Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities

Brittany A. Seibert; Michael K. Keel; Terra R. Kelly; Roger A. Nilsen; Paula Ciembor; Denise Pesti; Christopher R. Gregory; Branson W. Ritchie; Michelle G. Hawkins

doi:10.1371/journal.pone.0258500

Peer Review History

Original SubmissionJune 26, 2021
7 Sep 2021 Decision Letter - Deborah Dean, Editor PONE-D-21-20941Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilitiesPLOS ONE Dear Dr. Hawkins, 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 address each issue raised by the reviewers in a point-by-point response with corrections to the text/tables/figures where indicated. Please submit your revised manuscript within Oct 22 2021 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. 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Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 6. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The authors thank the Wildlife Health Center Fellowship grant program for their financial support of the study and the veterinarians and staff from the following rehabilitation centers for their sample collection and processing: California Raptor Center, California Wildlife Center, Pacific Wildlife Care, Monterey SPCA, Living Desert. We would also like to thank the Infectious Disease Laboratory, University of GA for the generous donation of the qPCR testing, EBA testing and chlamydial cultures and the Avian and Wildlife Laboratory, University of Miami for partial donation of IFA testing.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. 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If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 8. Please upload a new copy of Figure 1 as the detail is not clear. Please follow the link for more information: " ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/. 9. 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. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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: The findings of this study are new and interesting as they appear shortly after publication of the species Chlamydia buteonis. Samples for serology and qPCR were collected from 263 birds of prey, representing 18 species. Chlamydial DNA was found in 4.18 % and anti-chlamydial Abs detected in 3.14 % of tested birds. As could be expected, a serologic response was typically observed in adults. Nevertheless, the inclusion of serology in parallel to DNA testing is an important asset of the present work. The manuscript is concise and well written. Data processing and presentation are adequate. A few points remain unclear and should be revised. 1. Line 114: Plasma samples were divided into two aliquots and tested using two serologic assays. Please, indicate the chlamydia species of the hawk isolate used in the assay at the IDL. 2. Line151-52: a) It is unclear from the text whether both positive cultures from SWHA (IDL16- 5840_SH) and RTHA (IDL17_4553_RTH) were genome sequenced or only one. How many sequences will be (or have been) deposited at NCBI? b) It seems that complete assembly of the raw sequences has not been achieved as 742 clusters are reported. If so, a reason should be given. 3. Lines 157-59: The qPCR assay for C. buteonis used here is probably new and has not been published previously. If so, the authors should include data showing the specificity of that assay. Besides, the protocol of a specific and sensitive assay can be found in reference 10. 4. Line 207: The authors claim that the clade containing their own C. buteonis isolates is distinctly different from other chlamydial species. However, there is only one more species, C. psittaci, on Fig. 1. The substantiate the statement, at least one more species should appear in the figure, e.g. C. trachomatis as a non-avian Chlamydia sp. 5. In reference 10, only hawks (genus Buteo) were mentioned as known hosts of C. buteonis. The present study revealed new observations on host specificity of this species that should be summarized and included in the text as a major conclusion. Reviewer #2: The authors present an analysis of detection of chlamydial DNA and/or antibodies in raptors presenting to wildlife rehabilitation centres. The analysis is clear and easy to follow, and the trends apparent related to age, different species are also clearly documented. The sample size of 263 from 18 species is good size, and across several distinct centres. The evidence of increasing serological responses in age and chlamydial DNA associated with disease supports the conclusion of the potential for risk and the prevalence of the organism in these wild populations. The sequence analysis confirming similarity to a recently described c.buteonis adds confidence to the findings. Overall this study is a valid and useful addition to the literature on chlamydia in wildlife. ****** 6. 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: Yes:** Willa Huston [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0258500.r001
Revision 1
20 Sep 2021 Author Response Responses to Editorial Comments: 1. We have worked to ensure that all of the formatting conforms to PlosOne. 2. No animals were “sacrificed,” therefore nothing was included in the methods regarding this. 3. I apologize for not initially providing complete information on the minimum dataset, as this is my first PlosOne submission. I have reviewed the entire manuscript and all data necessary to complete statistical analyses are included within the manuscript in the tables and figures. We have no ethical or legal issues with sharing our data. Specifically these 5 items are addressed: 1) Sequences (SWHA (IDL16-5840_SH) and RTHA (IDL17_4553_RTH) were submitted to NCBI BioProject accession number PRJNA613727 (Lines 174, 175 in manuscript) 2) Data for the primers for the qPCR for C. buteonis are within the manuscript (Line 186, 187 in manuscript) 3) Fig 1. Maximum likelihood phylogenetic tree showing that the SWHA and RTHA isolates were most similar to C. buteonis. 4) Tables 1 and 2 provide the raw data for chlamydial DNA and serology (elementary body agglutination; EBA and immunofluorescent antibody; IFA) by raptor species, therefore providing the raw data used for the statistical analyses. 5) Fig 2 and 3. Map of the significant spatial cluster for raptors testing positive for Chlamydia spp. DNA by rt-qPCR in northern California was developed using methods described within the manuscript and using the raw data using the qPCR results from table 2. 4. It is stated that the grant information provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. I updated the “funding information” section but it was not a grant, rather in-kind donation of the qPCR, EBA serology and chlamydial cultures. As several authors are from this laboratory I was unsure how else to clarify? Our new financial disclosure should read “This study was financially supported through the Karen C. Drayer Wildlife Health Center Fellowship Grant Program, School of Veterinary Medicine, Univ. of California, Davis, Davis, CA and the Infectious Disease Laboratory, College of Veterinary Medicine, University of Georgia, Athens, GA.” 5. I would like to update the information regarding the role of funders for this research. The Karen C. Drayer Wildlife Health Center funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Infectious Disease Laboratory, University of GA donated the costs of the qPCR testing, EBA testing and chlamydial cultures and were instrumental in data analysis, decision to publish and preparation of the manuscript. Multiple authors are affiliated with the laboratory (Ritchie, Nilsen, Ciembor, Pesti, Gregory) but only Ritchie and Nilsen were involved in manuscript preparation. We have removed the Avian and Wildlife Laboratory at the Univ. of Miami, as this is a research price given to all researchers and therefore we do not believe now is a source of funding. Please let me know if you disagree? 6. Acknowledgements of funding within the manuscript have been removed (Lines 447-449). The corrected statement reads: “The authors thank the veterinarians and staff from the following rehabilitation centers for their sample collection and processing: California Raptor Center, California Wildlife Center, Pacific Wildlife Care, Monterey SPCA, Living Desert. 7. This work was presented at the 2016 ExoticsCon conference in Portland, OR by my Master’s student Seibert (first author) and was published for association members in their proceedings for that year as a 250 word abstract. The abstract is only reviewed by one person for approval to present at the conference. This is the abstract “Chlamydia psittaci is a reportable, zoonotic bacterium with health implications for birds and humans. Due to its zoonotic potential, veterinary staff should be updated on the prevalence and clinical presentations of Chlamydia spp. infections in raptors. A retrospective study evaluating raptors testing positive for Chlamydial infections was conducted through the UC Davis Veterinary Medical Teaching Hospital medical records from 2004 –2016. A confirmed or probable case was based on the National Association of Public Health Veterinarians criteria for C. psittaci.1 Specific C. psittaci diagnostic testing included real-time PCR (RT-PCR) from a combination mucosal swab, elementary body agglutination testing, indirect fluorescent antibody testing, immunofluorescent antibody staining for Chlamydia spp. antigen, and isolation of the organism. Signalment (sex, age, seasonality and species) and clinicopathologic results from positive cases were analyzed. A Chi-squared test was used to statistically evaluate the data, and significance was defined as p ≤ 0.05. Forty eight red-tailed hawks (RTHA; Buteo jamaicensis) and 1 Swainson’s hawk (B. swainsoni) were identified as confirmed or probable cases. A 22.9% prevalence was identified from the total number of raptors tested for C. psittaci and 1.9% prevalence was identified from the total number of all raptors admitted to the VMTH. There was a significant difference in prevalence between age (p0.005), seasonality (p0.001) and species (p0.001). Tissues most commonly identified as positive were lung, liver, air sac, heart and kidney. Genetic sequencing of tissue samples from two RTHA with conflicting IFA/EBA results suggests that these infections may be caused by a novel Chlamydial-like organism.” We did not feel this 250 word abstract for a conference constituted previous publication. 8. I put figure 1 into the PACE system and it did not find any image problems but did increase the dpi to 300 x 300 and created a proper TIF. I hope this is acceptable? Please let me know if it is not. 9. We reviewed our references. None have been retracted. Responses to Reviewers: Thank you for giving us the opportunity to re-submit a revised manuscript of “Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities” for PlosOne. We appreciate yours along with the reviewers’ time and effort spent providing feedback and insightful comments to help improve our manuscript. We have incorporated most of the suggestions made by you and the reviewers. Please see below a point-by-point response to the reviewers’ comments and concerns. Line numbers refer to the revised manuscript file with tracked changes. 5. Review Comments to the Author Reviewer #1: The findings of this study are new and interesting as they appear shortly after publication of the species Chlamydia buteonis. Samples for serology and qPCR were collected from 263 birds of prey, representing 18 species. Chlamydial DNA was found in 4.18 % and anti-chlamydial Abs detected in 3.14 % of tested birds. As could be expected, a serologic response was typically observed in adults. Nevertheless, the inclusion of serology in parallel to DNA testing is an important asset of the present work. The manuscript is concise and well written. Data processing and presentation are adequate. A few points remain unclear and should be revised. 1. Line 114: Plasma samples were divided into two aliquots and tested using two serologic assays. Please, indicate the chlamydia species of the hawk isolate used in the assay at the IDL. Author Response: Thank you, the species of the hawk isolates used were added. 2. Line151-52: a) It is unclear from the text whether both positive cultures from SWHA (IDL16- 5840_SH) and RTHA (IDL17_4553_RTH) were genome sequenced or only one. How many sequences will be (or have been) deposited at NCBI? Author Response: Thank you, both positive cultures were sequenced, and this information was added. Two sequences have been deposited at NCBI under the BioProject accession number PRJNA613727. b) It seems that complete assembly of the raw sequences has not been achieved as 742 clusters are reported. If so, a reason should be given. Author Response: Thank you, the Anvi'o software determined 742 single copy genes that were distinguishable as homologous between all the genomes and determined to be single copy genes in all the genomes; and therefore, used for the phylogenetic analysis. This was not a measure of completeness of any genome, just the number of genes that were used in the phylogenetic analysis. The description of the methods was updated in the manuscript. 3. Lines 157-59: The qPCR assay for C. buteonis used here is probably new and has not been published previously. If so, the authors should include data showing the specificity of that assay. Besides, the protocol of a specific and sensitive assay can be found in reference 10. Author Response: Thank you, we expanded on the flow of the C. buteonis identification in the positive samples within the method section. The samples were first analysed using the C. psittaci specific qPCR with a melting curve analysis following a previously described method. However, the positive samples from this study showed a different melting curve signature compared to C. psittaci positive control samples. Thus, those samples were amplified using C. buteonis specific primers described in the method section and then Sanger sequenced to confirm C. buteonis identity. 4. Line 207: The authors claim that the clade containing their own C. buteonis isolates is distinctly different from other chlamydial species. However, there is only one more species, C. psittaci, on Fig. 1. To substantiate the statement, at least one more species should appear in the figure, e.g. C. trachomatis as a non-avian Chlamydia sp. Author Response: Thank you, we understand that the conclusion suggested results showing more than 1 species and therefore edited the results section of Figure 1 to state the following “This analysis supported the inclusion of the SWHA and RTHA isolates in the clade containing C. buteonis as determined by Laroucau et al, which is a phylogenetically intermediate between C. psittaci and C. abortus.” 5. In reference 10, only hawks (genus Buteo) were mentioned as known hosts of C. buteonis. The present study revealed new observations on host specificity of this species that should be summarized and included in the text as a major conclusion. Author Response: Thank you. This finding was emphasized in the abstract, discussion and conclusion. Reviewer #2: The authors present an analysis of detection of chlamydial DNA and/or antibodies in raptors presenting to wildlife rehabilitation centres. The analysis is clear and easy to follow, and the trends apparent related to age, different species are also clearly documented. The sample size of 263 from 18 species is good size, and across several distinct centres. The evidence of increasing serological responses in age and chlamydial DNA associated with disease supports the conclusion of the potential for risk and the prevalence of the organism in these wild populations. The sequence analysis confirming similarity to a recently described c.buteonis adds confidence to the findings. Overall this study is a valid and useful addition to the literature on chlamydia in wildlife. Thank you so much for your review. Attachments Attachment Submitted filename: 2021_PlosOne_Response to Reviewers FINAL.docx https://doi.org/10.1371/journal.pone.0258500.r002
29 Sep 2021 Decision Letter - Deborah Dean, Editor Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities PONE-D-21-20941R1 Dear Dr. Hawkins, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Deborah Dean, M.D., M.P.H. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0258500.r003
Formally Accepted
4 Oct 2021 Acceptance Letter - Deborah Dean, Editor PONE-D-21-20941R1 Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities Dear Dr. Hawkins: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Deborah Dean Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258500.r004

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