the sentence in lines 195-196. I needed to reread the sentence a few times to understand what the subject of the sentence was.

Line 218: please indicate here that the observations were from 15 "unique" adult moose.

Line 247: Here you state there is a peak in temperature at the end of July, yet the trendline continues to increase. I am not seeing a peak. Can you clarify this?

Lines 295-298: This really cool interpretation belongs in the discussion, in my opinion.

First paragraph of discussion: Much of the information about blackfly phenology comes from a manuscript in review. Are there other sources in the literature that can be used?

Line 325: Again here, I don't see the peak.

Line 328: "Only a few of the moose..." but still, it is 25% of those sampled, so I don't think this should be discounted.

Line 354: Please check throughout the use of "black flies" vs. "blackflies" and standardize if appropriate.

Line 360: "never" is a strong word...maybe soften to "never to our knowledge"

Congratulations on the discovery of an uncharacterized Onchocerca species! I hope these comments prove useful!

Sincerely,

-William (Bill) Severud

Reviewer #2: Benedict et al. ‘Sores of boreal moose reveal a novel species of parasitic nematode within the genus Onchocerca.’

The authors used PCR and histology to describe lesions on the hind legs of moose and showed evidence of a genetically unique Onchocerca sp to be associated with lesions in adult moose. Molt patterns and thermal imaging were used to support the hypothesis that sores develop later in the year as hair is sparse during molt. The manuscript is reasonably well-written and provides useful and interesting information.

Most of my suggestions are to expand on sections of the methods and results to improve clarity, as some methods of scoring were not very objective or well-described. Minor suggestions are also suggested at the bottom.

Terminology changed throughout the manuscript and became confusing to follow at times. Finally, the title implies that this is a novel species and while that may be true, a thorough discussion on the challenges of characterizing filarids is warranted considering isolation and morphological evaluation of these nematodes is challenging. The authors use ‘previously uncharacterized’, ‘novel operational taxonomic unit’ and ‘novel species’ throughout the text and title. Since these have different meanings and implications, I strongly suggest a more detailed discussion paragraph about what is explicitly unique about these nematodes, why the OTU terminology is used, and what else needs to be done to further emphasize if this is a ‘novel species.’ The terminology in the abstract seems the most appropriate: ‘previously unknown genetic lineage.’

Lines 106-109: Has this molt score been used in the literature or is this a novel scoring system? Is it based on known progression of molt? If so, include a reference similar to that in the calf molt paragraph.

Line 110: Without context, assessing the ‘level of inflammation’ grossly seems extremely subjective and, in my opinion, without much merit in the absence of histology. If ulceration, size of individual lesions, ‘swelling’ (as is sometimes used in the text), extent of hemorrhage or hyperkeratosis, etc. is what is being assessed, I suggest saying this specifically here in the methods or describing what is meant by ‘level of inflammation’ to the same detail as the molt score. When consistent terminology is defined, also consider rephrasing at lines 233 and 234. If ‘scoring’ inflammation is not important for statistics or other aspects of the results, you could also remove these modifiers completely.

Table 1: hock scores did not appear to be introduced in the text and lacks context in the methods. If line 110 is meant to refer to the level of ‘inflammation’ or meant to introduce the ‘hock score’, I recommend being more explicit.

Lines 295-298 and 345: See comment above regarding novel sequence, OTU, and species.

Results lines 285-289: It seems there are two goals here: 1: to compare PCR prevalence against time to support that later in the year with reduced hair cover, PCR prevalence increases, and 2: to show PCR prevalence in lesions vs non lesions. The first point seems to be appropriately addressed, but not the second point. A simple descriptive statistic on the percentage of ‘sores’ with onchocerca DNA present compared to the percentage of ‘normal tissue’ with onchocerca DNA present would be useful to provide more evidence of causation.

Other important discussion points not included:

1: why there is a lack of sores in other hairless areas of the body ‘eyes, perianal region, etc.’ particularly since flies are known to be present here

2: biopsies from horses with ‘summer sores’ often lack nematodes in section, yet we still assume they are the cause similar to nematodes in this study. This point will provide more support for causation.

Minor suggestions

Line 39: plural subject � ‘are important barriers between the animal…’

Line 46: ‘often have been documented with round sores….’

Lines 82-87: ‘tracked’ vs ‘track’, etc. to keep in past or present tense

Line 94-97: Are these moose captive or free ranging, since both terminology is used? The implications are very different, so I suggest being clearer in this section

Line 168: ‘Top’ of the leg is a little confusing; suggesting using ‘proximal’ or ‘lateral aspect of exposed leg’ or something similar for clarity

Line 192: 4 um ‘thick’ sections, for clarity

Line 219, 265: August 1st and June 30th, etc. to be consistent with how other dates are in the text

Line 227: a reminder of when the ‘end of the study’ occurred may be useful

Figure 4: could the symbols be included in the text figure key with the solid/dashed lines?

Figure 5B: Is the pale staining dermis necrosis or autolysis? If necrosis, that seems to be an important modifier here.

Line 308: While this is probably true, I don’t think you ‘confirmed’ that this is the reason that moose calves did not develop sores ,and I suggest hedging a little bit on this statement.

Line 289: I am unclear what is meant by ‘same moose skin but different lesions.’

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Reviewer #1: Yes: William J Severud

Reviewer #2: No

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PONE-D-22-25378R1Sores of boreal moose reveal a previously unknown genetic lineage of parasitic nematode within the genus OnchocercaPLOS ONE

Dear Dr. Benedict,

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.

The reviewers acknowledged a great improvement of the manuscript. It can be accepted following some small edits and clarifications, as suggested by Reviewer 2.

Please submit your revised manuscript by Jan 06 2023 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,

Angela Monica Ionica, Ph.D.

Academic Editor

PLOS ONE

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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.

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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)

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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: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

Reviewer #2: 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: (No Response)

Reviewer #2: 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: (No Response)

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 #1: (No Response)

Reviewer #2: Benedict et al. sores of boreal moose....

The authors have drastically improved the manuscript. I have a few minor suggestions below to help improve clarity of some sentences. Nice study with interesting findings.

Track changed version; 114-116; new sentence isn’t clear to me. Perhaps it’s the use of ‘what’ in line 115?

Track changed version line 137; can thermal imagery capture inflammation? Perhaps ‘ to track hair loss as a possible indicator of inflammation’?

Track changed version line 204-207; do these sentences say the same thing?

Track changed version line 338; need complete sentence after semicolon

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Reviewer #1: Yes: William J Severud

Reviewer #2: No

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Sores of boreal moose reveal a previously unknown genetic lineage of parasitic nematode within the genus Onchocerca

PONE-D-22-25378R2

Dear Dr. Benedict,

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,

Angela Monica Ionica, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: