Hunting and Stray Dogs as Reservoirs of Echinococcus Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Hunting and stray dogs as reservoirs of Echinococcus species" provides data on the occurrence of Echinococcus species in dogs from Serbia.

The topic is worthy of investigation due to the relevance of this parasite for public health.

The study is scientifically sound.

The manuscript is overall clear.

Therefore, I do recommend acceptance for publication in Animals, following some minor revisions.

Lines 119-176: Please, divide the Materials and Methods section into subparagraphs, e.g., sample collection, area description, DNA extraction, molecular analyses, statistical analysis, etc.

Lines 120-128: Do you have any other epidemiological data on dogs?

Lines 165-170: Please, provide details on DNA purification and sequencing, and/or any other method you use for Echinococcus species and genotype identification.

Lines 177-196: The presentation of results needs some revisions. I suggest dividing it into sub-paragraphs, i.e., screening PCR for detection, PCR for species/genotype characterization, statistical analysis, etc. 

Author Response

Comments 1: 

The manuscript "Hunting and stray dogs as reservoirs of Echinococcus species" provides data on the occurrence of Echinococcus species in dogs from Serbia. The topic is worthy of investigation due to the relevance of this parasite for public health. The study is scientifically sound. The manuscript is overall clear. Therefore, I do recommend acceptance for publication in Animals, following some minor revisions.

Response 1: 

We would like to thank the reviewer for their effort to thoroughly review the manuscript and suggest important improvements. We have made the suggested changes. 

Comment 2: 

Lines 119-176: Please, divide the Materials and Methods section into subparagraphs, e.g., sample collection, area description, DNA extraction, molecular analyses, statistical analysis, etc.

Response 2:

We thank the reviewer for this suggestion. We have made the requested revisions. The MM section now contains four sub-headings. (L128: 2.1 Fecal sample collection, L153: 2.2 Isolation and enrichment of taeniid eggs from fecal samples, L169-170: 2.3 DNA extraction and molecular detection of cyclphyllidean, E. granulosus s. l. complex and E. multilocularis DNA, L195 Statistical analyses)

Comment 3:

Lines 120-128: Do you have any other epidemiological data on dogs?

Response 3: 

We have made an effort to cite pertinent studies from Serbia with relevant information for this manuscript. Our selection was based on studies which used fecal samples of owned and shelter/stray dogs and parasitological examination with findings of taeniid eggs (with or without species identification). We were unable to find studies from Serbia which used molecular methods to identify Echinococcus spp. eggs. We also did not include studies which present findings of other parasites in fecal samples (protozoa, roundworms, etc.) as we did not consider these reports necessarily relevant for the manuscript. We hope that the reviewer can accept our response.  

Comment 4:

Lines 165-170: Please, provide details on DNA purification and sequencing, and/or any other method you use for Echinococcus species and genotype identification.

Response 4:

We would like to respectfully point the reviewer to L173-174 in the current version, which are unchanged from the previous manuscript version:

DNA was extracted from 30 μL of the egg pellet using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

There was no subsequent purification protocol, the DNA was used after Trizol extraction. In our experience, DNA extracted using Trizol reagent does not need additional purification. Also, as the extraction was done from taeniid eggs, not the original fecal samples, we believe that the resulting DNA was quite pure, based on the quality of the PCR reactions and the currently ongoing sequencing. Sequencing has not been done for purposes of confirmation here, the methodology was validated instead. Sequencing is currently ongoing but for phylogenetic studies, using different targets - the targets amplified here are useful for detection primarily, but not entirely useful for phylogenetic studies. The results will be published in due time, we will be preparing a manuscript in the coming weeks. If the reviewer is referring to sequencing mentioned in L182-183 now (Positive (E. multilocularis DNA confirmed by sequencing) and negative (nuclease free water) controls were included in each run), the sequencing of the DNA used as a positive control was not done in the scope of this study. It was done as a routine method when validating positive controls in preparation for implementation of protocols. This occurred much earlier before the study was even conceived. To validate the positive control, standard Sanger sequencing was applied with primers for the entire COX1 gene (sequenced in the forward and reverse directions). The positive control was obtained from another laboratory, it was not originally isolated from a host from Serbia. For the current study, we have identified the species and genotype as described in references 21 (Maksimov et al., 2020) and 22 (Štefanić et al., 2004). Maksimov et al., have developed a Taqman probe based multiplex PCR which identifies species of the E. granulosus s. l. complex. The probes can precisely identify the species, however, the probes do not precisely identify the genotype for some species, like E. canadensis - a single probe is used which does not distinguish between G6, G7, G8 and G10. For E. granulosus sensu stricto, a single probe identifies G1 and G3. For all the samples analyzed in our study, we have thus indicated multiple genotypes. Precise identification of the genotypes is data that we also plan to publish in the upcoming manuscript. The species identification however is very precise and this multiplex PCR method has been extensively validated by the authors and used in published literature. E. multilocularis was detected by primers designed by Štefanić et al., which are specific for this species and have been used in the literature. We have confirmed the specificity of these primers on our validated positive control and we have sequenced the resulting amplicon. The primers do indeed only detect E. multilocularis. We also point to the current results from the manuscript under review which show that we did not detect any co-infections with different Echinococcus species, despite the fact that all samples were analyzed for the presence of cyclophyllidean, E. granulosus s. l. complex and E. multilocularis DNA.  We hope that the reviewer can accept our explanation.

Comment 5:

Lines 177-196: The presentation of results needs some revisions. I suggest dividing it into sub-paragraphs, i.e., screening PCR for detection, PCR for species/genotype characterization, statistical analysis, etc. 

Response 5:

We respectfully point out to the reviewer that the Results section is very short, thus it would be rather difficult to make subdivisions. We did however take the reviewer’s point and we have sub-divided the Discussion section instead into three sections to improve readability - L242: 4.1 Detection of cyclophyllidean DNA in fecal samples, L270: 4.2 Detection of Echinococcus spp. DNA in fecal samples, L309: 4.3 Dogs as reservoirs of Echinococcus spp. eggs and implications for public health

We hope that the reviewer can accept our revisions.

Reviewer 2 Report

Comments and Suggestions for Authors

The article in subject is devoted to a problem involving both veterinary and public health. Therefore its actuality is out of any doubt.

However, I have some comments that (I hope) would contribute to improving of this manuscript.

The Title “Hunting and stray dogs as reservoirs of Echinococcus species” sounds a bit self obvious. I would suggest adding a real novelty and input in it as well as a location of the survey (Serbia?). Something like: “First molecular evidence of Echinococcus species in hunting and stray dogs in Serbia (via fecal testing)”.

The Simple Summary can be (but not mandatory) more simple and non-specialist-reader-friendly. Presently it contains Latin names, Echinococcus strain numbers and so on (which is not simple for average person), and can serve as a professional abstract.

The Abstract contains outlines of background, M&M, results, discussion and conclusion.

Lines 39-40: There is an explained abbreviation (CE), but it does not appear further on the text of the abstract. So, I suggest deleting it. 

Keywords: if there is no requirement about an alphabetic order, then OK. However, present set of keywords looks too general. I would suggest specifying it.

Introduction.

Line 57:” Cestodes (tapeworms) are parasites in the gastrointestinal (GI) tract of dogs. ” sounds too controversial.

Not ALL cestodes are parasites of dogs, obviously.

Moreover, not all cestodes in dogs are parasites of the gastrointestinal tract — Spirometra larval stage can cause both non- and proliferative sparganosis within various organs and tissues of intermediate host, and a dog can be that host (see Miller LR, Harley SK, Pollock IL, Amerman HK, Sobotyk C. Proliferative sparganosis caused by Spirometra sp. 3 in a dog. J Vet Diagn Invest. 2026 Apr 22:10406387261438437. DOI: 10.1177/10406387261438437). Therefore, I suggest rewriting the first intro sentence more precisely.

Line 58: “comparatively few” — how many? Which ones exactly?

Line 59: “Tapeworm infection(s) in dogs” kindly make a choice — infection or infections.

Lines 59-60: In the light of sparganosis you cannot state that “Tapeworm infection(s) in dogs result from ingestion of infected intermediate hosts”.

The Introduction contains a full background regarding both Echinococcus infections and its status in Serbian dogs. The aim is clearly declared (yet “Cyclophillidean tapeworm infection” sounds too general. Maybe Taeniidae?)

Materials and Methods.

Kindly, add time period when your research took place. Coordinates of the regions involved are desirable also (in decimal degrees).

What was the age range of the dos sampled? What were their breeds (weight)? Were both sexes represented in fecal sampling? If yes, were they presented equally?

Line 144: “The samples were centrifuged at 1600 × g for 30 min with the brake off, the supernatant” are you sure that centrifugation lasted half an hour (not 30 sec)?

Results.

Tables facilitate reading, yet figures would be nice also.

Discussion is comprehensive enough and highlights its limitations also which is good.

Conclusions corresponds to the aim and results.

References are almost all from the XXI century. However, they are relevant and numerous enough.

Overall the Paper would win if supplementary files like filled questionnaires (or at least blank questionnaire), sequences obtained, etc. would be ready available, not just “from the corresponding author”.

 

 

Author Response

Comments 1: 

The article in subject is devoted to a problem involving both veterinary and public health. Therefore its actuality is out of any doubt. However, I have some comments that (I hope) would contribute to improving of this manuscript.

Response 1:
We would like to thank the reviewer for the helpful suggestions and comments to improve the manuscript. We have made an effort to accept the suggestions.

Comment 2:

The Title “Hunting and stray dogs as reservoirs of Echinococcus species” sounds a bit self obvious. I would suggest adding a real novelty and input in it as well as a location of the survey (Serbia?). Something like: “First molecular evidence of Echinococcus species in hunting and stray dogs in Serbia (via fecal testing)”.

Response 2:

We appreciate the reviewers suggestion, however, we are unable to change the title of the manuscript now, as the preliminary results of this study have already been presented under the same title at several conferences. We therefore would like to preserve the title, to provide continuity. Another (more compelling reason) to provide continuity is the fact that the study is part of a national project and any dissemination of the project's results have to be tracked by the local funders - thus retaining the title helps a lot. We hope that the reviewer can accept our explanation.

Comment 3:

The Simple Summary can be (but not mandatory) more simple and non-specialist-reader-friendly. Presently it contains Latin names, Echinococcus strain numbers and so on (which is not simple for average person), and can serve as a professional abstract.

Response 4:

We understand the reviewer's concern. Unfortunately, we cannot simplify the summary any more to avoid using species and genotypes (we agree, it is not 'simple'), as that would necessitate a much more lengthy summary. According to the journal instruction, the summary has to be brief. Thus we have to retain the species names and genotypes.

Comment 5:

Lines 39-40: There is an explained abbreviation (CE), but it does not appear further on the text of the abstract. So, I suggest deleting it.

Response 5:

We would like to thank the reviewer for noticing. It has been removed. 

Comment 6:

Keywords: if there is no requirement about an alphabetic order, then OK. However, present set of keywords looks too general. I would suggest specifying it.

Response 6:

We thank the reviewer for this suggestion. We have now put the keywords in alphabetical order and we have replaced two keywords to add specificity (we have added E. granulosus sensu lato and E. multilocularis and removed Canidae and Cyclophyllidea). Please see L55-56 now.

Comment 7:

Line 57:” Cestodes (tapeworms) are parasites in the gastrointestinal (GI) tract of dogs. ” sounds too controversial. Not ALL cestodes are parasites of dogs, obviously. Moreover, not all cestodes in dogs are parasites of the gastrointestinal tract — Spirometra larval stage can cause both non- and proliferative sparganosis within various organs and tissues of intermediate host, and a dog can be that host (see Miller LR, Harley SK, Pollock IL, Amerman HK, Sobotyk C. Proliferative sparganosis caused by Spirometra sp. 3 in a dog. J Vet Diagn Invest. 2026 Apr 22:10406387261438437. DOI: 10.1177/10406387261438437). Therefore, I suggest rewriting the first intro sentence more precisely.

Response 7:

We thank the reviewer for this suggestion. We have re-written the sentence to avoid ambiguity. Please see L 59-60 now:
The class Cestoda consists of different species of tapeworms some of which are parasites of the gastrointestinal (GI) tract of dogs.

We would however respectfully like to point out that to the best of our knowledge, sparganosis is a rare disease in dogs. The prevalence of infection with Spirometra species (with gastrointestinal localization of the adult tapeworm) varies geographically (it is higher in Asia than in Europe). We have reviewed the suggested reference which concerns proliferative sparganosis, an extremely rare condition that is less frequent than non-proliferative sparganosis. Taking into account cases of proliferative and non-proliferative sparganosis alike, to the best of our knowledge and capacity to search the pertinent literature, dogs are far more frequently definitive hosts for Spirometra species which implies gastrointestinal localization of the (adult) tapeworm. Similarly, there are cases of echinococcosis in dogs, but these are infrequent as compared to gastrointestinal infections (without overt disease). For the sake of conciseness, we have decided not to describe uncommon or infrequent cases of infections in dogs with tapeworm localization outside of the gastrointestinal tract. We hope that the reviewer can find our revision in L59-60 satisfactory.

Comment 8:

Line 58: “comparatively few” — how many? Which ones exactly?

Response 8:
We thank the reviewer for the suggestion. We have revised the sentence and specified the species. Please see L60-65 now:
Several tapeworm species found in dogs are zoonotic, but infections with comparatively few, such as Taenia multiceps, T. crassiceps and T. serialis, Echinococcus granulosus sensu lato (s. l.) complex and E. multilocularis, resulting from accidental ingestion of parasite eggs found in feces, as well as Spirometra spp. from consumption of infected dog tissues, can cause severe symptoms and/or disease in humans.
Again, we are focusing on the common/usual species for the sake of brevity. We hope that the reviewer can accept our revisions.

Comment 9:

Line 59: “Tapeworm infection(s) in dogs” kindly make a choice — infection or infections.

Response 9:

We have revised as “infections” (L65 now).

Comment 10:

Lines 59-60: In the light of sparganosis you cannot state that “Tapeworm infection(s) in dogs result from ingestion of infected intermediate hosts”.

Response 10:

We respectfully like to point out that dogs are infected by procercoid and plerocercoid larvae found in tissues of intermediate hosts, which in the former case implies copepods. Despite the fact that sparganosis is sometimes referred to as a waterborne disease, infection occurs after accidentally ingesting copepods that live in the water while drinking. To become infected, dogs must consume a primary or secondary intermediate host which already carries the larvae (i.e. an infected host) to become definitive hosts (commonplace), or possibly become intermediate hosts themselves and develop sparganosis (rare). For the sake of precision, we have now revised L65 and L67 to specify that infection can occur after eating a "primary or secondary" (L65) intermediate host and specifically "amphibians, reptiles and crustaceans (for Spirometra species)" (L67). We hope that the reviewer can accept our revisions.

Comment 11:

The Introduction contains a full background regarding both Echinococcus infections and its status in Serbian dogs. The aim is clearly declared (yet “Cyclophillidean tapeworm infection” sounds too general. Maybe Taeniidae?)

Response 11:

We would like to point out that we used “Cyclophillidean tapeworm infections” (L123-124 now) to reflect the fact that the tapeworm egg isolation and enrichment method does not exclude Mesocestoides eggs. We did not perform microscopy screening, thus we do not have visual confirmation that indeed only taeniid eggs were collected. The methodology we have used primarily targets taeniid eggs and has been used widely for the collection and enrichment of Echinococcus eggs from fecal samples, but it still cannot entirely exclude eggs of similar size and/or density. Mesocestoides eggs are heavy and we have used a flotation solution specifically designed for heavy eggs. Additionally, the primers used for molecular detection are not specific for Taeniidae, but can detect a wide range of Cyclophillidean tapeworms, including Mesocestoides. We hope that the reviewer can accept this explanation. 

Comment 12:

Kindly, add time period when your research took place. Coordinates of the regions involved are desirable also (in decimal degrees).

Response 12:

We have added the year of collection, 2025 (L130 now). The samples were collected over a one week period and processed in batches. We did not specify exact dates, we hope that the reviewer can accept that. As to the coordinates, we unfortunately cannot provide such data as it would violate our sampling agreement and informed consent. The geolocations are in fact addresses. We would kindly ask the reviewer for understanding. To provide at least some geographical references, we have in the original submission in L137-142 identified only the names of townships which are in the vicinity of the collection sites. These lines remain unchanged in the revised manuscript:

Hunting dogs originated from four locations in eastern Serbia (Šuvajić, Srednjevo, Tribrode, Veliko Gradište) in the Danube valley and one location in central Serbia (Smederevska Palanka). Stray dogs originated from various localities (mostly villages) surrounding the township of Ub, southwest of Belgrade, a farming and agricultural area in which several large abattoirs are located.

Comment 13:

What was the age range of the dos sampled? What were their breeds (weight)? Were both sexes represented in fecal sampling? If yes, were they presented equally?

Response 13:

We would respectfully like to point out to the reviewer that we are not trying to ascertain risk factors for infection or looking at possible effects of infection in this study. As such, the collection of biometric data from dogs was not vital for this study. We wish to point out that for stray dogs, age and breed could only be estimated, not identified, thus the information is not precise. To the best of our knowledge, the weight of stray dogs is likely to be the result of a multitude of factors, not parasitic infection alone. We hope that the reviewer can accept our explanation.

Comment 14:

Line 144: “The samples were centrifuged at 1600 × g for 30 min with the brake off, the supernatant” are you sure that centrifugation lasted half an hour (not 30 sec)?

Response 14:

We wish to confirm that centrifugation does indeed take 30 min with the brake off. This is the required time to ensure proper separation of eggs from debris.

Comment 15:

Tables facilitate reading, yet figures would be nice also.

Response 15:

Respectfully, we believe that the tables are much more informative and we hope that the reviewer can accept our explanation.

Comment 16:

Overall the Paper would win if supplementary files like filled questionnaires (or at least blank questionnaire), sequences obtained, etc. would be ready available, not just “from the corresponding author”.

Response 16:

We would like to point out to the reviewer that we have obtained only informed consent for this study. The ‘questionnaires’ appear in the acknowledgement, but their purpose was not to collect biometric data for analysis. We apologize for any confusion this may have caused. The answers concern anti-cestode treatment, outdoor or indoor lifestyle and origin (whether any of the hunting dogs were recently adopted from pet shelters or are rescued strays), which we consider to be important for sample quality control. As the questionnaires were not analyzed as part of this study, we believe that it is not necessary to publish a blank copy. A blank copy of the informed consent form however has been submitted to the journal as per the Editor's request. We hope that the reviewer can accept our explanation. We would like to clarify that sequencing was done for the purposes of this study. 

 

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract

• Lines 41–42: consider using “a more severe disease” instead of “the more severe disease”.
• Lines 44–45: sentence is slightly overloaded and could be split for clarity.
• Line 45: unnecessary comma after sample size should be removed.
• Ensure consistent formatting of sample sizes (“n = 29” vs “n=29”).
• Minor inconsistencies in percentages compared to Results should be checked.

Introduction

• Line 59: “Tapeworm infection(s)” → use either singular or plural consistently.
• Line 61: “Tenidae” should be corrected to “Taeniidae”.
• Lines 71–75: sentence is too long and could be split for readability.
• Lines 92–93: sentence could be slightly simplified for clarity.

Materials and Methods

• Line 120: “strays” → consider “stray dogs” for clarity.
• Lines 121–123: sentence is somewhat complex and could be simplified.
• Lines 129–130: wording regarding treatment could be clarified.
• Line 139: “tea strainers” → consider a more formal/scientific term if available.
• Line 168: “μl” should be standardized to “μL”.
• Ensure consistent formatting of units and spacing throughout the section.
• Please clarify the sampling strategy (e.g., convenience vs systematic sampling).
• It is unclear how sample independence was ensured, particularly for stray dogs housed in shared environments.
• Additional information on sampled animals (age, sex, health status, deworming history) would strengthen the study or should be acknowledged as a limitation.
• It should be clarified whether PCR detection reflects:
  • active infection or
  • passive passage of ingested parasite DNA
• Please indicate whether positive samples were confirmed by sequencing or discuss this as a limitation.

Results

• Line 178: confidence interval formatting should be standardized (e.g., “95% CI = ...”).
• Lines 179–180: minor inconsistencies in percentages compared to Abstract should be checked.

Discussion

• Lines 209–211: sentence is quite long and could be split for clarity.
• Line 217: “in concordance with” → consider “consistent with”.
• Lines 237–238: sentence is slightly heavy and could be simplified.
• Line 244: wording could be made more concise.
• Lines 267–270: sentence is long and could be split for readability.
• The statement that:
  • “stray dogs are a reservoir”
    should be presented more cautiously, as PCR detection alone does not confirm reservoir status.
• Similarly, statements regarding:
  • “important link in the transmission chain”
    may be somewhat overstated and should be either supported or softened.
• The limitation regarding the inability to distinguish between:
  • true infection and environmental contamination/transient passage
    should be clearly acknowledged.
• The discussion could be strengthened by including ecological and behavioral factors (dog management, feeding habits), not only methodological differences.

Statistical Analysis

• The statistical approach is acceptable. Authors may clarify whether additional analyses (e.g., risk factor analysis) were considered.

Conclusions

• Lines 316–318: statements may be slightly strong relative to the presented data and could be phrased more cautiously.

Author Response

Comment 1:

• Lines 41–42: consider using “a more severe disease” instead of “the more severe disease”.
• Lines 44–45: sentence is slightly overloaded and could be split for clarity.
• Line 45: unnecessary comma after sample size should be removed.
• Ensure consistent formatting of sample sizes (“n = 29” vs “n=29”).
• Minor inconsistencies in percentages compared to Results should be checked.

Response 1:

We than the reviewer for these helpful suggestions, we have made the requested changes: please see L42 (a more severe..), L44-46 (In an ongoing screening of definitive hosts for the presence of Echinococcus spp. to discover reservoirs of E. multilocularis, fecal samples of hunting (n=29) and stray dogs (n=66) were examined), the sentence could not be meaningfully split, but we have tried to simplify it instead and hope that this is acceptable. The comma was removed. We have standardized formatting throughout the manuscript and made sure that the percentages are consistent. We thank the reviewer for noticing the mistakes.

Comment 2:

• Line 59: “Tapeworm infection(s)” → use either singular or plural consistently.
• Line 61: “Tenidae” should be corrected to “Taeniidae”.
• Lines 71–75: sentence is too long and could be split for readability.
• Lines 92–93: sentence could be slightly simplified for clarity.

Response 2: 

We thank the reviewer for these suggestions. We have revised the sentences accordingly, please see L65 now (infections), L68 (Taeniidae), L69-72 and 72-74: 

Risk factors for infection identified by the European Scientific Counsel Companion Animal Parasites (ESCCAP) and various scientific studies include: lifestyle (e.g. pet, hunting, shelter, stray), time spent outdoors and diet. Additional important factors are hunting behavior and contact with other animals (including wildlife) and especially the status, practice and frequency of ecto- and endoparasite treatment [2-6].

We apologize to the reviewer, we could not simplify the sentence without prolonging the introduction. We hope that the reviewer can accept our other revisions.

Comment 3:

• Line 120: “strays” → consider “stray dogs” for clarity.

Response 3:

We have accepted the suggestion, please see L120 (stray dogs)

Comment 4:

• Lines 121–123: sentence is somewhat complex and could be simplified.

Response 4:

Accepted. Please see L123-126:

The aim of this study was to assess the molecular occurrence of Cyclophillidean tapeworm infections in hunting and stray dogs and specifically those caused by E. multilocularis and E. granulosus s. l. complex.

Comment 5:

• Lines 129–130: wording regarding treatment could be clarified.

Response 5:

Accepted. We have made an effort to clarify. Please see L143-145:

Based on the information obtained from owners and/or shelter staff at the time of sample collection, none of the dogs were treated with antihelminthics or formulations effective against cestodes prior to sampling.

Comment 6:

• Line 139: “tea strainers” → consider a more formal/scientific term if available.

Response 6: 

Revised. Please see L157-158: nylon mesh strainers with pore size of 0.5 mm...

Comment 7:

• Line 168: “μl” should be standardized to “μL”.

Response 7:

We thank the reviewer for noticing. We have standardized the formulation.

Comment 8:

• Ensure consistent formatting of units and spacing throughout the section.

Response 8: 
We thank the reviewer for noticing. We have standardized the units/spacing.

Comment 9:

• Please clarify the sampling strategy (e.g., convenience vs systematic sampling).

Comment 9:

We have clarified the strategy now. Please see newly added L132-134:

Sampling was based on convenience and contingent upon signed informed consent (for owners and shelter staff) and signed cooperation agreements (for dog shelters). 

Comment 10:

• It is unclear how sample independence was ensured, particularly for stray dogs housed in shared environments.

Response 10:

We have clarified as requested. Please see newly added L137-138:

To ensure sample individuality in shared environments at the shelter, samples were collected immediately after defecation.

Comment 11:

• Additional information on sampled animals (age, sex, health status, deworming history) would strengthen the study or should be acknowledged as a limitation.

Response 11:

We would respectfully like to point out to the reviewer that the collection of biometric data was actually not within the scope of this study and such data was not collected. We are not considering this to be a limitation of the study, as the dogs here are treated only as definitive hosts of Echinococcus spp. in context of public health. We are not concerned with evaluating risk factors for infection or assessing characteristics of infection in dogs - we are primarily concerned with detecting and identifying Echinococcus spp. in dog feces. Additionally, accuracy of biometric data from stray dogs is not guaranteed, while deworming status cannot be ascertained - these dogs only recently arrived at the shelter, as stated in L132. We hope that the reviewer can accept our explanation.

Comment 12:

Please indicate whether positive samples were confirmed by sequencing or discuss this as a limitation

 Response 12:

We respectfully disagree that lack of sequencing confirmation is a limitation of the present study and we are in fact highly concerned by the reviewer's request. We believe that by declaring that the lack of sequencing confirmation is a limitation of our study, it may suggest to some readers that our results are suspect and possibly invalid. However, we acknowledge the reviewer's concern regarding specificity of the detection methodology. We therefore would like to first point to the fact that the detection methodology itself (including the primer sequences) was published and cited by several subsequent papers -Štefanić et al., 2004 cited 236 times, Maksimov et al., 2020 cited 34 times, Martini et al., 2022 cited 21 times. Based on our reading of the original papers and a number of the citing papers, the methodology has been exhaustively validated in different laboratories. Importantly, the PCR detection methodology has been used primarily with fecal samples and/or eggs purified from fecal samples. We strongly believe that once a methodology itself has been validated, confirmation of every positive finding by sequencing is not necessary. To validate PCR detection in house, we use positive controls or we sequence a select number of amplicons. To validate the multiplex qPCR, we have used positive controls (sequenced DNA) which originated from livestock samples collected earlier for other studies. To validate the EM-H15 and EM-H17 primers, we used a validated positive control obtained from another laboratory. We sequenced amplicons obtained with these primers from the positive control to confirm specificity. The cyclo primers detect multiple cestodes, thus implying non-specificity. Thirdly, we would like to additionally point to the data from the current study. We have stated in L275-277 that all of the samples in which Echinococcus spp. DNA was detected were also positive for Cyclophyllidean DNA and in L206-207 that “co-infections with different Echinococcus spp. were not detected”. We believe this supports our claim that the primers and probes used are in fact detecting tapeworms and that these tapeworms are of the Echinococcus genus and that they do in fact distinguish between different Echinococcus species. We hope that the reviewer can accept our explanation.

Comment 13:

• Line 178: confidence interval formatting should be standardized (e.g., “95% CI = ...”)

Response 13:

We thank the reviewer for noticing. We have done as suggested.

Comment 14:

• Lines 179–180: minor inconsistencies in percentages compared to Abstract should be checked.

Response 14:

We thank the reviewer for noticing. We have checked the percentages and adjusted them accordingly.

Comment 15:

• Lines 209–211: sentence is quite long and could be split for clarity.

Response 15:

Accepted and revised. Please see L230-233 now.

Comment 16:

• Line 217: “in concordance with” → consider “consistent with”.

Response 16:

Accepted. Revised. Please see L246 now.

Comment 17:

• Lines 237–238: sentence is slightly heavy and could be simplified.

Response 17:

Accepted. Revised. Please see two sentences now L258-262.

Comment 18:

• Line 244: wording could be made more concise.

Response 18:

Accepted and revised. Please see L268.

Comment 19:

• Lines 267–270: sentence is long and could be split for readability.

Response 19:

We were unable to split the sentence in a meaningful way, we hope the reviewer can accept this.

Comment 20:

• The statement that: “stray dogs are a reservoir” should be presented more cautiously, as PCR detection alone does not confirm reservoir status.

Response 20:

We would like to respectfully point out that the DNA used as a template for PCR detection was extracted after flotation and mesh filtration of the fecal samples to collect taeniid eggs. It thus implies that Echinococcus eggs were in fact present in the feces of some dogs. This finding suggests that there indeed were adult Echinococcus worms in the gastrointestinal tract of these dogs, which implies infection. As infected definitive hosts, dogs are reservoirs of infection for other hosts in our opinion. 

Comment 21:

• Similarly, statements regarding: "important link in the transmission chain” may be somewhat overstated and should be either supported or softened.

Response:

We are not sure that we follow the reviewer's line of argumentation here which we take to be an extension of comment 20. If that is the case, we interpret the reviewer's comments to mean that the reviewer is not convinced that detection of Echinococcus DNA in the egg pellet recovered from dog feces implies infection, thus challenging the reservoir designation and possibility of transmission. We apologize in advance if we are providing a reply to a concern which we did not correctly interpret. We do not believe that there is a high likelihood that the occurrence of Echinococcus spp. eggs in dog feces could be the result of anything but infection, which implies that adult tapeworms are in the GI tract. We cannot know this for certain, as live dogs were sampled. Another possibility referred to by the reviewer in a later comment is the passive transition of ingested Echinococcus spp. eggs from the environment. We would like to briefly address this comment here and more detailed when appropriate but we do not believe that using the methodology, i.e. flotation and mesh filtration, would in fact be able to recover an accidentally ingested egg, as this would imply that it passes through the entire GI tract without loss of structural integrity. We believe this is unlikely. To address transmission, we refer to L51-53: “E. multilocularis DNA was detected only in stray dog feces, suggesting that strays are a reservoir and possibly important link in the transmission chain”. The sentence syntax itself implies uncertainty, which we believe is warranted as we are only reporting the presence of Echinococcus spp. eggs (detected by PCR) in feces, but we do not have proof of actual transmission which has occurred from the dogs analyzed by this study. We simply imply that dogs may be an important link in the transmission chain in general terms, as they are definitive hosts.  

Comment 22: 

• The limitation regarding the inability to distinguish between: true infection and environmental contamination/transient passage should be clearly acknowledged.

Response 22:

We would like to refer the reviewer to our previous reply that the methodology used in this study is based on the isolation (by flotation) and enrichment (by sieving) of taeniid eggs. The methodology has been validated and other studies have shown that the pellet which is recovered after sieving and centrifugation indeed contains taeniid eggs. We ourselves have confirmed by microscopy that taeniid eggs are present in the pellets. The DNA which was used as a template for molecular detection was in fact extracted from the egg pellet. We have previously commented that structural integrity of the eggs is necessary for recovery by the flotation/sieving method and that we believe that it is unlikely that an egg which has passed through the entire GI tract will remain intact. We point to the fact that ingestion of eggs by dogs can in rare cases lead to aberrant infection which results in disease, which would imply that eggs have hatched and released larvae which have exited the GI tract. Hatching is facilitated by the environmental conditions inside the stomach and GI tract and implies a loss of the egg integrity. This suggests that it is unlikely that an intact egg would passively transit the stomach and GI tract and be recovered by flotation and mesh filtration from the feces. We do acknowledge the fact that it is not possible to completely rule out transient passage, however, we would like to maintain that is an exceedingly low probability, while the probability that adult worms in the GI tract (which implies infection) are the source of the eggs we have recovered is much higher in comparison. Environmental contamination cannot be ruled out entirely, but we have not considered it as an important factor for several reasons:
1.  All of the hunting dogs reside in individual kennels, which are regularly cleaned. Even if a kennel is contaminated with eggs, the eggs originate from the occupying dog, which has no impact on the results.
2. Kennels (individual and community) at the shelter are also regularly cleaned and disinfected. While we cannot guarantee with absolute certainty that all eggs are destroyed or eliminated by the cleaning process, we can claim that the eggs certainly originate from a stray dog. Considering the likelihood that the eggs originate from the current occupants as opposed to previous occupants, we cannot be sure, but we do believe that it is more likely that they in fact originate from the current occupants, due to the frequency of the cleaning.
3. Fecal samples collected from the shelter’s dog run were indeed recovered from the ground, but immediately after defecation, primarily to ensure that individual dogs were not sampled twice. As a precautionary measure enforced by the shelter, there are always only a few dogs out in the run at the same time, which made tracking of individual dogs when sampling relatively easy. While Echinococcus spp. eggs could indeed have been present in the soil of the dog run, they could only have originated from the shelter’s dogs, as the dog run is fenced in. Thus we can certainly claim that the eggs originated from stray dogs, but we could not with complete certainty claim that they originated from the currently sampled dogs.
Finally, we would like to point out to the reviewer that we do appreciate the suggestions, however, based on our data, the number of dogs from which Echinococcus spp. eggs were recovered in this study is fairly low. This finding suggests that, even if present, environmental contamination is not widespread. It stands to reason that in confined spaces, like dog shelters especially, environmental contamination should have a noticeable impact on the findings, thus we would expect to see a large number of positive samples. As this was not the case, we strongly believe that we are indeed dealing with genuine infection. As far as acknowledging possibilities other than genuine infection, we believe that the suggested alternatives are highly speculative and frankly far less likely than genuine infection.

Comment 24:

The discussion could be strengthened by including ecological and behavioral factors (dog management, feeding habits), not only methodological differences

Response 24:

We would like to point out to the reviewer that most of our samples originated from stray dogs. These stray dogs were sampled prior to administration of anti-cestode treatment shortly (days) after arrival at the shelter. We therefore strongly suspect that the stray dogs could not have been infected at the shelter. We can only confirm that the dogs were roaming freely, at least for some time, prior to their arrival at the clinic. All other aspects of their lives prior to arrival are in fact unknown to us and cannot be inferred from the samples we have collected. Any discussion regarding ecological or behavioral factors as suggested would be only speculative, while management and feeding habits apply only after some time spent at the shelter but again have no bearing on the infection with Echinococcus spp. We hope that the reviewer can accept our explanation.

Comment 25: 

• The statistical approach is acceptable. Authors may clarify whether additional analyses (e.g., risk factor analysis) were considered.

Response 25:
No risk factor analysis was considered or performed. All statistical approaches have been mentioned.

Comment 26:

• Lines 316–318: statements may be slightly strong relative to the presented data and could be phrased more cautiously.

Response 26:
We have addressed the reviewer's concerns with 'reservoir status' in previous comments. We hope the reviewer can accept our responses.