PONE-D-19-20232

Canine distemper in Nepal's Annapurna Conservation Area

PLOS ONE

Dear Ms Ng,

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.

In addition to comments made by Reviewer 1, would you please consider the following suggestions:

- Line 178: "Where there were litters of pups below 12 weeks, only one individual was sampled as exposure to CDV is likely identical for litter-mates". How many of these pups tested positive (ELISA and PCR) ? Has maternal antibodies transfer been documented for CDV ? Do we know anything about the duration of immunity and how this may have affected your results ? (assuming some pups tested positive) ?

- Line 184: "The tubes were refrigerated at 5 °C in the field before being delivered to the National Zoonoses and Food Hygiene Research Centre (NZFHRC) in Kathmamdu". Were all samples shipped at the same time, in the same batch ? Were samples frozen or kept refrigerated on there journey to Kathmamdu ? Given the distance between Manang and Kathmamdu, I'm assuming that this may take several hours. Additional information on sample conservation would be welcome.

- Line 186: "The latter was stored in a Viral Transport Medium". Please provide information on the type of medium.

- Line 228: "Permission to conduct this study was granted by the Manang Nyeshang VDC Mayor’s Office (Permit no.: (074/075) 261, 2075/01/19)". Have you obtained permission from other villages ? From a governmental institution in Nepal ?

- Line 330: "Sera from 68 dogs were analysed with ELISA assay to detect CDV antibodies, and 70.6% were seropositive". How many dogs were sampled, tested, and positive, in each location ? This is a bit unclear throughout the manuscript, but critical for the interpretation of the data as location seems to be the most important variable. This could be presented in a figure presenting the seroprevalence per location (with 95% confidence intervals).

- Line 332: "Blood samples from 58 (81.7%) dogs were sufficient for PCR analysis, and of these, the target CDV P-gene was detected in nine (12.7%) individuals". Same remarks as above. In addition, I'm guessing that you targeted only a small portion of the P-gene with PCR system (you may provide the size in the method section). Was sequencing done on the positive samples ? If any, it would be great to, at least, include a brief genetic analysis of the detected viruses: level of similarity between sequences, phylogenetic analysis, etc. Given your results support that there is no population structure for the host (dogs), I wonder how this could affect the genetic diversity and spatial structure of CDV.

- Line 400: "Owners reported sourcing their dogs from locations as far away as Kathmandu". Any idea why ? Regarding the implementation of vaccination programs, wouldn't it be sustainable to target the major "dog sources", as detected in your study, rather than performing mass vaccination in remote locations ?

- Line 424: "The high proportion of dogs in ideal body condition recorded in this study was contrary to expectations for dogs in a developing country". Can this have affected your PCR detection rate ? Were there healthy dog shedding CDV in your study ? Might be interesting to have a look at the potential effect of dog body condition on viral shedding ?

- Finally, I also agree with Reviewer 1 that the dog-wildlife-livestock interaction part may be removed from the manuscript (or presented in the supplementary material section ). More information on the number of participant reporting predator would be needed, and potential biases affecting the response may need to be considered (do they own livestock ? amount of time spent watching for predators ? etc.). Types of interactions between dogs and each type of predators would also need to be considered (direct, indirect, allowing transmission of CDV or not, etc.).

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PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: I have two major concerns with the research methods described in this paper.

1) Most of the dogs surveyed in this study were free-roaming, and the authors state that this was not an unexpected finding (Line 394). Indeed, a vast majority of dogs in Nepal (and the rest of the Indian subcontinent) are free-roaming, regardless of the ownership status. Furthermore, in these regions, the concept of dog ownership differs from that of the western world - dogs are loosely owned (village dogs/community dogs) and are not subject to any healthcare interventions like vaccination/deworming/neutering. In this context, the authors should explain how the 71 dogs they have included in this study ('belonging' to dog owning households) are different from the village dogs not included in this study. How representative these 71 dogs are of the larger village dog population? Can data obtained through convenience sampling (as per the information provided by the authors Lines 122-134) be extrapolated to infer about the regional dog population (e.g. Line 384 "The high CDV seroprevalence among domestic dogs...")?

The modeling/analysis used here also needs further explanation - for instance, Line 230: "..using Akaike Information Criterion (47) to explain variation in number of dogs per household and CDV seroprevalence": if 86% of the 'owned' dogs are free-roaming (Table 1), is it necessary to explore for factors like number of dogs per household to explain CDV seroprevalence? You are setting up a weak argument, and using statistical analysis/modeling to refute it. See L432 in your manuscript: "Few predictors of CDV were found, and the high degree of movement and mixing of domestic dogs could explain this."

2) The dog-wildlife-livestock interaction part in this manuscript is methodologically problematic (based on information derived from questionnaire surveys where the respondent perceptions and recall biases affect the response) and should be removed form this manuscript.

Minor comments: (L386) "Seropositive dogs reportedly interacting with wild carnivores suggests that this dog population could pose a risk of transmission of CDV to wild carnivores." Actually seropositive dogs do not play any role in the transmission of CDV - they are immune due to prior exposure. Dogs recovering from natural infection due to CDV develop a lifelong immunity to this pathogen (see Schultz, R., Thiel, B., Mukhtar, E., Sharp, P. & Larson, L. Age and long‐term protective immunity in dogs and cats. J. Comp. Pathol. 2010.142, S102–S108) and such dogs do not play any current or future role in the transmission of CDV. Reconsider line 469 as well: if dogs are exposed to CDV in young age, the survivors are actually immune (for life) and play no role in CDV transmission.

Provide confidence intervals wherever you report seroprevalence (e.g. L328)

L446: "No dogs presented clinical signs of CDV. The healthy body condition of most dogs could

have enabled them to resist the effects of disease (63)." Remove this as it is conjectural and not supported by your data.

Line 458: "To address this, measures to promote vaccination and limit number of dogs per household and their roaming behaviour could be implemented. " Do you think such recommendations are implementable?

Reviewer #2: I found this manuscript to be well written, clear and concise. This is novel and original research. The statistics are detailed and appropriate for the study. The design of the study is more than adequate and answers the questions posed by the investigators as to the epidemiology of an important viral pathogen in domestic dogs.

The conclusions are valid and supported by the data. The results and discussion section explain adequately how these conclusions were reached. The article is organized well and illustrates clearly the importance of this pathogen and the potential for its threat to wildlife in the region.

The study was done in an ethical manner and appears to have complied with all requirements for its performance. In addition, the raw data is available for scrutiny.

The Introduction reviews the published literature and presents it in a suitable manner for posing the questions asked. The methodology is adequately explained and appears to be complete and inclusive of the necessary information. In the Results section, Table 1 logically presents the data for assessment by the reader.

The only concern I have is the depth of the statistical analyses make assessment of the study and its conclusions a challenge for those unfamiliar with the methods described. Having said that, I believe the authors have adequately explained this methodology as outlined in the requirements.

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Canine distemper in Nepal's Annapurna Conservation Area: Implications of dog husbandry and human behaviour for wildlife disease.

PONE-D-19-20232R1

Dear Dr. Ng,

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With kind regards,

Camille Lebarbenchon

Academic Editor

PLOS ONE

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