Health Status of the Eastern Grey Squirrel (Sciurus carolinensis) Population in Umbria: Results of the LIFE Project ‘U-SAVEREDS’
, Silvia Crotti 1, Daniele Paoloni 2, Valentina La Morgia 3, Andrea Felici 1, Paola Papa 1, Gian Mario Cosseddu 4,5, Livia Moscati 1 and Paola Gobbi 1
Round 1
Reviewer 1 Report
The study by Cruciani et al. collects the results obtained throughout a LIFE project related to the conservation of the red squirrel. During the last decades, the trade in exotic animals and their adaptation to new ecosystems have posed a threat in many cases to native biodiversity, as is the case of the Florida pond turtle, the American crab, and the Eastern gray squirrel. In my opinion, all projects dedicated to the eradication of these invasive species should always carry out a health study, since the spread of exotic diseases in ecosystems may be more important than the competition for the ecological niche with native species. Therefore, congratulations on this work.
However, before publishing it I would like to suggest some modifications to the authors:
The cardinal points in English are always capitalized, so the name of Sciurus carolinensis should be spelled Eastern gray squirrel. Please review the text to modify it.
Throughout the text, you write about the danger that gray squirrel populations can pose to humans due to their behavior and because they can be reservoirs of zoonoses. I agree with the authors, however, I think it should be softened since after reading the full text one of the conclusions that the reader can draw is that the gray squirrel should be extinct. If you carry diseases in Italy, why not in the USA? I think it would be more appropriate to focus more on the ecological damage that these disease carriers can do by introducing mortal pathogens into the ecosystem, although it is mentioned that of course some are zoonotic and can cause a negative impact on public health.
L. 32: "human health, wild and domestic animals", sounds weird, change it to "human and domestic and wild animals health".
L. 50: in my opinion, it is better not to put acronyms in keywords unless they are used frequently, such as AMR (antimicrobial resistance). It's just a suggestion, but maybe it would be better to change IAS to "invasive animal species".
L. 61-65. This sentence gives the impression that SARS, MERS, and the rest of the epidemics/pandemics had been caused by IAS and therefore systematic sampling of only IAS is important. Actually, this is not the case, for example, MERS had its origin in the dromedary, an animal native to Saudi Arabia, the main country affected. I think that what the authors wanted to express is that the development of these diseases has highlighted the importance of monitoring ecosystems, including IAS, as an early warning signal against possible epidemics. Please reword the sentence to better understand.
L. 74. "50 km2", please raise the "2".
L. 84. "Squirrels host many other pathogens", they do not always carry pathogens, please modify the sentence: "Squirrels can host many other pathogens".
L. 111-112. "and their behavior with humans have critical implications on public health", please modify the sentence: "could have critical implications...".
L. 112-114. I believe that this sentence about environmental education should be in the discussion.
Material and methods. Several European and Italian regulations are mentioned, please add their references in the text and in the references section.
L. 146. Please put Sciurus carolinensis in italics.
Tables 1 and 2. I think they could be merged and make a single table. In the case of agents detected by PCR, I suggest adding an extra column to indicate the reference used to obtain the PCR protocol.
Bacteriological and virological examinations. It is generally very underdeveloped. This section should indicate all the steps performed to be able to reproduce the analyses. However, there are very few details. In fact, there is only one sentence about virology tests. I suggest that you expand on the information on this point.
L. 176. Please change "specimens" to "samples".
L. 204-205. Please justify the frame.
Molecular analysis. In this section, you have described the PCRs carried out to detect the pathogens in Table 1, but not those in Table 2. Although the pathogens in Table 2 were initially detected by serology, the additional molecular analyzes carried out have not been included in this section (Toxoplasma, Leptospira, Francisella and Chlamydia). When adding it, you must include references to the protocols used for each pathogen and specify in greater detail the methodology used. The sentence that is written about it is not enough (L241-242).
L. 243-244. Puumala virus and Dobrava virus (common name), or Puumalavirus and Dobravavirus (scientific name). Please check the name of the viruses in the text.
L. 279. It is indicated that Escherichia coli was isolated, but the number of positive samples is not indicated. Were those Escherichia coli strains characterized to learn more about them?
L. 280. Please change "examinations" to "analysis".
L. 286. "to guarantee human health"... please reformulate the sentence. This information not only serves to reinforce the surveillance of important pathogens for humans but also for domestic animals and livestock (West Nile, Babesia, Borrelia, Leptospira...) and of course wild populations of native fauna (Adenovirus, Poxvirus, West Nile, Chlamydia, Francisella...). And that is the objective of One Health: that the information obtained is used in all areas.
L. 324-326. These phrases should be in the Results section.
L. 335. "has been previously reported in squirrels."
L. 348. Please, if you write the full name of Borrelia lusitaniae, write also the full name of Coxiella burnetii
L. 354. "could be related to dogs."
L. 386-389. Please include a reference that confirms this data.
There is no conclusion section. Please review the publisher's guide carefully to confirm if an exclusive section for conclusions is mandatory or if it is not necessary.
Author Response
Response to Reviewer 1 comments
Dear Reviewer 1,
thank you for your generous comments on the manuscript and your revisions and suggestions. The revisions have been approved by all authors and the changes are marked using the “Track Changes” function as requested. The revised manuscript has been uploaded.
Point 1:
The cardinal points in English are always capitalized, so the name of Sciurus carolinensis should be spelled Eastern gray squirrel. Please review the text to modify it.
Response 1: Done.
Point 2: Throughout the text, you write about the danger that gray squirrel populations can pose to humans due to their behavior and because they can be reservoirs of zoonoses. I agree with the authors, however, I think it should be softened since after reading the full text one of the conclusions that the reader can draw is that the gray squirrel should be extinct. If you carry diseases in Italy, why not in the USA? I think it would be more appropriate to focus more on the ecological damage that these disease carriers can do by introducing mortal pathogens into the ecosystem, although it is mentioned that of course some are zoonotic and can cause a negative impact on public health.
Response 2: The section “5. Conclusion” has been added to the manuscript and here the authors improved the concept of the ecological damage.
Point 3: L. 32: "human health, wild and domestic animals", sounds weird, change it to "human and domestic and wild animals health".
Response 3: Done.
Point 4: L. 50: in my opinion, it is better not to put acronyms in keywords unless they are used frequently, such as AMR (antimicrobial resistance). It's just a suggestion, but maybe it would be better to change IAS to "invasive animal species".
Response 4: Done.
Point 5: L. 61-65. This sentence gives the impression that SARS, MERS, and the rest of the epidemics/pandemics had been caused by IAS and therefore systematic sampling of only IAS is important. Actually, this is not the case, for example, MERS had its origin in the dromedary, an animal native to Saudi Arabia, the main country affected. I think that what the authors wanted to express is that the development of these diseases has highlighted the importance of monitoring ecosystems, including IAS, as an early warning signal against possible epidemics. Please reword the sentence to better understand.
Response 5: The sentence has been modified how suggested.
Point 6: L. 74. "50 km2", please raise the "2".
Response 6: Done.
Point 7: L. 84. "Squirrels host many other pathogens", they do not always carry pathogens, please modify the sentence: "Squirrels can host many other pathogens".
Response 7: Done.
Point 8: L. 111-112. "and their behavior with humans have critical implications on public health", please modify the sentence: "could have critical implications...".
Response 8: Done.
Point 9: L. 112-114. I believe that this sentence about environmental education should be in the discussion.
Response 9: The sentence has been also exploited in the “Conclusions” section.
Point 10: Material and methods. Several European and Italian regulations are mentioned, please add their references in the text and in the references section.
Response 10: Authors guideline do not give any details about regulations’ format. Despite this you can deduce from the manuscript their number/ID.
Point 11: L. 146. Please put Sciurus carolinensis in italics.
Response 11: Done.
Point 12: Tables 1 and 2. I think they could be merged and make a single table. In the case of agents detected by PCR, I suggest adding an extra column to indicate the reference used to obtain the PCR protocol.
Response 12: Done.
Point 13: Bacteriological and virological examinations. It is generally very underdeveloped. This section should indicate all the steps performed to be able to reproduce the analyses. However, there are very few details. In fact, there is only one sentence about virology tests. I suggest that you expand on the information on this point.
Response 13: The authors have improved this paragraph.
Point 14: L. 176. Please change "specimens" to "samples".
Response 14: Done.
Point 15: L. 204-205. Please justify the frame.
Response 15: Done.
Point 16: Molecular analysis. In this section, you have described the PCRs carried out to detect the pathogens in Table 1, but not those in Table 2. Although the pathogens in Table 2 were initially detected by serology, the additional molecular analyzes carried out have not been included in this section (Toxoplasma, Leptospira, Francisella and Chlamydia). When adding it, you must include references to the protocols used for each pathogen and specify in greater detail the methodology used. The sentence that is written about it is not enough (L241-242).
Response 16: The additional molecular analyzes were not performed because all the serological investigations gave negative results. Therefore, the authors deleted the sentence (L241-242) and cleared the “Additional investigations” field in the Table 1.
Point 17: L. 243-244. Puumala virus and Dobrava virus (common name), or Puumalavirus and Dobravavirus (scientific name). Please check the name of the viruses in the text.
Response 17: Done.
Point 18: L. 279. It is indicated that Escherichia coli was isolated, but the number of positive samples is not indicated. Were those Escherichia coli strains characterized to learn more about them?
Response 18: Done.
Point 19: L. 280. Please change "examinations" to "analysis".
Response 19: Done.
Point 20: L. 286. "to guarantee human health"... please reformulate the sentence. This information not only serves to reinforce the surveillance of important pathogens for humans but also for domestic animals and livestock (West Nile, Babesia, Borrelia, Leptospira...) and of course wild populations of native fauna (Adenovirus, Poxvirus, West Nile, Chlamydia, Francisella...). And that is the objective of One Health: that the information obtained is used in all areas.
Response 20: Done.
Point 21: L. 324-326. These phrases should be in the Results section.
Response 21: Done.
Point 22: L. 335. "has been previously reported in squirrels."
Response 22: Done.
Point 23: L. 348. Please, if you write the full name of Borrelia lusitaniae, write also the full name of Coxiella burnetii
Response 23: Done.
Point 24: L. 354. "could be related to dogs."
Response 24: Done.
Point 25: L. 386-389. Please include a reference that confirms this data.
Response 25: the authors changed “data not shown” in “data not yet published”. In the uploaded version, this sentence is in the “Conclusion” section.
Point 26: There is no conclusion section. Please review the publisher's guide carefully to confirm if an exclusive section for conclusions is mandatory or if it is not necessary.
Response 26: The authors added “Conclusion” section.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The Authors present an interesting paper describing the negative consequences of inadvertent introduction of Sciurus carolinensis into a new ecosystem (Perugia). The potential and disastrous risks associated with such human-triggered species introductions are manifold and well-documented in multiple examples.
Thus, the Authors lay a solid groundwork for the caveats they propose. Moreover, a good deal of the supporting data tend to reinforce the operative hypotheses conjectured herein.
However, the Reviewer finds absent a seemingly key facet in the Author's articulation of hypothesis that must be addressed. To begin, it is axiomatic that both introduced and indigenous mammalian species may carry and transmit zoonotic vector-borne organisms. Here, and pointedly, the Authors contrast S. carolinensis & S vulgaris noting [confident behavior with humans -- "line 27, 44 & elsewhere"] yet apparently without any cite supporting this seemingly crucial aspect of zoonotic transmission.
Further, line 285, 286 describe a "potential role as a zoonotic carrier for the eastern grey squirrel" but again without citing evidence of host to human transmission. Is this not a crucial piece in the puzzle being described? Even if such evidence is missing - though the Reviewer believes it is available - such should be articulated.
In sum, zoonoses constitute a "chain of events" leading pathogenic infection from host to human. The Authors are describing most of this chain but have failed to incorporate a key link. In the Reviewer's assessment, this must be positively addressed.
Author Response
Response to Reviewer 2 comments
Dear Reviewer 2,
thank you for your generous comments on the manuscript and your revisions and suggestions. The revisions have been approved by all authors and the changes are marked using the “Track Changes” function as requested. The revised manuscript has been uploaded.
The authors responded to Reviewer comments and suggestions as follows.
Point 1:
However, the Reviewer finds absent a seemingly key facet in the Author's articulation of hypothesis that must be addressed. To begin, it is axiomatic that both introduced and indigenous mammalian species may carry and transmit zoonotic vector-borne organisms. Here, and pointedly, the Authors contrast S. carolinensis & S vulgaris noting [confident behavior with humans -- "line 27, 44 & elsewhere"] yet apparently without any cite supporting this seemingly crucial aspect of zoonotic transmission.
Response 1: The authors exploited the concept as suggested.
Point 2: Further, line 285, 286 describe a "potential role as a zoonotic carrier for the eastern grey squirrel" but again without citing evidence of host to human transmission. Is this not a crucial piece in the puzzle being described? Even if such evidence is missing - though the Reviewer believes it is available - such should be articulated.
Point 3: In sum, zoonoses constitute a "chain of events" leading pathogenic infection from host to human. The Authors are describing most of this chain but have failed to incorporate a key link. In the Reviewer's assessment, this must be positively addressed.
Response 2 and 3: To enhance the concept a new reference was added (Lewis et al. 1975).
Author Response File: Author Response.docx
