Molecular epidemiology of Leptospira spp. among wild mammals and a dog in Amami Oshima Island, Japan

So Shinya; Yukinori Muraoka; Daigo Negishi; Nobuo Koizumi

doi:10.1371/journal.pone.0249987

Peer Review History

Original SubmissionJanuary 14, 2021
5 Mar 2021 Decision Letter - Axel Cloeckaert, Editor PONE-D-21-01288 Molecular epidemiology of Leptospira spp. among wild mammals and dogs in Amami Oshima Island, Japan PLOS ONE Dear Dr. Shinya, 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 consider the comments of the reviewers to improve the manuscript. Please submit your revised manuscript by Apr 17 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. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Axel Cloeckaert Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 3. We note that Figure 1 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: 3.1. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” 3.2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/ [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 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 ****** 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: Shinya et al. manuscript, “Molecular epidemiology of Leptospira spp. among wild mammals and dogs in Amami Oshima Island, Japan” describes for the first time the genetic variability of Leptospira spp. in a region with high risk of infection in Japan. The study is of local importance since there is an epidemiological context that benefits Leptospira spp. infection and nevertheless the population of this pathogen has not been previously studied. Authors evaluated samples from 42 black rats (Rattus rattus), 12 Ryukyu wild boars (Sus scrofa riukiuanus), 2 goats (Capra aegagrus hircus) and 1 rabbit (Pentalagus furnessi) from different location within Amami Oshima Island. Serogroup, species (single locus strategy) and intra-species variability (MLST or MLVA) were evaluated. Authors described that Leptospira spp. were isolated from 3 Ryukyu wild boars, 2 black rats, 1 rabbit and 1 dog. Intra-species variability was evaluated by 2 different strategies according to the species. L. borgpetersenii was evaluated by MLST #1 and L. interrogans by MLVA as previous studies of this group. Data accession: only flaB sequences were deposited in DDBJ, MLST and MLVA sequences should also be available. General suggestions by the reviewer: Previous studies by the authors included MLVA typing of L. borgpetersenii and L. interrogans isolates from different regions of Japan (https://doi.org/10.1016/j.meegid.2015.08.013). On the other hand, pubmlst database includes 30 isolates from Japan with information about STs variability according to the same scheme applied in this work (https://pubmlst.org/bigsdb?db=pubmlst_leptospira_isolates&page=query, search: country=Japan). Among these isolates the database show 15 STs with 4 STs assigned to L. borgpetersenii and 10 to L. interrogans. In this context, my suggestion is to include MLST typing to L. interrogans isolates to better understand the population genetics proposed in the study as MLST strategy is based on loci under negative selection. Furthermore, MLST typing should be included in the pubmlst database. On the other hand, MLVA is more accurate to study a specific location in a specific period of time since these markers variate more frequently. Therefore, these results is adequate for the Amami Oshima Island as a picture of genetic variability during the study. Overall, the study is missing a phylogenetic study in order to better understand the relations among the new isolates as commented in the discussion section. Please add this analysis in the context of previous data. Specific changes suggested by the reviewer: Page 7, line 139: the first reference in the line should be 19, not 20. Page 8, line 165: delete “(DDBJ 165 accession number LC596935– LC596941)” as this is already mentioned in methods section. Page 12, line 210: replace “..black rats are an important reservoir of L. interrogans for dogs on Amami Oshima Island” to “…black rats may be an important reservoir of L. interrogans for dogs on Amami Oshima Island”. The number of isolates studied is too small to make a statement. Page 13, line 213: authors cannot speak of prevalence because the study does not include a sampling design with a distribution analysis. Change prevalence to frequency Page 13, line 216: change various to different Table 2 and supplementary table 1: both tables should be merged. Supplementary table specifies as reference “this study” for isolates not described in the manuscript, please correct this. Reviewer #2: This a well written and straight forward manuscript providing valuable epidemiological information on Leptospira prevalence supported by isolation and molecular characterization, in Amami Oshima island in Japan. Authors must add approval information for animal use protocol used in this study Line 83 What do you mean by breeding places? ****** 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: Yes:** Paula Ruybal Reviewer #2: No [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.0249987.r001
Revision 1
26 Mar 2021 Author Response Response to Editorial Office and Reviewers Comments from Editorial Office Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Reply: We revised our manuscript according to the PLOS ONE's style requirements. We also had our revised manuscript checked by professional proofreading. Comment 2: We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Reply: We prepared a Supporting Information file (S1 Table) for the laboratory data on the dog with leptospirosis in Amami Oshima Island in 2018 that was described as “data not shown” in our previous manuscript. We also prepared a Supporting Information file (S2 Table) for the laboratory data on D-KS19-7K. We also cited the URL for the report on leptospirosis patients in Japan in 2016 in our revised manuscript for the “unpublished data” in our previous manuscript. Comment 3: We note that Figure 1 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). Reply: We prepared Figure 1 using pictures published by the Geospatial Information Authority of Japan that are provided under the CC BY 4.0. Comments from Reviewer #1 Comment 1: only flaB sequences were deposited in DDBJ, MLST and MLVA sequences should also be available. Reply: We deposited the isolate information and MLST profiles in the PubMLST database. We added the following sentence in P7L141. Information on the isolates analyzed in this study and new STs (ST328, ST329, and ST331) obtained in this study have been deposited in the database. We also deposited genome sequences containing VNTR determined in this study in DDBJ (accession numbers LC619028–LC619057). We added the following sentence in P7L154. The genome sequences containing VNTR determined in this study have been deposited in a public database (DDBJ accession numbers LC619028–LC619057). The MLVA profiles for the Leptospira strains isolated in Okinawa Prefecture had previously been determined by the comparison of those from Leptospira strains in our previous study [Ref 14: Koizumi et al., Infect Gen Evol. 2015]. Comment 2: Previous studies by the authors included MLVA typing of L. borgpetersenii and L. interrogans isolates from different regions of Japan (https://doi.org/10.1016/j.meegid.2015.08.013). On the other hand, pubmlst database includes 30 isolates from Japan with information about STs variability according to the same scheme applied in this work (https://pubmlst.org/bigsdb?db=pubmlst_leptospira_isolates&page=query, search: country=Japan). Among these isolates the database show 15 STs with 4 STs assigned to L. borgpetersenii and 10 to L. interrogans. In this context, my suggestion is to include MLST typing to L. interrogans isolates to better understand the population genetics proposed in the study as MLST strategy is based on loci under negative selection. Furthermore, MLST typing should be included in the pubmlst database. On the other hand, MLVA is more accurate to study a specific location in a specific period of time since these markers variate more frequently. Therefore, these results is adequate for the Amami Oshima Island as a picture of genetic variability during the study. Overall, the study is missing a phylogenetic study in order to better understand the relations among the new isolates as commented in the discussion section. Please add this analysis in the context of previous data. Reply: We performed MLST for L. interrogans serogroups Australis and Hebdomadis strains isolated in Kyushu Main Island, Okinawa Prefecture, and Taiwan. STs determined were included in Table 2 in our revised manuscript. We also prepared the phylogenetic tree based on the concatenated sequences of the seven genes as a supplementary figure (S1 Fig). For these, we modified the paragraph of MLST in the Materials and Methods section as follows: (Previous) MLST was performed for the Leptospira borgpetersenii isolate using seven housekeeping genes, glmU, pntA, sucA, tpiA, pfkB, mreA, and caiB, as previously described [20]. Sequence type (ST) was assigned through the PubMLST database [20]. (Revised) MLST was performed for the Leptospira strains isolated in this study, for those isolated in Kyushu Main Island, and those isolated in Okinawa Prefecture (Table 2) using seven housekeeping genes, glmU, pntA, sucA, tpiA, pfkB, mreA, and caiB, as previously described [20]. Sequence type (ST) was assigned using the PubMLST database [21]. Information on the isolates analyzed in this study and new STs (ST328, ST329, and ST331) obtained in this study have been deposited in the database. Phylogenetic relationships of the concatenated sequences (5ʹ-glmU-pntA-sucA-tpiA-pfkB-mreA-caiB-3ʹ) determined in this study and deposited in the MLST database (PubMLST, https://pubmlst.org/organisms/leptospira-spp) were determined by reconstructing a phylogenetic tree by the maximum likelihood method using IQ-TREE [22] with 1,000 ultrafast bootstrap replicates. We described the results of MLST in the Results section as follows: P10L181. The rat and dog isolates of L. interrogans serogroup Australis (AMM-012 and D-KS19-7K) belonged to ST105, which was detected in Okinawa Prefecture and Taiwan, whereas the L. interrogans serogroup Australis strain isolated from Kyushu Main Island belonged to the phylogenetically distant lineage, ST120 (Table 2 and S1 Fig). Two STs were identified in L. interrogans serogroup Hebdomadis isolates (Table 2). Two of the three Ryukyu wild boar isolates (AMM-042 and AMM-043) showed the novel ST, ST328, which was phylogenetically related to other STs of serogroup Hebdomadis strains in Japan (S1 Fig). The other wild boar isolate (AMM-047) and the Amami rabbit isolate (AMM-057) belonged to ST140, which has been identified in L. interrogans serogroup Hebdomadis strains on Kyushu Main Island (Table 2). We also modified the descriptions of MLVA results as follows: (Previous) MLVA was employed for L. interrogans because MLVA using 11 loci has a higher discriminatory power and was more concordant with serotyping than MLST using seven housekeeping genes for L. interrogans [13, 14]. As a result, the rat and dog isolates of L. interrogans serogroup Australis (AMM-012 and KS19-7K) showed the same MLVA profile (MLVA type) (Table 2). The profile was almost identical to that of the isolates from Okinawa Prefecture and Taiwan (identity: 10/11, S1 Table), whereas it was different from those from Kyushu Main Island (identity: ~1/11) [13, 14]. Two MLVA types were identified in L. interrogans serogroup Hebdomadis isolates from Ryukyu wild boars, one of which (AMM-047) was identical to that from the Amami rabbit (Table 2). One of the MLVA types (AMM-047 and AMM-057) was identical to that of the isolates from Kyushu Main Island, whereas it was different from those from Okinawa Prefecture (identity: ~3/11, S1 Table) [13, 14]. Another MLVA type (AMM-042 and AMM-043) was different from those of the isolates from Kyushu Main Island and Okinawa Prefecture (identity: ~4/11, S1 Table) [13, 14]. (Revised) MLVA was further employed for L. interrogans isolates because MLVA using 11 loci has higher discriminatory power and was more concordant with serotyping than MLST using seven housekeeping genes for L. interrogans [13,14]. The rat and dog isolates of L. interrogans serogroup Australis (AMM-012 and D-KS19-7K) showed the same MLVA profile (MLVA type) (Table 2). The profile was almost identical to that of the isolates from Okinawa Prefecture and Taiwan (identity: 10/11, Table 2), whereas it was different from those from Kyushu Main Island (identity: ~1/11) (Table 2) [13,14]. As with MLST, two MLVA types were identified in L. interrogans serogroup Hebdomadis isolates: the wild boars and rabbit isolates (AMM-047 and AMM-057) showed identical MLVA profiles, which were identical to those of the isolates from Kyushu Main Island, whereas they were different from those from Okinawa Prefecture (identity: ~3/11, Table 2) [13,14]. The MLVA types of AMM-042 and AMM-043 were different from those of the isolates from Kyushu Main Island and Okinawa Prefecture (identity: ~4/11, Table 2) [13,14]. For the interpretation of MLST results, we added the following sentences in the Discussion section. P19L303. On the other hand, the same ST, ST118, was identified in human and dog patients both in Kyushu Main Island and Okinawa Prefecture (Table 2), suggesting that there is an unidentified maintenance animal(s) that carries this ST in these areas. This may also be true for the MLVA type/ST140 of the serogroup Hebdomadis strain. In addition, novel ST/MLVA profiles were identified in wild boar isolates of the serogroup Hebdomadis. Further research is needed to clarify the phylogenetic relationship of L. interrogans serogroup Hebdomadis strains in Japan. We included the results of MLST in the Abstract of our revised manuscript too. (Revised) Leptospirosis is a worldwide zoonosis caused by the pathogenic Leptospira spp. Canine and human leptospirosis sometimes occur on Amami Oshima Island, located in the Nansei Archipelago, southwestern Japan; however, information on the causative Leptospira spp. on this island is quite limited. This study aimed to investigate the molecular and serological characteristics of Leptospira spp. isolated from wild animals and a dog in Amami Oshima Island. We obtained seven Leptospira strains by culturing kidney tissues of wild animals, such as black rats (2), wild boars (3), and rabbit (1) as well as blood from a symptomatic dog. Using flaB sequencing and microscopic agglutination test with antisera for 18 serovars, the isolates were identified as Leptospira borgpetersenii serogroups Javanica (black rat), L. interrogans serogroup Australis (black rat and dog), and L. interrogans serogroup Hebdomadis (wild boar and rabbit). The sequence type (ST) of L. borgpetersenii serogroup Javanica was determined to be ST143 via multilocus sequence typing (MLST) using seven housekeeping genes. For L. interrogans, MLST and multiple-locus variable-tandem repeat analysis (MLVA) revealed identical ST and MLVA types in rat and canine isolates, whereas two STs and MLVA types were identified in wild boar isolates. The STs and MLVA types of rabbit and one of the wild boars were identical. Bacterial culture and flaB-nested polymerase chain reaction demonstrated a high rate of Leptospira infection in wild boars (58.3%, 7/12), whereas Leptospira spp. were detected in 4.8% of black rats (2/42). This study revealed diverse Leptospira genotype and serotype maintenance in wild mammals on Amami Oshima Island. MLST and MLVA indicated that black rats were a source of canine infection. Wild boars carry L. interrogans and are considered an important maintenance host because antibodies against serogroup Hebdomadis were detected in human and canine leptospirosis patients on this island. Comment 3: Page 7, line 139: the first reference in the line should be 19, not 20. Reply: Thank you for the reviewer’s indication. We corrected the reference number. Comment 4: Page 8, line 165: delete “(DDBJ 165 accession number LC596935– LC596941)” as this is already mentioned in methods section. Reply: According to the reviewer’s comment, we deleted the phrase in our revised manuscript. Comment 5: Page 12, line 210: replace “..black rats are an important reservoir of L. interrogans for dogs on Amami Oshima Island” to “…black rats may be an important reservoir of L. interrogans for dogs on Amami Oshima Island”. The number of isolates studied is too small to make a statement. Reply: We changed the sentence as the reviewer suggested. Comment 6: Page 13, line 213: authors cannot speak of prevalence because the study does not include a sampling design with a distribution analysis. Change prevalence to frequency. Reply: According to the reviewer’s comment, we changed the word “prevalence” to “frequency”. Comment 7: Page 13, line 216: change various to different Reply: According to the reviewer’s comment, we changed the word “various” to “different”. Comment 8: Table 2 and supplementary table 1: both tables should be merged. Supplementary table specifies as reference “this study” for isolates not described in the manuscript, please correct this. Reply: According to the reviewer’s comment, we prepared the new Table 2 by merging the previous Table 2 and S1 Table. As mentioned above, we added data on L. interrogans serogroups Australis and Hebdomadis strains isolated in Kyushu Main Island in the revised Table 2. Comments from Reviewer #2 Comment 1: Authors must add approval information for animal use protocol used in this study Reply: The rats were captured as the program of the Ministry of the Environment and this investigation was approved by the Ministry. We modified the sentence as follows: (Previous) A total of 42 black rats (Rattus rattus) were captured as a bycatch under the extermination program of invasive feral cats conducted by the Ministry of the Environment. (Revised) A total of 42 black rats (Rattus rattus) were captured as a bycatch under the extermination program of invasive feral cats conducted by the Ministry of the Environment, and this investigation was approved by the Ministry. Cultivation of Leptospira spp. from dead Amami rabbits was also approved by the Ministry of the Environment. We added the following sentence in our revised manuscript. P5L92. The cultivation of Leptospira spp. from dead Amami rabbits was also approved by the Ministry. In Japan, there is no ethics committee available for private-practice animal hospitals. The investigation on canine leptospirosis was conducted according to the ethical codes of the Japan Veterinary Medical Association. We obtained the oral consent for collection of blood for Leptospira cultivation from owners (P5L99). Comment 2: Line 83 What do you mean by breeding places? Reply: We used “breeding place” as the places where the dogs enrolled in this study were kept by their owners. We change the word “breeding place” into “place of residence” in the revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0249987.r002
30 Mar 2021 Decision Letter - Axel Cloeckaert, Editor Molecular epidemiology of Leptospira spp. among wild mammals and dogs in Amami Oshima Island, Japan PONE-D-21-01288R1 Dear Dr. Shinya, 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, Axel Cloeckaert Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0249987.r003
Formally Accepted
1 Apr 2021 Acceptance Letter - Axel Cloeckaert, Editor PONE-D-21-01288R1 Molecular epidemiology of Leptospira spp. among wild mammals and a dog in Amami Oshima Island, Japan Dear Dr. Shinya: 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. Axel Cloeckaert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0249987.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .