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PLOS One

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Additional Editor Comments:

Please revise the manuscript as per the comments raised.

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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?

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Comments To Authors:

This paper is generally well written and describes the different genotypes of S. neurona identified in California sea lions with protozoal-related polyphasic myositis. The finding of many different genotypes associated with this condition is novel and publication of these results is warranted. Though this was a relatively small sample size these results serve as an important basis for developing further research projects examining the pathogenesis of this important protozoal-associated disease. The below comments and suggestions would improve clarity of the manuscript.

- Throughout suggest changing “captive” to “managed care” as the later terminology is currently more widely accepted

- Around Line 80 – It would be good to mention the specific geographic area that TMMC covers for these stranded CSLs for general reference. Also, I am assuming that the 25 CSLs that were investigated stranded throughout the range of coast that TMMC covers, however, it would be good to state that there was no geographic clustering of cases.

- Around line 83 – you could combine B and C as there isn’t much difference between these two categories of cases. Listing 3 different categories for this may lead to more confusion and is unnecessary for this paper.

- Figure Legend Line 130 – It would be good to orientate to where exactly the muscle in the gross picture is located. Is this thoracic wall?

- Figure Legend Line 131 – Can you tell for sure this is a “mature” sarcocyst given the smaller size? If not leaving out “mature” is likely fine for this paper.

- In the genotype table it would be good label the columns loci where appropriate as the abbreviations can be confusing. Similar with “MS” – microsatellite type. Can the table be formatted longitudinally for this journal so as to create more space for the headers?

- If there were two different genotypes of S. neurona present in these cases could that have been detected with the methods used? And if so and no cases had more than one genotype it would be good to state in the paper as potentially CSLs could be exposed to different genotypes of S. neurona at different time points in their lives.

- In the beginning of the Discussion around line 159 disease severity is mentioned several times, however, the specific severity of the 25 selected cases was not described. Given the large number of genotypes identified, it does not appear that genotype relates to severity of the polyphasic myositis, however, it may be good to state that the 25 selected cases all had moderate to severe polyphasic myositis if this is appropriate. This could be accomplished by stating that you only selected cases with histopathologic evidence of moderate to severe polyphasic myositis.

- Line 169 – Recommend changing to “disease being immune-mediated” Removing “more”

- Line 184 – This sentence needs to be edited or split into two sentences as the difference between site fidelity and prey is confused between otters and CSLs.

- Line 191 – Edit for grammar and punctuation

- For the Discussion section, some discussion is needed on how these results may differ from the genotype noted in some pinniped species in the Pacific NW associated with severe meningoencephalitis. And the implications of this considering the different manifestation of infection noted in these CSLs.

Reviewer #2: Dear Authors,

Your report describes the genotypes associated with a subset of S. neurona-infected California sea lions with polyphasic rhabdomyositis. You identified nine new Sn genotypes and discovered that there does not seem to be an association between genotype and presence of polyphasic rhabdomyositis. In addition, you investigated some comparisons between Sn infection risk factors in CSL and other marine intermediate hosts. Overall, I found your manuscript to be generally well-written, clear, and concise, with a few areas that I thought could use some elaboration or re-organization. Great work!

On line 32, consider changing “fifteen” to “15”.

On lines 61-63, you could consider adding information from Barbosa et al., which lists severe Sn protozoal encephalitis as a direct cause of death in a Steller sea lion as well as in several Pacific harbor seals (so, further evidence for this species), although I suppose it doesn’t specify “meningo-“ encephalitis, but rather just encephalitis.

On line 67, you first mention the “unique syndrome of severe polyphasic rhabdomyositis…”. It would be great to have a more specific definition of this term here, and especially including the significance of “polyphasic” as opposed to simply “rhabdomyositis”.

On line 69, you mention “a recent increase in severe, fatal muscular disease since 2015”, citing Seguel et al. as your reference. I think this sentence is not well-enough supported by the data, and I would recommend removing it all together. In the Seguel et al. paper, which describes polyphasic rhabdomyositis cases between 2001-2016, there is only reference to the years 2006 and 2008 having significantly higher case numbers (nothing about increases in cases during 2015 or 2016). In the Whoriskey et al., paper, it is noted that during the study period (Sept 2015-Dec 2017) the number of cases that met the inclusion criteria for the study was approximately 6 times the annual number of cases recorded during the preceding 15 years. I’m thinking this is the paper you had meant to reference there. However, I still have a little bit of a concern. First, it is not specified whether this increase is statistically significant, and second, I’m not seeing evidence that there has been a sustained increase “since 2015”, especially given that we have seen previously (in Seguel et al.), that increases can occur and then case numbers can subsequently return to baseline (i.e., I’m not seeing any data from the years 2018-present that suggest the increase is still present, and has been present “since 2015” as opposed to just fluctuating with higher case numbers in some years).

On line 101, you note that a modification was made to the manufacturer’s instructions for DNA extraction. Could you perhaps briefly specify/include why such a modification was made?

On line 121-122, you state, “All 25 selected cases had severe, polyphasic rhabdomyositis with intralesional sarcocysts affecting multiple skeletal muscles…”. Does this mean multiple skeletal muscle types across the overall sample set or multiple skeletal muscle types in each individual? If the prior (this is my guess), could I request that you reword the sentence to be a little more clear? And if the latter, please add information to the sentence starting on line 96 (“Skeletal muscle samples from these 25 CSLs…” to confirm that multiple skeletal muscle sample types from each individual were collected and analyzed.

On line 127, I think it would be pertinent to add “intermediate” between “other” and “host”, such that it reads, “…other intermediate host…”.

On line 134 (the Table 1 title), it would be ideal to have the title be more explanatory, such as “Sarcosystis neurona genotypes identified among a population of California sea lions with clinical and histopathological evidence of S. neurona-associated rhabdomyositis”, or something similar.

For your Table, it might be best to have it sideways to better fit all the title words. But perhaps it will look different in final formatting.

On line 136, for consistency, it would be great to change “types” to “genotypes”.

On lines 141-142, you note that six of the identified genotypes have been documented in southern sea otters. Later, you note additional species in which these genotypes have been documented, and I think it would be ideal to mention that here in this sentence as an overall intro, such as, “The remaining six genotypes characterized in these CSLs have been documented in southern sea otters, and in some cases, other marine and/or terrestrial species as well”, or something similar.

On lines 142-150, for each of your examples of sarcocystis in other species, could you please include the year, the general location, and whether any muscle pathology/rhabdomyositis was noted in individuals (or what the pathogenesis was)? You have some of this information for each example, but it would be great to keep it consistent, and I was definitely curious about the presence of muscle pathology in the other cases, for comparison’s sake. Additionally, I believe this entire section, beginning on line 142 at, “The Ia and Ib/c/d/gg genotypes identified in 4%...” and through the end of the paragraph, “…harbor seals, and porpoises” on line 150, belongs in the discussion section, since this content does not include your results, but rather comparison to others’ results. If this section is moved to the discussion, expanding the sentence on lines 141-142 as I recommended above, will be even more appropriate. Also, you give examples in this section for the Ia, Ib/c/d/gg, and IIg/j genotypes, and if they exist, it would be ideal to have examples for the III l/m/o, Viy, and IIi genotypes as well.

On line 146, please add the scientific name for raccoons.

On line 149, you mention “terrestrial mammals, including…”, however on line 150, you include in this list “…harbor seals, and porpoises”, which of course are not terrestrial. �

On line 151, (the Figure 2 title), it would be ideal to have it be more explanatory, such as “Sarcosystis neurona genotype frequencies in California sea lions with S. neurona-associated rhabdomyositis”, or something similar.

I think lines 166-174 (starting with, “The wide diversity…”) could be removed from here and would be better suited for the latter part of the discussion section since it’s a “future study” type statement.

In your paragraphs on lines 175-211, there is quite a bit of redundancy and also, some topics, such as diet differences between sea otters and CSLs are mentioned in two different areas. I think this whole paragraph could be tightened up a bit and reorganized such that first habitat risk factors, and then dietary risk factors are described and compared. At the end of the comparison, your sentence on line 187 starting with “The risk factors that make sea otters highly susceptible to protozoal infections…” could be a great wrap-up to that discussion; then discuss "future studies".

On line 184, I believe the “CSLs” after “California” is a typo (i.e., it should be removed).

On line 198, you reference “risk factors for fatal sarcocystosis” and I think it seems a bit out of place since your paper is focusing on polyphasic rhabdomyositis. Perhaps a simple modification to “risk factors for various forms of sarcocystis infection” or something similar.

On line 212 you also mention “fatal infections” and I was curious how many of your cases had fatal Sn-associated polyphasic rhabdomyositis? I could see how it is inferred that it was all of your cases, but I think it would be great to state that more clearly, earlier, in the results section (if the cases were all fatalities as a direct result of Sn-associated polyphasic rhabdomyositis), and how that was determined.

On line 214, I highly suggest removing “that emerged in the past decade” from the sentence (and consider removing “recently described” as well), since we know it was described at least as early as 2012 per Carlson-Bremer et al., (more than a decade ago), and the 2019 Seguel et al. paper notes that polyphasic rhabdomyositis had been a consistent but uncommon cause of stranding during the prior 15 years.

On line 216, I would change “infected” to “clinically-infected” to better differentiate these from “incidentally-infected”.

Finally, for Figure 2, I was trying to determine whether it adds useful information to your manuscript, especially since the data presented in the figure is already listed in your text, and since a black-and-white copy does not allow for color distinction. I would recommend either removing the figure all together, or keeping it but removing overlapping data from your manuscript. For example, removing the % of each genotype identified and just keeping in the “n=#”.

Overall, despite my long-winded review, I think this is a very important piece of the puzzle to have analyzed in the search for factors leading to variations in Sn presentation. Your methods were sound and analyses were straight-forward. Thank you for this contribution and I look forward to seeing your updated submission and/or publication.

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what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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Molecular characterization of Sarcocystis neurona genotypes in California sea lions (Zalophus californianus) with polyphasic rhabdomyositis

PONE-D-26-19817R1

Dear Dr. Shapiro,

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.

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Kind regards,

Vikrant Sudan, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: All of my comments and suggestions have been addressed. The edits to the comments strengthen the paper.

Reviewer #2: Thank you for your thorough responses to my previous questions/concerns/recommendations. Everything has been quite well addressed.

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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

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