Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See attachement

Comments for author File: Comments.pdf

Author Response

Point 1: Title: I suggest the title should be rephrased. What do the authors mean by natural identification?

My suggestion would be- Microscopic and molecular identification of Sarcocystis species in wild brown rats (Rattus norvegicus) in Lithuania and Latvia

Response 1: Thank you for your valuable comment. We changed.

  Point 2: Line 36. Mention the four genetic loci amplified in the study

Response 2: Thank you for your worthwhile comment. We inserted.

  Point 3: Line 38. The authors should provide a sentence stating the significance of the findings in terms or veterinary, public health or environment/conservation.

Response 3: Thank you for your comment. We have added a sentence indicating the significance of the study.

Point 4: Line 122- delete- ‘’remaining’’

Response 4: Thank you. We deleted.

Point 5: Fig 1. There are total of 26 dots (9 red and 17 green) representing 27 samples. One is missing, please cross-check and correct accordingly.

Response 5: Thank you for your meaningful comment. We inserted an incorrect map, and it has now been corrected.

Point 6: Line 126- Replace ‘isolated’ with ‘’recovered’’ or any other suitable word.

Response 6: Changed.

Point 7: Line 135- 2.2. ‘Molecular Analysis’: change to ‘’DNA extraction’’

Response 7: Thank you for your valuable comment. We changed.

Point 8: Reconcile line 138 what samples? Do you mean DNA or ???

Response 8: Yes, DNA. We added.

Point 9: Line 140: provide a subtitle here. 2.3 Molecular identification of Sarcocystis species

Response 9: Thank you for your valuable comment. We provided.

Point 10: Line 177- Change- “3.1. Sarcocystis spp. Identification in Intestine Samples of Brown Rat’’ to- ‘Microscopic identification of Sarcocystis spp. in Intestine Samples’

Response 10: Thank you. We changed as asked.

Point 11: Line 196-197. Please rephrase this sentence for clarity.

Response 11: Thank you for your worthwhile comment. The sentence has been rephrased to improve clarity and readability.

Point 12: Line 203. Replace- ‘help of’ with ‘’primers’’

Response 12: Thank you. We replaced.

Point 13: I suggest that the authors should provide a phylogenetic tree(s) based on one or two of the gene targets amplified in this study

Response 13: We thank the reviewer for this valuable suggestion. In response, we generated phylogenetic trees and included them in the revised manuscript (Fig. 3). The Materials and Methods and Results sections were supplemented accordingly. Due to differences in the genetic regions analyzed, sequence lengths, and primer binding sites, separate phylogenetic reconstructions were performed to ensure the most appropriate treatment of each dataset. These analyses support the species identifications obtained in this study, while some sequences remain unresolved at the species level.

Point 14: Line 205- delete ‘’high’’

Response 14: Corrected.

 Point 15: Tables 2 and 3 in the present form are not clear to me. I guess the essence of using several primers is to enable the authors to resolve the true identities of the parasites. Therefore, I expected them to select the results that provide the best evidence of species identification rather than providing range of similarities as presented in the present Table 2. I have made a suggestion for merging the two tables for clarity and reader friendliness. If the suggested format is acceptable, authors should populate the table with the data on all the 27 samples used in the study accordingly. However, the authors are at liberty to modify/merge the tables as they dim most appropriate, but it should be clean and clear! Other sections of the results should be reduced as the modified table will eliminate some excess. The aim, overall is to limited the length of the material to the most essential. This way readers will easily appreciate the novelty in the results, which will otherwise be lost in the tortuosity.

Response 15: We thank the reviewer for this detailed and constructive comment. In response, we have merged the former Tables 2 and 3 into a single table to improve clarity and reader friendliness. Although the text was modified accordingly.  

Point 16: Line 254-255. I suggest that the authors should tone down the language of this sentence. Remember, the sample size of 27 is not sufficient for drawing such strong and authoritative wordings.

Response 16: Thank you for your worthwhile comment. We agree with the reviewer and have toned down the wording to avoid overgeneralization. The revised sentence now emphasizes that the findings provide preliminary evidence rather than a definitive global assessment.

Point 17: Lines 256, 299- delete- (Table 2)

Response 17: Thank you. We deleted.

Point 18: Authors should kindly reconcile the statements in Lines 290-191 and 296-297 and adjust accordingly.

Response 18: Thank you for your worthwhile comment. We deleted that: “They rarely engage in predatory behavior; instead, their intake of meat reflects opportunistic consumption of available resources.“

 Point 19: Line 322- delete-(Table 3)

Response 19: Thank you. We deleted.

 Point 20: Line 325- Change ‘’Taking’’ to ‘Taken’

Response 20: Thank you for your meaningful comment. We changed.

 Point 21: Line 329. The authors should provide a statement highlighting the significance of the findings in terms or veterinary, public health or environment/conservation.

Response 21: We thank the reviewer for this valuable comment and have added a sentence highlighting the veterinary, public health, and environmental relevance of our findings by emphasizing the potential role of synanthropic rodents in Sarcocystis transmission at the wildlife–domestic animal interface.

Reviewer 2 Report

Comments and Suggestions for Authors

Review

The manuscript entitled “Firs natural identification of apicomplexan Sarcocystis parasites in intestines of wild brown rat (Rattus norvegicus)” qualifies and is of sufficient quality to be published in Animals. The authors have extensive experience with Sarcocystis spp., and this manuscript rigorously describes the discovery of stages (oocysts and sporocysts) in fecal/intestine samples from Rattus norvegicus in Europe, with a detailed description of the possibility that this rodent species is the definitive host of Sarcocystis species, using appropriate methodology, microscopy, nested PCR, and sequencing of four different genetic markers. In addition to having developed numerous primer pairs to differentiate between the wide variety of species of this parasite, providing novel and interesting information for the scientific community related to parasitology. Likewise, minor corrections and suggestions that the authors and the editor should consider are proposed for incorporation into the manuscript to improve its understanding.

Kind regards,

 

Suggestions

Simply summary

Line 15. If S. cymruensis is the valid scientific name, it should be written first: S. cymruensis (syn. S. rodentifelis).

Abstract

Line 35. Please, indicate the number of positive cases after the percentage, e.g.: 25.9% (7/27).

Line 36. “Four genetic loci”. Please, indicate the names of the genetic markers analyzed.

Line 45. Please, confirm it should be written “Phylum Apicomplexa” instead of “order”.

Lines 91, 142, 193 and Table 1. ITS1 should not be in italics, as it is not a gene. Please, correct.

Line 91. Please, change 28S ribosomal (rRNA) to 28S ribosomal RNA gene.

Materials and methods.

Line 120. How did the researchers obtain the rodent carcasses? Did they go on field trips, capture the rodents with traps, or were the animal donated by a pest control company or similar? Please provide a detailed explanation.

Line 127. Please, specify the model of the microscope used for the microscopic examination.

Figure 1. Please specify the countries on the map. Eg.: “ (…) sampling sites in Lithuania (colored in yellow) and Latvia (colored in pink)” for a better understanding for non-European readers.

Table 1. It seems that the authors used different primer pairs in the second round of nested PCR, but in most cases, the primer pair used in the first round of nested PCR was the same. Please specify this in the text.

Line 174 and 175. Please specify the genetic marker associated with the accession number. Similar to the Data Availability Statement section.

Line 193. Sarcocystis should be in italics.

Table 2. Indicate the number of positive samples for each sequence for better understanding.

Table 2. Please explain why the Ziu1 and Ziu3 sequences cannot be designated as known species if they share 100% identity to the species S. myodes and S. rileyi, respectively.

Line 231. Were 12 or 15 pure DNA sequences obtained? Please correct If necessary.

Discussion.

Line 256. Please, add the number of positive between parentheses after 25.9%.

Line 260. Please change to S. cymruensis, it is not necessary to indicate again S. rodentifelis.

Additional considerations:

I believe that phylogenetic analysis would help to understand the phylogenetic position and identity of the different sequences obtained, especially the new unidentified sequences. The authors should consider the possibility of creating different phylogenetic trees.

 

Author Response

Point 1: The manuscript entitled “Firs natural identification of apicomplexan Sarcocystis parasites in intestines of wild brown rat (Rattus norvegicus)” qualifies and is of sufficient quality to be published in Animals. The authors have extensive experience with Sarcocystis spp., and this manuscript rigorously describes the discovery of stages (oocysts and sporocysts) in fecal/intestine samples from Rattus norvegicus in Europe, with a detailed description of the possibility that this rodent species is the definitive host of Sarcocystis species, using appropriate methodology, microscopy, nested PCR, and sequencing of four different genetic markers. In addition to having developed numerous primer pairs to differentiate between the wide variety of species of this parasite, providing novel and interesting information for the scientific community related to parasitology. Likewise, minor corrections and suggestions that the authors and the editor should consider are proposed for incorporation into the manuscript to improve its understanding.

Kind regards,

Response 1: Thank you very much for such a positive evaluation.

Suggestions

Simply summary

Point 2: Line 15. If S. cymruensis is the valid scientific name, it should be written first: S. cymruensis (syn. S. rodentifelis).

Response 2: Thank you for your valuable comment. We have changed as suggested.

Abstract

Point 3: Line 35. Please, indicate the number of positive cases after the percentage, e.g.: 25.9% (7/27).

Response 3: Thank you, we indicated.

Point 4: Line 36. “Four genetic loci”. Please, indicate the names of the genetic markers analyzed.

Response 4: Thank you for your worthwhile comment. We inserted.

Point 5: Line 45. Please, confirm it should be written “Phylum Apicomplexa” instead of “order”.

Response 5: Yes, we confirm and changed. Thank you.

Point 6: Lines 91, 142, 193 and Table 1. ITS1 should not be in italics, as it is not a gene. Please, correct.

Response 6: Thank you for your worthwhile comment. Changed.

Point 7: Line 91. Please, change 28S ribosomal (rRNA) to 28S ribosomal RNA gene.

Response 7: Corrected.

Materials and methods.

Point 8: Line 120. How did the researchers obtain the rodent carcasses? Did they go on field trips, capture the rodents with traps, or were the animal donated by a pest control company or similar? Please provide a detailed explanation.

Response 8: Thank you for your meaningful comment. Twenty-seven brown rats, were snap-trapped captured between 2022 and 2025 in various regions of Lithuania and in the Daugavpils municipality of Latvia. We added the missing words.

Point 9: Line 127. Please, specify the model of the microscope used for the microscopic examination.

Response 9: Thank you for your useful comment. We added the missing information.

Point 10: Figure 1. Please specify the countries on the map. Eg.: “ (…) sampling sites in Lithuania (colored in yellow) and Latvia (colored in pink)” for a better understanding for non-European readers.

Response 10: Thank you for your accurate comment. We have incorporated your suggestion.

Point 11: Table 1. It seems that the authors used different primer pairs in the second round of nested PCR, but in most cases, the primer pair used in the first round of nested PCR was the same. Please specify this in the text.

Response 11: We thank the reviewer for this comment. Sentences were added to the Methods section to clarify how many primer pairs were used in each PCR round at different loci.

Point 12: Line 174 and 175. Please specify the genetic marker associated with the accession number. Similar to the Data Availability Statement section.

Response 12: Thank you for your comment. We added the missing information.

Point 13: Line 193. Sarcocystis should be in italics.

Response 13: Corrected.

Point 14: Table 2. Indicate the number of positive samples for each sequence for better understanding.

Response 14: Thank you. We thank the reviewer for this comment. Table 2 has been revised and reformatted, and we believe that the presentation of the data now allows the number of positive samples for each sequence to be more easily discerned.

Point 15: Table 2. Please explain why the Ziu1 and Ziu3 sequences cannot be designated as known species if they share 100% identity to the species S. myodes and S. rileyi, respectively.

Response 15: Thank you for your useful comment. The sequences of Sarcocystis sp. Ziu1, Sarcocystis sp. Ziu2, and Sarcocystis sp. Ziu3 were obtained from genetic regions that are insufficient for species-level discrimination (Lines 212–214). Because these regions are highly conserved among multiple Sarcocystis species, the sequences cannot be reliably assigned to a single known species and were therefore designated as Sarcocystis sp. Furthermore, phylogenetic analysis did not provide a clear classification.

Point 16: Line 231. Were 12 or 15 pure DNA sequences obtained? Please correct If necessary.

Response 16: Thank you for your valuable comment. A total of 15 pure Sarcocystis DNA sequences were obtained from 12 individual brown rats. To improve clarity, we have slightly revised the ambiguous sentence so that it more clearly reflects that 12 animals yielded pure sequences.

Discussion.

Point 17: Line 256. Please, add the number of positive between parentheses after 25.9%.

Response 17: Thank you. We added.

Point 18: Line 260. Please change to S. cymruensis, it is not necessary to indicate again S. rodentifelis.

Response 18: Corrected.

Additional considerations:

Point 19: I believe that phylogenetic analysis would help to understand the phylogenetic position and identity of the different sequences obtained, especially the new unidentified sequences. The authors should consider the possibility of creating different phylogenetic trees.

Response 19: Thank you for your valuable comment. We agree with the reviewer’s assessment that phylogenetic analyses are useful for understanding the phylogenetic position and identity of the obtained sequences, particularly the unidentified ones. Accordingly, due to differences in the gene regions analyzed, sequence lengths, and primer targets, we performed separate phylogenetic analyses to ensure the most appropriate treatment of each dataset. The resulting phylogenetic trees (Fig. 3a-g), now incorporated into the revised manuscript, clarify the phylogenetic relationships of the obtained sequences. However, for some sequences, species-level identification remains unresolved despite these analyses.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

figure 1, Line 140. Sampling sites in Lithuania (colored in yellow) and Latvia (colored in pink). Does this refer to the whole map of the countries or just sampling sites? Please modify it accordingly.

Author Response

Point 1:

Figure 1, Line 140. Sampling sites in Lithuania (colored in yellow) and Latvia (colored in pink). Does this refer to the whole map of the countries or just sampling sites? Please modify it accordingly. 

Response:

Thank you for your valuable comment, we have corrected the mistake. Figure 1 caption was changed “Figure 1. Geographic distribution of rodent sampling sites in Lithuania and Latvia created with Quantum Geographic Information System (QGIS, https://www.qgis.org). The territory of Lithuania is shown in yellow and that of Latvia in pink. Green circles indicate brown rat individuals positive for Sarcocystis spp., while red circles represent negative individuals. “