Occurrence of Spirometra mansoni in Domestic Dogs from Rural Ecuador and Its Public Health Relevance

Response to Reviewer Comments One.

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections. The corrections made are highlighted in red.

I would like to thank the reviewer for their relevant comments and suggestions, which will improve the article.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

The previously requested corrections were made.

Are all the cited references relevant to the research?

Yes

Corrections were made to the references

Is the research design appropriate?

Yes

The requested corrections were made to improve the research design.

Are the methods adequately described?

Yes

The requested corrections were made to improve the methods appropriately.

Are the results clearly presented?

Yes

The requested corrections were made to improve the methods appropriately.

Are the conclusions supported by the results?

Yes

The requested corrections were made to support the results with the conclusions.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

·       The article requires a major revision.

·       The submitted manuscript is disorganized and difficult to understand; it lacks structure and clarity.

·       The figures need to be reorganized and captions added.

·       Not all results are presented.

·       The English language editing is essential.

·       It is important to distinguish between the results and the discussion.

·       Rewrite the "Conclusions" section and format the "References" section according to the journal's guidelines.

·       Essentially, some parts of the article need to be rewritten.

·       The authors state that molecular analyses were performed using PCR with ITS2 primers; however, the corresponding results are not presented.

·       Were the obtained sequences deposited in a public database?.

·       Was any intraspecific variation detected, and what level of similarity was observed compared with other published S. mansoni sequences?.

·       Such statements Regarding the PCR products, 67 of the 67 samples were obtained using specific primers.

·       All DNA samples yielded a PCR product of approximately 120 bp. Are not the kind of findings that should be included in a scientific publication without further analysis.

·       In general, the article must be fundamentally revised and then reconsidered for publication in Animals.

Response 1:

We agree with this comment. Therefore, we have made the corrections to the manuscript highlighted in red.

The article underwent a very thorough review.

The manuscript was reorganized for greater clarity. A new structure was implemented.

The figures were reorganized and captions were added.

All results were presented.

The necessary corrections were made to the English language.

The results and discussion chapters were separated.

The "Conclusions" section was rewritten.

The "References" section was formatted according to the journal's guidelines.

Some parts of the article were rewritten.

New molecular analyses were performed using PCR with ITS2 primers. In addition, very interesting new results are presented.

The obtained sequences were deposited in GenBank.

The obtained sequences were analyzed and compared with others deposited in GenBank.

The entire section on PCR was rewritten.

All DNA samples produced a PCR product.

Molecular characterization and phylogeny were also performed.

Comments 2:

The title is too long. I would suggest shortening the title to: Occurrence of Spirometra mansoni in Domestic Dogs from Rural Ecuador and Its Public Health Relevance.

Response 2:

The title was changed to the requested one. It can be found in number 2 (located to the right of the article).

Comments 3:

Abstract: Line 35: Spirometra (S.) mansoni causes…… why is there,, (S) “if later you still write in full versión of Spirometra mansoni? I would suggest to delete it

Response 3:

The requested correction was made (the change is in number 38).

Comments 4:

Abstract: Line 35-39: In my opinion, this sentence is too long and could be divided into two separate sentences.

Response 4:

The requested correction was made (the changes is between numbers 38-42).

Comments 5:

Abstract: Line 45-48: Again too long sentence. The statement made at the end of this section is not fully supported by the data presented in the manuscript. I would suggest moderating the wording of this conclusion.

Response 5:

The requested correction was made (the changes is between numbers 49-51).

Comments 6:

Introduction: Line 65-66: Sparganosis is a metacestodiasis or larval cestodiasis caused by

Spirometra mansoni infection [1]. is being repeated from lines 53-54.

Response 6:

The requested correction was made (the changes is between numbers 57-58).

Comments 7:

Introduction: Line 79: Are you sure want to state that?. There are more than 64 described species of Spirometra Spirometra?.

Response 7:

The requested correction was made (the changes is between numbers 83-84).

Comments 8:

Introduction: Line 37-100: I would suggest breaking the goal down into specific points based on the tasks to make it clearer. For example 1) to describe Spirometra mansoni in domestic dogs through stool analysis; 2) to analyze its prevalence in a riverine zone of Ecuador, and etc.

Response 8:

The suggested objectives were broken down (the changes are between numbers 57-58).

Comments 9:

Introduction: Figure 1 must be in Materials and Methods section. Furthermore, the map needs to be edited to include place names, the meanings of the circles, and their labels.

Response 9:

Figure 1 was moved to the Materials and Methods section. In addition, the map was edited and the names of the places, the meaning of the circles, and labels were added (the changes are between numbers 126-129).

Comments 10:

Materials and Methods: Line 107-107: all these places ,, Loma Larga in the city of Nobol; Santa Rosa in the city of Daule and Las Cañas in the city Lomas de Sargentillo “could be added into map.

Response 10:

The places studied were added to the map. (the changes are between numbers 126-129).

Comments 11:

Materials and Methods: Line 109-111: This sentence should be moved to the discussion page.

Response 11:

The sentence:

“Furthermore, it is important to highlight that the sites studied had temperatures ranging from 20 °C to 37 °C, with a tropical savanna climate. was moved from materials and methods to the discussion chapter”

was moved from materials and methods to the discussion chapter.

Comments 12:

Materials and Methods: Line 112-116: To be honest, I don't really understand the point of this paragraph here-is it really necessary? Personally, I think it's the part of a scientist's planning that stays in the draft; of course, one might disagree.

Response 12:

The suggested corrections were made.

Comments 13:

Materials and Methods: Line 117-124: This section is very interesting, but it delves into social/economic or even other types of research, where, in my opinion, specific questions should be presented, and the results should include a detailed interpretation and analysis of the answers. In the methodology section, this part should be separated and presented in the appendices, specifying exactly which aspects were evaluated; then the methods should describe what will be done with this data, which statistical methods will be used, and so on. I am not an expert in this type of research, so it might be worth consulting with specialists in this field.

Response 13:

This section was restructured.

Comments 14:

Materials and Methods: Lines 125-138: The situation is similar to that described in the previous paragraph, except that here everything must be presented from a veterinary perspective, while maintaining structure and clarity.

Response 14:

This section was restructured.

Comments 15:

Materials and Methods: Section 2.2. Transportation and analysis of samples from domestic dogs This section should be divided into subsections, as it constitutes the main part of the study. I would suggest two subsections: 2.2 Transportation and analysis of samples from domestic dogs. 2.3 Coproparasitological methods. A brief description of the methods used (direct examination, flotation (Willis), and sedimentation with centrifugation using saline solution) is needed. The cited book and other source are very general, so the details of the study remain unclear.

Response 15:

This section was restructured (the changes are between numbers 161-184).

Comments 16:

Materials and Methods: Line 145: 40X please write: ,, x400 magnification.

Response 16:

The requested corrections were made (the changes are between numbers 177-178).

Comments 17:

Materials and Methods: Line 146: How many blood smaples have been taken from how animals?.

Response 17:

Sixty-seven dogs that tested positive underwent further blood and fecal examinations (the changes are between numbers 179-184).

Comments 18:

Materials and Methods: Line 159: How many samples were positive?

Response 18:

67 samples were positive.

Comments 19:

Results and Discussion: Please format this section according to the journal's guidelines and separate the "Results" section from the "Discussion" section.

I suggest considering swapping Sections 3.1 and 3.2. Presenting the infection data first would improve the flow of the Results section and help readers better understand the subsequent analyses. At present, questions arise regarding the number of infected animals, the number of positive cases, infection intensity, and related findings before this information is provided.

Response 19:

The "Results" section was separated from the "Discussion" section and the suggested corrections were made.

Comments 20:

Results and Discussion: Line 187-188: What this,, 60-187 µm long and 30-40 µm “is? Average? Or min-max? How many eggs, n?.

Response 20:

These are criteria described by Bowman and Alvarado et al., where it is mentioned that the eggs of S. Mansoni present: A lid at the distal end of the egg with a flattened shape, equatorial swelling, brown in color, between 60 and 70 µm long and 30–40 µm wide; elliptical, rounded or convex in shape and the presence of morulated structures inside. (the changes are between numbers 187-191).

Comments 21:

Results and Discussion: Line 190-191: ,,morulated structures were observed inside the eggs” ------Can you show that by arrow in Figure 2? Is this a distinctive diagnostic Spirometra mansoni feature? Do other Spirometra species have different ones?

Response 21:

The morulate structures within the identified eggs are not a distinguishing feature between Spirometra species.

Comments 22:

Results and Discussion: Line 191-192: ,, These characteristics corresponded to those reported for Spirometra mansoni [7,18] – This sentence should be moved to the discussion page.

Response 22:

The requested correction was made.

Comments 23:

Results and Discussion: Figure 2. Why isn't there a ruler?

Response 23:

The requested correction was made.

Comments 24:

Results and Discussion: Line 200-210: This paragraph gets ahead of itself and discusses DNA test results that will be presented later. Furthermore, this paragraph contains many sentences that should be part of the discussion rather than the results, for example, This species has been reported in frogs and domestic cats in the United States [6], domestic cats in Mexico [20], coyotes, domestic dogs and cats from Costa Rica [7], crab foxes (Cerdocyon thousand) in Colombia [21] and domestic cats in Ecuador [8] This section needs to be rewritten.

Response 24:

The Results and Discussion were separated.

Comments 25:

Results and Discussion: Line 211-219: This paragraph is the discussion part.

Response 25:

The Results and Discussion were separated.

Comments 26:

Results and Discussion: Line 222: Please indicate in parentheses how many of the cases studied were positive.

Response 26:

The suggested corrections were made. There were 67 positive cases.

Comments 27:

Results and Discussion: Section 3.3. Molecular identification of Spirometra mansoni in fecal samples from domestic dogs using the PCR technique.

First, is the ITS2 region alone sufficient to reliably identify and confirm the species as Spirometra mansoni? I could not find adequate information in the Introduction regarding the molecular tools and markers used for the identification of Spirometra species, nor a justification for the choice of ITS2. Second, the molecular results supporting the identification of S. mansoni are not presented in sufficient detail. Were the obtained sequences deposited in a public database? Was any intraspecific variation detected among the samples? What level of sequence similarity was observed compared with other published S. mansoni sequences? Furthermore,statements such as “Regarding the PCR products, 67 of the 67 samples were obtained using specific primers. All DNA samples yielded a PCR product of approximately 120 bp. constitute sufficient evidence for species identification without additional sequence analysis and interpretation. The manuscript should include the molecular findings that support the identification of S. mansoni. Finally, after these statements, the text appears to return to the Discussion rather than presenting additional Results. The distinction between the Results and Discussion sections should be clarified.

Response 27:

Although there is limited information on the molecular identification of Spirometra mansoni, especially using the ITS molecular marker, the presence of S. mansoni was confirmed by PCR. The resulting sequence was analyzed and compared with other sequences deposited in GenBank, and further details were added.

Regarding the Introduction on the tools and molecular markers used for the identification of Spirometra species using the ITS2 molecular marker, this was added to the aforementioned section (numbers 100-103). Regarding the PCR products obtained, this section was modified for clarity.

New PCR and molecular sequencing analyses were performed for Spirometra mansoni, obtaining very interesting results, which were described in the papers.

Comments 28:

Results and Discussion: Sections 3.4. Determination of clinical characteristics in domestic dogs from the studied sectors and 3.5. Determination of clinical characteristics in people from the studied sectors.

As I mentioned earlier, I believe the manuscript would benefit from the involvement of an expert who could provide professional guidance on the methodology and assist with the analysis and interpretation of these results.

Response 28:

The suggested corrections were made.

Comments 29:

Conclusions: This section presents a condensed version of the results rather than conclusions. Please rewrite it.

Response 29:

The suggested corrections were made.

Comments 30:

References list: [11] is it: ,, Georgis' Parasitology for Veterinarians 11th Edition

Please format it according to the journal's requirements.

Response 30:

The suggested corrections were made.

4. Response to Comments on the Quality of English Language

Point 1:

Comments 31:

I would suggest that English language could be reviewed by a native or fluent speaker.

Response 31: The grammar and semantics of the article's content in English were improved.

5. Additional clarifications

Thank you so much for your support for my country. Your comments and suggestions greatly helped the manuscript.

They improved the work.

Response to the second reviewer's comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections. The corrections made are highlighted in red.

I would like to thank the reviewer for their relevant comments and suggestions, which will improve the article.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

The previously requested corrections were made.

Are all the cited references relevant to the research?

Yes

Corrections were made to the references

Is the research design appropriate?

Yes

The requested corrections were made to improve the research design.

Are the methods adequately described?

Yes

The requested corrections were made to improve the methods appropriately.

Are the results clearly presented?

Yes

The requested corrections were made to improve the methods appropriately.

Are the conclusions supported by the results?

Yes

The requested corrections were made to support the results with the conclusions.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1

The statistical analysis is inadequate and potentially inappropriate. Student’s t-test is reported for differences in prevalence among sectors, but the outcome is categorical and should be analyzed using the chi-square test, Fisher’s exact test, or logistic regression. Exact p-values, confidence intervals, and test statistics are missing.

Response 1:

The requested corrections were made

Comments 2:

The conclusions regarding public health significance are overstated. Human risk was inferred only from surveys and visual inspection; no serological, imaging, or parasitological diagnostic methods were performed in humans.

Response 2:

The requested corrections were made, especially in the conclusions.

Comments 3:

The title is too long and should be shortened for greater precision.

Response 3:

The title was corrected, in accordance with the first reviewer's instructions.

Comments 4:

L27. The term “infestation” should be replaced with “infection” throughout the manuscript.

Response 4:

The requested correction was made.

Comments 5:

L44. The abstract should report the exact number of positive samples, not only the percentage.

Response 5:

The requested correction was made.

Comments 6:

Figure 1 lacks sufficient explanation of the symbols and should include a clearer legend, geographic coordinates, and the data source.

Response 6:

The requested correction was made.

Comments 7:

Figure 2 should include a scale bar and a clearer caption indicating the staining status and measurement method.

Response 7:

The requested correction was made.

Comments 8:

The phrase “observed via Lugol light microscopy” should be reworded for clarity; Lugol is a stain, not a microscopy method.

Response 8:

The sentence was reformulated more clearly.

Comments 9:

The diagnostic exclusion of other parasites should be interpreted cautiously because no detailed assessment of the sensitivity of the methods used is provided.

Response 9:

The request was excluded.

Comments 10:

Several grammatical and typographical errors should be corrected, including “aplicable,” “morbitidy,” “tinction,” and poorly constructed sentences.

Response 10:

The requested correction was made.

Comments 11:

Materials and Methods: Line 112-116: To be honest, I don't really understand the point of this paragraph here-is it really necessary? Personally, I think it's the part of a scientist's planning that stays in the draft; of course, one might disagree.

Response 11:

The suggested corrections were made.

Regarding the molecular study, new PCR and molecular sequencing analyses were performed for Spirometra mansoni, obtaining very interesting results, which were described in the papers.

4. Response to Comments on the Quality of English Language

Point 1:

Comments 4:

I would suggest that English language could be reviewed by a native or fluent speaker.

Response 1: The grammar and semantics of the article's content in English were improved.

5. Additional clarifications

Thank you so much for your support for my country. Your comments and suggestions greatly helped the manuscript.

They improved the work.

Response to the third reviewer's comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections. The corrections made are highlighted in red.

I would like to thank the reviewer for their relevant comments and suggestions, which will improve the article.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

The previously requested corrections were made.

Are all the cited references relevant to the research?

Yes

Corrections were made to the references

Is the research design appropriate?

Yes

The requested corrections were made to improve the research design.

Are the methods adequately described?

Yes

The requested corrections were made to improve the methods appropriately.

Are the results clearly presented?

Yes

The requested corrections were made to improve the methods appropriately.

Are the conclusions supported by the results?

Yes

The requested corrections were made to support the results with the conclusions.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1

The content of the manuscript does not adequately justify the title.

Response 1:

The requested corrections were made (The changes are in line 2, taking into account the suggestion of the first reviewer).

Comments 2:

The manuscript does not provide detail methods for the morphometry. It is expected that a subset of helminth eggs from the 67 positive samples should be measured and the mean values be presented. Equally, eggs with characteristic features should be photographed and presented.

Response 2:

The criteria used for morphometrics were taken from Alvarado et al. [7] and Bowman [19]. In addition, the average values ​​of the 67 positive eggs were presented. Furthermore, eggs with distinctive characteristics (modifications between lines 254 and 259) were also presented.

Comments 3:

The PCR was conducted on only microscopy positive samples. This limits the true prevalence of S. mansoni in the study population considering the fact that microscopy may fail to detected some positive samples. Importantly, the authors presentation of the PCR method is insufficient; from DNA extraction, primer selection and base pair size, and the interpretation of the gel result. Gel pictures of positive samples and controls were not shown, and there was no sequencing results and BLAST search to confirm if amplified products were really S. mansoni DNA.

Response 3:

The requested correction was made. The corrections regarding PCR were made throughout the text. New PCR and molecular sequencing analyses were performed for Spirometra mansoni, obtaining very interesting results, which were described in the papers.

Comments 4:

The description of sampled dogs and the analysis of demographic data to determine risk factors was not explicit. This needs to be addressed especially using the appropriate methods of determining some parameters and the use of correct terminologies.

Response 4:

The requested correction was made.

Comments 5:

Same observation in 4 above applies to the section on ‘’Determination of clinical characteristics in dog owners.

Response 5:

The requested correction was made.

Comments 6:

Lines 192-194. The statement on the microscopic examination of the 406 fecal samples using several methods (direct examination, flotation, and sedimentation with centrifugation using saline solution) is unusual. I am surprised that all the dogs did not harbor any other helminth eggs (Cystoisospora sp., Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Trichuris sp., Taenia sp., Strongyloides sp., and Capillaria sp) except S. mansoni as stated by the authors. Moreso, the authors mentioned that the dogs did not receive any veterinary health care (see lines 273-276).

Response 6:

The requested correction was made. Those related to Cystoisospora sp., Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Trichuris sp., Taenia sp., Strongyloides sp., and Capillaria sp. were removed.

Comments 7:

Lines 285-286: ‘’No cases of subcutaneous lesions suggestive of sparganosis were recorded during the physical examination of subjects’’. This comment needs to interpreted with caution. Authors did not present details on how the dog owners were assessed, hence there could be lapses with the evaluation method. Authors need to addressed this in the M&M section.

Response 7:

The requested correction was made.

Comments 8:

Authors should provide the limitations of the study.

Response 8:

At the end of the discussion, the limitations of the study were added.

Comments 9:

Line 20- Change ‘’an’’ to ‘of’---both pathologies are an animal and public health concern to-- both pathologies are of animal and public health concern.

Response 9:

The requested correction was made.

Comments 10:

Line 27- Rephrase to use the actual number of infected dogs instead of ‘’17%’’

Response 10:

The percentage value of positive cases was reformulated throughout the text.

Comments 11:

Line 32: be specific with dogs, ‘’pets’’ is general and may include non-carnivores.

Response 11:

The suggested corrections were made.

Comments 12:

Line 55. Change-‘Spirometra mansoni’ to ‘’S. mansoni’’ here and elsewhere in the manuscript except the first use or at beginning of a sentence.

Response 12:

The suggested corrections were made in all the text.

Comments 13:

Line 83. ‘South Africa’ is not a continent, change to ‘’Africa’’

Response 13:

The suggested corrections were made.

Comments 14:

Figure 1. Provide a key for the interpretation of the results. What do the colors on the pie chart represent?.

Response 14:

The suggested corrections were made in the Figure 1.

Comments 15:

Line 105. Rephrase-2.1. ‘’Collection of samples and physical analysis of participants’’ to 2.1. ‘Sample collection and physical examination of dogs’

Response 15:

The entire Materials and Methods section was reformulated.

Comments 16:

Lines 117-135. This section needs reordering and rephrasing for clarity. Authors need to use appropriate terminologies.  It is vague in the present form.

Response 16:

This segment was reorganized and reformulated.

Comments 17:

Lines 126-127. The weight estimation of dogs based on visual assessment is not scientifically justified. Why not use an accurate method of measurement?

Response 17:

This statement was improved.

Comments 18:

Line 136-138. How- was this done, who- did the examination, where- in the hospital or in the dog owners house. Authors need to be explicit about the procedure.

The whole of the section needs to be reorganized for flow-first, period of study, followed by the selection of study area and inclusion and exclusion criteria of dogs. Then, owner education and seeking for informed consent and questionnaire administration. Next, fecal sampling collection and physical examination of dogs and other demographic data etc.

Response 18:

The suggested corrections were made. Inclusion criteria were added.

Comments 19:

Line 144. What is ‘’Lugol’’ name of the microscope or what? Explain or delete-‘’Lugol’’ . Do you need Lugol Iodine for egg detection? See line 199 where you stated not with Lugol. So what is ‘Lugol light microscopy’.

Response 19:

The suggested corrections were made.

Comments 20:

Line 166-167. Mention the expected band size of the target gene amplified by the ITS primers. What do you mean by ‘A sample generously provided---‘’  be specific.  DNA of S. mansoni or what?.

Response 20:

The suggested corrections were made.

Comments 21:

Line 176-177: State how (in comparison to what?) the positive results were assessed based on the base pair size

Response 21:

The suggested corrections were made.

Comments 22:

Figure 2. There are not pointers to show the distinctive features. Author should provide detailed methodology of how the morphometry of representative eggs of S. mansoni detected in the study was conducted.

Response 22:

The suggested corrections were made.

Comments 23:

Figure 2. There are not pointers to show the distinctive features. Author should provide detailed methodology of how the morphometry of representative eggs of S. mansoni detected in the study was conducted.

Response 23:

The suggested corrections were made.

Comments 24:

Lines 200-201. The PCR results as currently presented did not provide confirmation of the S. mansoni. There are no clear gel pictures and no nucleotide sequences from the PCR products.  

Response 24:

The suggested corrections were made.

Comments 25:

Lines 225. The Table 1 did not show the- [with significant differences (p < 0.05 and t student test)].

Response 25:

The suggested corrections were made.

Comments 26:

The results and discussion should be presented separately.

Response 26:

The results and the discussion were separated.

4. Response to Comments on the Quality of English Language

Point 1:

Comments 4:

I would suggest that English language could be reviewed by a native or fluent speaker.

Response 1: The grammar and semantics of the article's content in English were improved.

5. Additional clarifications

Thank you so much for your support for my country. Your comments and suggestions greatly helped the manuscript.

They improved the work.

Response to the second reviewer's comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections. The corrections made are highlighted in red.

I would like to thank the reviewer for their relevant comments and suggestions, which will improve the article.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

The previously requested corrections were made.

Are all the cited references relevant to the research?

Yes

Corrections were made to the references

Is the research design appropriate?

Yes

The requested corrections were made to improve the research design.

Are the methods adequately described?

Yes

The requested corrections were made to improve the methods appropriately.

Are the results clearly presented?

Yes

The requested corrections were made to improve the methods appropriately.

Are the conclusions supported by the results?

Yes

The requested corrections were made to support the results with the conclusions.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1

The statistical analysis is inadequate and potentially inappropriate. Student’s t-test is reported for differences in prevalence among sectors, but the outcome is categorical and should be analyzed using the chi-square test, Fisher’s exact test, or logistic regression. Exact p-values, confidence intervals, and test statistics are missing.

Response 1:

The requested corrections were made

Comments 2:

The conclusions regarding public health significance are overstated. Human risk was inferred only from surveys and visual inspection; no serological, imaging, or parasitological diagnostic methods were performed in humans.

Response 2:

The requested corrections were made, especially in the conclusions.

Comments 3:

The title is too long and should be shortened for greater precision.

Response 3:

The title was corrected, in accordance with the first reviewer's instructions.

Comments 4:

L27. The term “infestation” should be replaced with “infection” throughout the manuscript.

Response 4:

The requested correction was made.

Comments 5:

L44. The abstract should report the exact number of positive samples, not only the percentage.

Response 5:

The requested correction was made.

Comments 6:

Figure 1 lacks sufficient explanation of the symbols and should include a clearer legend, geographic coordinates, and the data source.

Response 6:

The requested correction was made.

Comments 7:

Figure 2 should include a scale bar and a clearer caption indicating the staining status and measurement method.

Response 7:

The requested correction was made.

Comments 8:

The phrase “observed via Lugol light microscopy” should be reworded for clarity; Lugol is a stain, not a microscopy method.

Response 8:

The sentence was reformulated more clearly.

Comments 9:

The diagnostic exclusion of other parasites should be interpreted cautiously because no detailed assessment of the sensitivity of the methods used is provided.

Response 9:

The request was excluded.

Comments 10:

Several grammatical and typographical errors should be corrected, including “aplicable,” “morbitidy,” “tinction,” and poorly constructed sentences.

Response 10:

The requested correction was made.

Comments 11:

Materials and Methods: Line 112-116: To be honest, I don't really understand the point of this paragraph here-is it really necessary? Personally, I think it's the part of a scientist's planning that stays in the draft; of course, one might disagree.

Response 11:

The suggested corrections were made.

Regarding the molecular study, new PCR and molecular sequencing analyses were performed for Spirometra mansoni, obtaining very interesting results, which were described in the papers.

4. Response to Comments on the Quality of English Language

Point 1:

Comments 4:

I would suggest that English language could be reviewed by a native or fluent speaker.

Response 1: The grammar and semantics of the article's content in English were improved.

5. Additional clarifications

Thank you so much for your support for my country. Your comments and suggestions greatly helped the manuscript.

They improved the work.

Response to the third reviewer's comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections. The corrections made are highlighted in red.

I would like to thank the reviewer for their relevant comments and suggestions, which will improve the article.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

The previously requested corrections were made.

Are all the cited references relevant to the research?

Yes

Corrections were made to the references

Is the research design appropriate?

Yes

The requested corrections were made to improve the research design.

Are the methods adequately described?

Yes

The requested corrections were made to improve the methods appropriately.

Are the results clearly presented?

Yes

The requested corrections were made to improve the methods appropriately.

Are the conclusions supported by the results?

Yes

The requested corrections were made to support the results with the conclusions.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1

The content of the manuscript does not adequately justify the title.

Response 1:

The requested corrections were made (The changes are in line 2, taking into account the suggestion of the first reviewer).

Comments 2:

The manuscript does not provide detail methods for the morphometry. It is expected that a subset of helminth eggs from the 67 positive samples should be measured and the mean values be presented. Equally, eggs with characteristic features should be photographed and presented.

Response 2:

The criteria used for morphometrics were taken from Alvarado et al. [7] and Bowman [19]. In addition, the average values ​​of the 67 positive eggs were presented. Furthermore, eggs with distinctive characteristics (modifications between lines 254 and 259) were also presented.

Comments 3:

The PCR was conducted on only microscopy positive samples. This limits the true prevalence of S. mansoni in the study population considering the fact that microscopy may fail to detected some positive samples. Importantly, the authors presentation of the PCR method is insufficient; from DNA extraction, primer selection and base pair size, and the interpretation of the gel result. Gel pictures of positive samples and controls were not shown, and there was no sequencing results and BLAST search to confirm if amplified products were really S. mansoni DNA.

Response 3:

The requested correction was made. The corrections regarding PCR were made throughout the text. New PCR and molecular sequencing analyses were performed for Spirometra mansoni, obtaining very interesting results, which were described in the papers.

Comments 4:

The description of sampled dogs and the analysis of demographic data to determine risk factors was not explicit. This needs to be addressed especially using the appropriate methods of determining some parameters and the use of correct terminologies.

Response 4:

The requested correction was made.

Comments 5:

Same observation in 4 above applies to the section on ‘’Determination of clinical characteristics in dog owners.

Response 5:

The requested correction was made.

Comments 6:

Lines 192-194. The statement on the microscopic examination of the 406 fecal samples using several methods (direct examination, flotation, and sedimentation with centrifugation using saline solution) is unusual. I am surprised that all the dogs did not harbor any other helminth eggs (Cystoisospora sp., Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Trichuris sp., Taenia sp., Strongyloides sp., and Capillaria sp) except S. mansoni as stated by the authors. Moreso, the authors mentioned that the dogs did not receive any veterinary health care (see lines 273-276).

Response 6:

The requested correction was made. Those related to Cystoisospora sp., Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Trichuris sp., Taenia sp., Strongyloides sp., and Capillaria sp. were removed.

Comments 7:

Lines 285-286: ‘’No cases of subcutaneous lesions suggestive of sparganosis were recorded during the physical examination of subjects’’. This comment needs to interpreted with caution. Authors did not present details on how the dog owners were assessed, hence there could be lapses with the evaluation method. Authors need to addressed this in the M&M section.

Response 7:

The requested correction was made.

Comments 8:

Authors should provide the limitations of the study.

Response 8:

At the end of the discussion, the limitations of the study were added.

Comments 9:

Line 20- Change ‘’an’’ to ‘of’---both pathologies are an animal and public health concern to-- both pathologies are of animal and public health concern.

Response 9:

The requested correction was made.

Comments 10:

Line 27- Rephrase to use the actual number of infected dogs instead of ‘’17%’’

Response 10:

The percentage value of positive cases was reformulated throughout the text.

Comments 11:

Line 32: be specific with dogs, ‘’pets’’ is general and may include non-carnivores.

Response 11:

The suggested corrections were made.

Comments 12:

Line 55. Change-‘Spirometra mansoni’ to ‘’S. mansoni’’ here and elsewhere in the manuscript except the first use or at beginning of a sentence.

Response 12:

The suggested corrections were made in all the text.

Comments 13:

Line 83. ‘South Africa’ is not a continent, change to ‘’Africa’’

Response 13:

The suggested corrections were made.

Comments 14:

Figure 1. Provide a key for the interpretation of the results. What do the colors on the pie chart represent?.

Response 14:

The suggested corrections were made in the Figure 1.

Comments 15:

Line 105. Rephrase-2.1. ‘’Collection of samples and physical analysis of participants’’ to 2.1. ‘Sample collection and physical examination of dogs’

Response 15:

The entire Materials and Methods section was reformulated.

Comments 16:

Lines 117-135. This section needs reordering and rephrasing for clarity. Authors need to use appropriate terminologies.  It is vague in the present form.

Response 16:

This segment was reorganized and reformulated.

Comments 17:

Lines 126-127. The weight estimation of dogs based on visual assessment is not scientifically justified. Why not use an accurate method of measurement?

Response 17:

This statement was improved.

Comments 18:

Line 136-138. How- was this done, who- did the examination, where- in the hospital or in the dog owners house. Authors need to be explicit about the procedure.

The whole of the section needs to be reorganized for flow-first, period of study, followed by the selection of study area and inclusion and exclusion criteria of dogs. Then, owner education and seeking for informed consent and questionnaire administration. Next, fecal sampling collection and physical examination of dogs and other demographic data etc.

Response 18:

The suggested corrections were made. Inclusion criteria were added.

Comments 19:

Line 144. What is ‘’Lugol’’ name of the microscope or what? Explain or delete-‘’Lugol’’ . Do you need Lugol Iodine for egg detection? See line 199 where you stated not with Lugol. So what is ‘Lugol light microscopy’.

Response 19:

The suggested corrections were made.

Comments 20:

Line 166-167. Mention the expected band size of the target gene amplified by the ITS primers. What do you mean by ‘A sample generously provided---‘’  be specific.  DNA of S. mansoni or what?.

Response 20:

The suggested corrections were made.

Comments 21:

Line 176-177: State how (in comparison to what?) the positive results were assessed based on the base pair size

Response 21:

The suggested corrections were made.

Comments 22:

Figure 2. There are not pointers to show the distinctive features. Author should provide detailed methodology of how the morphometry of representative eggs of S. mansoni detected in the study was conducted.

Response 22:

The suggested corrections were made.

Comments 23:

Figure 2. There are not pointers to show the distinctive features. Author should provide detailed methodology of how the morphometry of representative eggs of S. mansoni detected in the study was conducted.

Response 23:

The suggested corrections were made.

Comments 24:

Lines 200-201. The PCR results as currently presented did not provide confirmation of the S. mansoni. There are no clear gel pictures and no nucleotide sequences from the PCR products.  

Response 24:

The suggested corrections were made.

Comments 25:

Lines 225. The Table 1 did not show the- [with significant differences (p < 0.05 and t student test)].

Response 25:

The suggested corrections were made.

Comments 26:

The results and discussion should be presented separately.

Response 26:

The results and the discussion were separated.

4. Response to Comments on the Quality of English Language

Point 1:

Comments 4:

I would suggest that English language could be reviewed by a native or fluent speaker.

Response 1: The grammar and semantics of the article's content in English were improved.

5. Additional clarifications

Thank you so much for your support for my country. Your comments and suggestions greatly helped the manuscript.

They improved the work.