Genetic Identification of Parasitic Giardia enterica in Three Wild Rodent Species from a Zoological Institution: First Host Records in Brazilian Porcupine (Coendou prehensilis) and Naked Mole Rat (Heterocephalus glaber), and Detection in Crested Porcupine (Hystrix cristata)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Title and Abstract

• The title is accurate but overly detailed. Consider simplifying by removing parenthetical family names (e.g., "Family Erethizontidae") to enhance readability.
• Abstract:

                  The phrase "understudied rodent hosts" is vague. Specify that Brazilian porcupines and naked mole rats are previously unreported hosts for Giardia enterica. The abstract lacks quantitative results (e.g., sequence similarity percentages, sample sizes).

2. Introduction

• Taxonomic Overload: Excessive focus on rodent systematics (e.g., suborder classifications) distracts from the core focus on Giardia. Condense or move non-essential details to supplementary material.
• Objective Clarity: The novelty of studying Brazilian porcupines and naked mole rats is underemphasized. Explicitly state that this is the first genetic characterization of Giardiain these species.
• Contextual Gaps: Limited discussion on why studying captive zoo animals is relevant to understanding zoonotic transmission risks.

3. Materials and Methods

• Sample Size Justification: The small sample size (2 porcupines per species, pooled mole rat feces) is a critical limitation. Justify why this is sufficient for genetic characterization or acknowledge potential biases.
• PCR Protocol Rationale: No explanation for selecting SSU-rRNA, bg, tpi, and gdh genes. Clarify why these markers were prioritized over others (e.g., *SSU-rRNA* failure rate).
• Microscopy Details: Insufficient information on cyst quantification (e.g., cysts per gram of feces).

4. Results

• SSU-rRNA Failure: The illegible SSU-rRNA sequences are mentioned but not critically discussed. Address potential causes (e.g., primer mismatches, PCR inhibitors).
• Data Presentation:
• Tables 2–4 are redundant with supplementary tables. Summarize key polymorphisms in the main text and relegate full alignments to supplements.
• Phylogenetic trees (Figure 2, S1–S3) lack clarity. Include bootstrap values for all major nodes in the main figure.
• GenBank Accessions: Accession numbers for sequences are listed but not hyperlinked or formatted per journal guidelines.

5. Discussion

• Epidemiological Speculation: The suggestion of different transmission routes for distinct G. enterica variants is unsupported by data. Remove speculative claims or frame them as hypotheses for future work.
• Zoonotic Implications: The public health relevance of finding G. enterica (a human pathogen) in zoo animals is underdeveloped. Discuss potential risks to zookeepers/visitors.
• Comparison with Prior Studies: Contrasting findings with Italian wild porcupines [14,15] lacks depth. Elaborate on how captive vs. wild environments might influence genotype diversity.

6. References

• Inconsistent Formatting: Some journal names are abbreviated (e.g., Vet. Parasitol.), others are spelled out (e.g., International Journal of Parasitology). Standardize per journal guidelines.
• Missing Citations: References for Table 1 (e.g., [16–19]) are incomplete or misaligned with the table entries (e.g., prairie dog entry cites [19], but [19] discusses Giardia in chipmunks).

7. Figures and Tables

• Figure 1: Image quality is unclear in the provided text. Ensure high-resolution images with scale bars in the final version.
• Supplementary Material: Critical data (full alignments, phylogenetic trees) are relegated to supplements. Include key supplementary figures/tables in the main text to support conclusions.

8. General Writing and Structure

• Repetition: The taxonomy of G. duodenalis sensu lato is redundantly explained in the Introduction and Discussion. Consolidate.
• Typos:
• "rutinary analysis" → "routine analysis" (p. 2).
• "manufactorer’s instructions" → "manufacturer’s instructions" (p. 5).

Abbreviations: Define all abbreviations (e.g., SSU-rDNA, bg) at first mention.

9. Ethical and Data Availability

• Ethical Statement: Although ethical approval is waived, clarify how animal welfare was ensured during sample collection.
• Data Accessibility: GenBank accession numbers are provided, but raw sequencing data (e.g., chromatograms) are not mentioned. Confirm availability upon request.

10. Conclusion

Impact Statement: The conclusion does not emphasize how this study advances the field. Highlight the importance of monitoring Giardia in captive wildlife for zoonotic surveillance.

• Recommendation: Major Revisions required to address methodological limitations, improve data presentation, and strengthen contextual relevance.

 

Author Response

We want to thank the reviewer for his/her critical comments that will help improve the quality of the manuscript. In the following lines, we response to his/her comments.

Comment 1. Title and Abstract

The title is accurate but overly detailed. Consider simplifying by removing parenthetical family names (e.g., "Family Erethizontidae") to enhance readability.

Response 1: Following this and the next comment, and also having into consideration comments by reviewer #2, we have modified the title as follows:

Genetic identification of parasitic Giardia enterica in three wild rodent species from a zoological institution: first host records in Brazilian porcupine (Coendou prehensilis) and naked mole rat (Heterocephalus glaber), and detection in crested porcupine (Hystrix cristata)

Comment 2: Abstract: The phrase "understudied rodent hosts" is vague. Specify that Brazilian porcupines and naked mole rats are previously unreported hosts for Giardia enterica. The abstract lacks quantitative results (e.g., sequence similarity percentages, sample sizes).

Response 2: We have modified the title (see Response 1) to address this comment. Regarding the abstract, and taking into account the comments from Reviewer 2 as well, we have added the sequence similarity percentages (line 26) and noted that the prevalence values reported in this study cannot be considered reliable due to the small size of the animal groups (lines 26-27).

Comment 3: Introduction

Taxonomic Overload: Excessive focus on rodent systematics (e.g., suborder classifications) distracts from the core focus on Giardia. Condense or move non-essential details to supplementary material.

Response 3: “Rodents” represent a highly diverse group of mammals that can be infected by several Giardia species; however, the particular species involved varied depending on the rodent group. As indicated in the manuscript (both in the main text and in table 1), species of the G. duodenalis complex (G. duodenalis, G. intestinalis, and G. enterica) have been reported across all rodent orders. In contrast, other Giardia species found in rodents appear to be restricted to a single suborder within Supramyomorpha. For readers unfamiliar with rodent taxonomy, we believe that a brief introductory paragraph of eight lines is not excessive and provides a helpful overview of rodent groupings. It is important to emphasize that the main objective of this manuscript is not solely focused on Giardia, but rather on Giardia infections in three rodent species housed in a zoological institution. Therefore, both the parasite and the hosts are central to the study. Readers of the journal may include parasitologists (who may benefit from understanding the taxonomic position of the hosts) and mammalogists, who may appreciate the background on Giardia taxonomy. We believe that retaining the introductory paragraph does not detract from the main objective of the manuscript, and we would kindly ask the reviewer to consider accepting its inclusion.

In addition, we have corrected Table 1 as the rodent classification used to organize the data in the table was previously presented incorrectly.

Comment 4: Objective Clarity: The novelty of studying Brazilian porcupines and naked mole rats is underemphasized. Explicitly state that this is the first genetic characterization of Giardia in these species.

Response 4: We have modified the title to emphasize this is the first report of Giardia in Brazilian porcupines and African naked mole rats. We have also made changes to the abstract, in accordance with comments from other reviewers, and have added (lines 28-29) the statement: “this is the first report identifying Giardia (G. enterica) in C. prehensilis and H. glaber”. Similarly, we have revised the introduction (lines 118-121) to state: “The aim of the present study is to genetically identify Giardia isolates from these hosts. To our knowledge, this is the first time that Giardia has been genetically identified to the species level in the Brazilian porcupine and the African naked mole rats.” This point is reiterated in lines 364-368, followed by an explanation for not performing cyst per gram counts (see response to comment 8): “the objective [of this study] was not to assess infection intensity or prevalence, but rather to achieve molecular identification of Giardia isolates from zoo-housed rodent species that had not previously been genetically characterized. Accordingly, no attempts were made to quantify cysts in fecal samples, and as previously discussed, reported prevalence values may be biased.”

Comment 5: Contextual Gaps: Limited discussion on why studying captive zoo animals is relevant to understanding zoonotic transmission risks.

Response 5: We added a new paragraph addressing the importance of studying captive animals in relation to zoonotic transmission risks (lines 70-80):

“Zoological institutions maintain a wide range of wild species under human supervision and care, facilitating closer contact between animals, zoo personnel, and visitors. These conditions can favor cross-species transmission of pathogens, particularly for parasites with direct life cycles, such as Giardia [8]. Monitoring gastrointestinal parasites in captive wildlife is therefore essential, not only for ensuring animal health but also for assessing zoonotic risks and preventing potential outbreaks [9]. This is especially relevant in zoological institutions, where the possibility of reverse zoonosis [9,10] may affect the health of captive populations and potentially impact wild populations if infect animals are released for reintroduction in the wild. Despite the importance of these issues, genetic characterization of Giardia isolates from captive zoo animals remains limited, particularly among lesser-studied rodent groups.”

Comment 6: Materials and Methods

Sample Size Justification: The small sample size (2 porcupines per species, pooled mole rat feces) is a critical limitation. Justify why this is sufficient for genetic characterization or acknowledge potential biases.

Response 6: Please note that this is not an epidemiological study aimed at estimating prevalence (i.e., a quantitative approach), but rather a qualitative study focused on the genetic identification of Giardia species infecting three rodent host species. Therefore, the number of individuals sampled is less critical than the fact that they tested positive repeatedly over time (except for naked mole rats, for which no additional positive samples were detected), suggesting consistent infection within the groups. The main limitation lies in the fact that only one sample per species was genetically analyzed, despite multiple positive detections over time. This leaves open the possibility that other Giardia species may have been present. However, even if such diversity existed and only one was detected, this does not diminish the relevance of our findings, which represent the first confirmed genetic identification of Giardia in Brazilian porcupines and naked mole rats and provide valuable reference data for future studies.

We added a new paragraph (lines 362-376) to justify the results are valid despite the small number of host individuals analyzed: “Although the number of animals in the groups analyzed in this study is small, the objective was not to assess infection intensity or prevalence, but rather to achieve molecular identification of Giardia isolates from zoo-housed rodent species that had not previously been genetically characterized. Accordingly, no attempts were made to quantify cysts in fecal samples, and as previously discussed, reported prevalence values may be biased. Notably, in both porcupine species, each group tested positive for Giardia on several occasions over time (although not always in the same individual), suggesting persistent infection within the group. Although only one positive sample per species was subjected to molecular analysis, and the possibility of additional genotypes circulating over time cannot be excluded, the sequence obtained from the crested porcupine matches those previously identified in wild individuals [17,18,30]. In contrast, the sequences from the isolates of Brazilian porcupine and naked mole rat represent the first available genetic data for Giardia in these host species. These findings provide a valuable reference for future comparative and epidemiological studies involving both captive and wild populations.”

Comment 7: PCR Protocol Rationale: No explanation for selecting SSU-rRNA, bg, tpi, and gdh genes. Clarify why these markers were prioritized over others (e.g., *SSU-rRNA* failure rate).

Response 7: The genetic markers used in this study (bg, gdh, and tpi) are those routinely applied for the characterization and identification of Giardia isolates (Capewell et al., Trends Parasitol. 2021, 37, 142-153; doi:10.1016/j.pt.2020.09.013). Other markers, such as elongation factor 1 alpha (ef-1α) (Monis et al., Mol. Biol. Evol. 1999, 16, 1135-1144; doi:10.1093/oxfordjournals.molbev.a026204) and the ribosomal ITS region (Beck et al., Vet. Parasitol. 2011, 175, 40-46; doi:10.1016/j.vetpar.2010.09.026), have also been used occasionally. Additional markers, such as microsatellites or repetitive DNA sequences, may be valid but appear to be underrepresented in the genome (Capewell et al., 2021).

Based on the available literature, we selected the commonly used bg, gdh, and tpi genes for genotyping and complemented this panel with the SSU rRNA gene to enhance isolate characterization. After obtaining the sequencing results, we initially considered omitting the SSU rRNA gene due to our failure to generate readable sequences, especially given that the results from the other three markers were clear and consistent. However, we ultimately decided to retain this marker in the manuscript because the analysis was performed and, importantly, negative results (such as unsuccessful sequencing) are often unpublished but can be valuable for other researchers by helping them avoid similar pitfalls. The SSU rRNA gene is not mentioned in the abstract or keywords. Nevertheless, if the reviewer feels that all mentions of this marker (in the Materials and Methods, Results, and Discussion) should be removed, we are open to doing so and would include instead a brief note in the Discussion stating that sequencing of this gene was attempted unsuccessfully and was therefore excluded from further analysis.

To clarify why these specific gene markers were selected, we have added the following sentence to the manuscript (lines 161-164):
“Following Capewell et al. (2011) [44], partial fragments of four genes commonly used for Giardia genotyping were amplified by nested or semi-nested PCR and subsequently sequenced: small subunit rRNA (SSU-rRNA), beta giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh).”

We encountered no issues during the sequencing of bg, gdh, and tpi amplicons, and the results enabled clear identification and differentiation of the isolates. In contrast, chromatograms corresponding to the SSU rRNA amplicons showed low signal intensity and unreadable peak patterns. While we cannot definitively explain the failure to obtain readable sequences for this marker, the successful sequencing of the single-copy genes suggests that problems related to sample quality (such as low DNA concentration, poor DNA quality, or PCR inhibitors) can be excluded. It is possible that the issue arose from low DNA concentration in the PCR products used for sequencing, high salt content, or suboptimal sequencing conditions.

We have added the following paragraph (lines 320-334) to reflect this and explain why the SSU rRNA gene was not further pursued:
“The reasons for the failure to obtain readable sequences of the SSU-rRNA gene remain unclear. Nested or semi-nested PCR approaches are commonly employed to enhance the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low concentrations of target DNA, and PCR inhibitors [44]. In each case, the second-round PCR (nested or semi-nested) used a new set of internal primers and the product from the first round as a template, thereby minimizing the likelihood of non-specific amplification [33, 41, 43, 44]. Given that high-quality sequences were obtained for the other markers (which are single-copy genes), it is unlikely that the SSU rRNA gene amplification failure was due to low DNA concentration, poor DNA quality, or the presence of PCR inhibitors. It is therefore more plausible that the issue was related to amplicon purification or suboptimal sequencing conditions. Since the bg, gdh, and tpi markers yielded clear and informative sequences for genotyping, additional attempts to sequence the SSU-rRNA gene were not pursued, particularly in light of its lower discriminatory power for differentiating assemblages or species within the G. duodenalis complex [44].”

Comment 8: Microscopy Details: Insufficient information on cyst quantification (e.g., cysts per gram of feces).

Response 8: This study was not designed to assess infection intensity or prevalence, but rather to achieve the genetic identification of Giardia isolates from zoo-housed rodent species that had not been previously characterized at the molecular level. For this reason, no attempt was made to quantify cysts in fecal samples. This is now stated in the text (lines 362-367):” the objective [of this study] was not to assess infection intensity or prevalence, but rather to achieve molecular identification of Giardia isolates from zoo-housed rodent species that had not previously been genetically characterized. Accordingly, no attempts were made to quantify cysts in fecal samples, and as previously discussed, reported prevalence values may be biased.”

Moreover, in our view, cyst quantification has limited value in protozoan infections such as Giardia. Unlike helminths, in which egg counts reflect the reproductive output of established adult stages, encystation in protozoa involves the elimination of trophozoites from the intestine in a process that is influenced by host-dependent physiological factors. As a result, cyst counts in feces do not reliably reflect the actual protozoan intestinal burden. High cyst output may reflect active clearance by the host rather than a high number of trophozoites remaining in the intestine, whereas low cyst counts do not preclude the presence of substantial trophozoite populations.

Comment 9: Results

SSU-rRNA Failure: The illegible SSU-rRNA sequences are mentioned but not critically discussed. Address potential causes (e.g., primer mismatches, PCR inhibitors).

Response 9: As noted in Response 7, we are unable to determine the reason for the failure to obtain readable sequences for the SSU rRNA gene. However, the other markers analyzed (bg, gdh, and tpi) were successfully sequenced, which rules out sample-related issues such as the presence of PCR inhibitors or a low number of cysts, as well as problems related to DNA extraction (e.g., low DNA concentration or poor DNA quality). While it would have been possible to repeat the analysis to attempt recovery of the SSU rRNA sequences, this was deemed unnecessary given the clarity and consistency of the results obtained from the other markers.

We have added the following text to the manuscript to reflect this (lines 324–335):
“The reasons for the failure to obtain readable sequences of the SSU-rRNA gene remain unclear. Nested or semi-nested PCR approaches are commonly employed to enhance the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low concentrations of target DNA, and PCR inhibitors [44]. In each case, the second-round PCR (nested or semi-nested) used a new set of internal primers and the product from the first round as a template, thereby minimizing the likelihood of non-specific amplification [36, 45, 47, 48]. Given that high-quality sequences were obtained for the other markers (which are single-copy genes), it is unlikely that the SSU rRNA gene amplification failure was due to low DNA concentration, poor DNA quality, or the presence of PCR inhibitors. It is therefore more plausible that the issue was related to amplicon purification or suboptimal sequencing conditions. Since the bg, gdh, and tpi markers yielded clear and informative sequences for genotyping, additional attempts to sequence the SSU-rRNA gene were not pursued, particularly in light of its lower discriminatory power for differentiating assemblages or species within the G. duodenalis complex [44].”

Comment 10: Data Presentation:

Tables 2–4 are redundant with supplementary tables. Summarize key polymorphisms in the main text and relegate full alignments to supplements.

Response 10: We believe that this comment and Comment 19 are somewhat contradictory. The full alignments are provided as supplementary material, as indicated, and the key polymorphisms are summarized in Tables 2-4, following the suggestion made in Comment 19. In our opinion, removing Tables 2-4 and instead describing all polymorphisms solely in the main text would make the information harder to follow and less accessible. For comparison, consider the clarity of the tables versus the following text version:

“The comparison between sequences obtained from the different host species revealed that they were not identical, and specific base differences were observed across all three molecular markers analyzed (bg, tpi, and gdh). In the bg alignment (Supplementary File S1), all three isolates differed from the reference G. enterica sequence (ACGJ01002392) at position 97 (G instead of R) and position 232 (C instead of T). In addition, the Brazilian porcupine and crested porcupine sequences showed a T at position 301, whereas the naked mole rat matched the reference with a C at this position.
The tpi marker showed more extensive divergence (Supplementary File S2). The Brazilian porcupine isolate differed from the reference sequences at two positions (76 and 387), while the crested porcupine exhibited notable divergence at eight positions (24, 30, 76, 153, 161, 195, 256, 265, and 282), suggesting it represents a more distinct variant. The naked mole rat sequence differed by a single base at position 519.
In the gdh gene alignment (Supplementary File S3), the Brazilian porcupine isolate showed changes at positions 120 (T vs. C), 405 (T vs. C), and 429 (Y vs. T). The crested porcupine also exhibited differences at positions 309 (A vs. C) and 429 (Y vs. T), while the naked mole rat differed at positions 333 (T vs. C) and 429 (Y vs. T). Notably, the ambiguous base Y (C/T) at position 429 was shared by all three isolates and may indicate either a true polymorphism or the presence of mixed base calls at this site.”

We kindly ask the reviewer to accept the current presentation of Tables 2-4, which we believe provide a clearer and more accessible summary of the key polymorphisms across the different gene markers.

Comment 11: Phylogenetic trees (Figure 2, S1–S3) lack clarity. Include bootstrap values for all major nodes in the main figure.

Response 11: Bootstrap values were already included at the nodes in all figures (both in the condensed tree of the bg gene presented in the main text as Figure 2, and in the full trees provided as supplementary files). We acknowledge that the quality of Figure 2 could be improved; however, if the manuscript is accepted for publication, higher-resolution images will be provided as required by the journal. Please note that embedding high-quality images in a text document can significantly increase the file size.

We have revised the phylogenetic trees to increase the font size of the bootstrap values for improved readability.

Comment 12: GenBank Accessions: Accession numbers for sequences are listed but not hyperlinked or formatted per journal guidelines.

Response 12: It is standard practice to submit sequences to GenBank and keep them in reserved status until the associated publication is accepted. In accordance with the journal’s guidelines, accession numbers are provided in the manuscript text and, where appropriate, in tables and figures. These sequences will be made publicly available upon acceptance of the paper. There is no requirement for the sequences to be freely accessible at the time of manuscript submission. Once released, the sequences will be hyperlinked in the published version of the article.

Comment 13: Discussion

Epidemiological Speculation: The suggestion of different transmission routes for distinct G. enterica variants is unsupported by data. Remove speculative claims or frame them as hypotheses for future work.

Response 13: We agree with the reviewer that the original statement was not adequately supported. We acknowledge that we did not express our intended meaning clearly, and the term "transmission route" was incorrect in this context. A more accurate term would be "transmission event." The word "route" may imply specific modes of transmission (e.g., via handling, food, water, soil, or passive vectors such as insects), and as the reviewer correctly pointed out, no data are currently available to identify the actual route(s) of transmission in this case.

As shown in the main text, tables, and figures, all Giardia isolates obtained from crested porcupine, Brazilian porcupine, and naked mole rats were identified as G. enterica. However, sequence differences across the three genetic markers (bg, tpi, and gdh) indicate that the isolates are genetically distinct. If all isolates had been identical, it could have suggested a single introduction of the parasite into the zoo, followed by subsequent transmission among host species. In contrast, the observed genetic differences, together with the fact that the infections occurred at different times, support the interpretation that separate transmission events likely occurred. We have revised the text accordingly (lines 378 and 388).

Comment 14: Zoonotic Implications: The public health relevance of finding G. enterica (a human pathogen) in zoo animals is underdeveloped. Discuss potential risks to zookeepers/visitors.

Response 14: In response to the reviewer’s comment, we have addressed the potential risks to zookeepers and visitors in the revised text (lines 390-396): “Transmission of the parasite from these animals to humans can be considered unlikely in the current zoo setting, as the animals are housed in isolated enclosures with minimal human contact. Only trained zookeepers enter the enclosures, and they do not directly interact with the animals except during veterinary check-ups. Enclosure cleaning is performed according to established protocols, and fecal material is disposed of under controlled conditions. Nevertheless, transmission risks cannot be completely excluded, and zoo animal-human transmission has been documented in other institutions [60].”

Comment 15: Comparison with Prior Studies: Contrasting findings with Italian wild porcupines [14,15] lacks depth. Elaborate on how captive vs. wild environments might influence genotype diversity.

Response 15: We have revised the text as follows (lines 341-354): “In the case of the crested porcupine, the sequences differed from those previously reported in wild animals from Italy [17, 18]. In natural environments, crested porcupines are exposed to a broad range of sympatric wildlife, including other reservoirs, and seasonal variation in habitat use and diet, all of which may influence both the risk of exposure and the selective pressures acting on circulating parasite strains. In contrast, zoo environments are characterized by increased host density, greater contact with humans (caretakers, visitors), artificial diets, reduced environmental microbial diversity, and potential cross-species transmission from other captive animals or contaminated sources. Such conditions may favor the establishment or persistence of different Giardia species and subspecific variants genotypes, or may facilitate the introduction and maintenance of variants not typically encountered in natural habitats. The present findings support this hypothesis and underscores the potential for captive environments to shape or concentrate specific parasite variants/genotypes of zoonotic relevance.”

Comment 16: References

Inconsistent Formatting: Some journal names are abbreviated (e.g., Vet. Parasitol.), others are spelled out (e.g., International Journal of Parasitology). Standardize per journal guidelines.

Response 16: We have revised the bibliography according to the journal guidelines.

Comment 17: Missing Citations: References for Table 1 (e.g., [16–19]) are incomplete or misaligned with the table entries (e.g., prairie dog entry cites [19], but [19] discusses Giardia in chipmunks).

Response 17: We have revised the references listed in Table 1. Please note that, in the revised version of the manuscript, the numbering has changed due to the inclusion of new references. To improve readability, we have also added dotted lines to separate each horizontal entry.

Comment 18: Figures and Tables

Figure 1: Image quality is unclear in the provided text. Ensure high-resolution images with scale bars in the final version.

Response 18: As noted in our response to Comment 11, we acknowledge that the quality of the figures included in the manuscript can be improved. If the manuscript is accepted for publication, high-resolution images will be provided upon request by the journal. Please note that embedding high-quality images in a text document can significantly increase the file size.

Comment 19: Supplementary Material: Critical data (full alignments, phylogenetic trees) are relegated to supplements. Include key supplementary figures/tables in the main text to support conclusions.

Response 19: Please also refer to our response to Comment 10. We have included a condensed version of the alignments in the main text, highlighting the key differences among sequences. For the phylogenetic analysis, we selected one gene (bg) as representative, since the results were consistent across all three markers, with the sequences from both porcupine species and the naked mole rat consistently clustering within the G. enterica group.

The full phylogenetic trees are large, as they include all published Giardia sequences from rodent hosts, and would occupy substantial space if included in the main text. For this reason, we chose to include one representative tree (bg), shown in a reduced format with branches corresponding to Giardia species other than G. enterica collapsed to improve readability. We believe that the main text contains all the necessary information to support the conclusions presented.

Comment 20: General Writing and Structure Repetition: The taxonomy of G. duodenalis sensu lato is redundantly explained in the Introduction and Discussion. Consolidate.

Response 20: The taxonomy of G. duodenalis sensu lato is explained in the Introduction, where we also indicate that, throughout the manuscript (i.e., in the text, tables, and figures), we refer to the species names rather than to the assemblages. However, in the Discussion, we include the former assemblage designations in two instances (lines 335 and 337) to facilitate comparisons with previously published studies that use assemblage nomenclature. While we prefer to retain these clarifications, we are happy to remove them if the reviewer considers them unnecessary or redundant.

Comment 21: Typos:

"rutinary analysis" → "routine analysis" (p. 2).

"manufactorer’s instructions" → "manufacturer’s instructions" (p. 5).

Abbreviations: Define all abbreviations (e.g., SSU-rDNA, bg) at first mention.

Response 21: We have revised the entire manuscript to improve the use of English, and numerous changes and corrections have been made throughout the text. In addition to addressing grammar and typographical issues, all abbreviations are defined at first mention and listed in a dedicated section at the end of the manuscript, prior to the references’ section.

Comment 22: Ethical and Data Availability

Ethical Statement: Although ethical approval is waived, clarify how animal welfare was ensured during sample collection.

Response 22: The ethical statement has been revised as follows: “Ethical approval was not required for this study. Fecal samples were collected non-invasively from the ground in the animals’ enclosures, as part of routine monitoring procedures conducted by the zoo veterinary staff. No animal was captured, handled, or subjected to any intervention for the purposes of this research. All procedures complied with the park’s institutional guidelines for animal welfare, ensuring minimal disturbance to the animals throughout the process.”

Comment 23: Data Accessibility: GenBank accession numbers are provided, but raw sequencing data (e.g., chromatograms) are not mentioned. Confirm availability upon request.

Response 23: We did not originally include a statement on this matter, as we considered it unnecessary. In our view, the availability of raw data is essential when different analytical approaches or interpretations can be applied to it (for example, morphometric datasets that can be subjected to a range of unidimensional and multidimensional analyses). However, data such as gel images showing a single band, or ELISA readings with predefined thresholds, typically do not require publication or public sharing, unless there are concerns regarding the integrity of the data or research process(something we trust is not considered the case here). The same rationale applies to chromatograms: a peak corresponding to an 'A' is simply a peak corresponding to an 'A', with little room for alternative interpretation. That said, we are fully willing to share the chromatograms obtained in this study upon reasonable request by other researchers. Accordingly, we have modified (lines 447-450) the Data Availability Statement as follows: “The original contributions presented in this study are included in the article, in the main text or as supplementary material. DNA sequences have been deposited in the GenBank/EMBL/DDBJ repositories under accession numbers PV391923-PV391931. Further inquiries about data supporting the conclusions of this article can be directed at the corresponding author.”

Comment 24: Conclusion

Impact Statement: The conclusion does not emphasize how this study advances the field. Highlight the importance of monitoring Giardia in captive wildlife for zoonotic surveillance.

Response 24: We have added a conclusion section (lines 409-423): “This study provides the first genetic identification of G. enterica in the Brazilian porcupine and the naked mole rat, and confirms the presence of this parasite in captive individuals of the crested porcupine. Although all isolates were identified as G. enterica through three independent genetic markers (tpi, gdh, and bg), the sequences differed among host species and from sequences available in GenBank, suggesting independent infection events and previously undescribed variants. These findings support the hypothesis that captive environments may influence the circulation and selection of specific Giardia genotypes, which may differ from those found in wild populations.

No clinical signs of giardiasis were observed in infected individuals, and in both porcupine species, repeated positive results over time suggest persistent colonization. Given the zoonotic potential of G. enterica, ongoing surveillance in both captive and wild rodent populations is warranted, particularly to clarify transmission dynamics and evaluate the role of these hosts as reservoirs. The genetic data generated in this study provide a valuable reference for future epidemiological, comparative, and taxonomic research on Giardia in porcupines and mole rats.”

 

Comment 25: Recommendation: Major Revisions required to address methodological limitations, improve data presentation, and strengthen contextual relevance.

Response 25: We sincerely thank the reviewer for the time and effort dedicated to reviewing this manuscript. The comments have significantly improved the clarity and overall quality of the work. We have addressed most of the points raised by making the requested modifications, while in a few instances, we have provided a reasoned justification for maintaining a different perspective.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Genetic identification of Giardia isolates from understudied rodent hosts: Brazilian porcupine, Coendou prehensilis (Family Erethizontidae); crested porcupine, Hystrix cristata (Family Hystricidae); and naked mole rat, Heterocephalus glaber (Family Heterocephalidae)” submitted to the Special Issue

The Long-Standing Problem of Parasitic Diseases in Zoo Animals: Current Challenges and Searching for Solutions

of the journal Journal of Zoological and Botanical Gardens.

 

Basic remarks

• The subject matter presented in the Manuscript corresponds to the subject areas of the JZBG and is particularly well fit to its special issue.
• Title should be corrected by excluding unnecessary punctuation and the words "Family" (Latin names are self-explanatory for biologists), but adding the epithet "parasitic" to the Giardia isolates.

Suggested title:

Genetic identification of parasitic Giardia isolates from rodent hosts: Brazilian porcupine Coendou prehensilis (Erethizontidae), crested porcupine Hystrix cristata (Hystricidae), and naked mole rat Heterocephalus glaber (Heterocephalidae)

• Abstract: remove redundant information (see attached pdf for proposed changes).
• Introduction provides some information about rodents, their biology and distribution, as well as possible contacts with humans, which is important for understanding their transmission of intestinal parasites; it also provides a description of the parasitic protozoa that affect rodents and details of their taxonomy, which explains for the need for additional research.

In zoological nomenclature, it is advisable to provide full Latin names of genera and species, including author and year, when they are first mentioned in the text. Subsequently, the generic name of the species should be abbreviated if the context makes it clear which genus is being referred to.

The minor changes I have suggested can be found in the attached pdf (lines 37, 50, 51, 62, 73, 74, 79, 97, and 107).

• Materials and Methods are described in sufficient detail, perhaps even too much detail.

The first paragraph (lines 112-117) is somewhat confusing. It is unclear what samples are being discussed: those studied in this work or some others? Please explain this more clearly.

Line 126: there is a reference that is incorrectly formatted and is not in the list of references.

Lines 138, 141, 167, and 174: PCR is an abbreviation for polymerase chain reaction, so the additional word reaction is not needed.

Line 139: What did you mean by "Fragments of four genes were partially amplified"?

Paragraphs 4-7 (147-176): Please explain to the readers why it was necessary to conduct PCR in two stages? Was there a second stage, the so-called re-PCR using the amplicons obtained in the first stage? There are many questions that require clarification.

Moreover, is it worth repeating the primer structures and PCR parameters if they are described in the relevant publications (references 33, 41-44) and you have not made any changes? In my opinion, the description of these procedures should be significantly shortened, but the general scheme of the experiment should be explained in more detail, as already stated above.

Last paragraph (186-196): It is unclear for what purpose the phylogenetic trees were reconstructed. To determine evolutionary relationships or to visualize species affiliation, which was determined using genetic distances? This point should be specified in the text.

• Results

Figure 1: Scale bars would be better to better to place in the lower right corner of each drawing.

Tables 2-4: I think that these tables should be removed and their contents easily explained in words. Otherwise, they clutter up the manuscript too much.

Tables 5-7 should be combined into one, which will make the material easier to understand.

	G. duodenalis	G. intestinalis	G. enterica-1	G. enterica-2
beta giardin
coendou
crested porcupine
naked mole rat
triose phosphate isomare
…
…
glutamate dehydrogenase
…

• Discussion contains many inaccuracies.

Line 272: Despite it… - change “it” to name of a parasite to make clear meaning.

Line 276: … due to reduced enclosure sizes change to “due to the limited life space”

Lines 278-279, 282: 50 % at the moment of positive samples – what did you mean?

Line 294: comma instead of semicolon

Line 295: need square bracket [ instead of round bracket (

Lines: 296-300: The text needs some correction. The English may not be correct, so the meaning is lost.

Line 303: use word morphology.

Line 304: organism? Did you mean parasite?

Lines 310-311: You should clarify whose unpublished data you are referring to.

Line 311: mole rats can not be negative. Avoid scientific jargon.

Line 313: see in attached pdf.

Line 315-316: So, you allow the transfer from humans to rodents (line 313), but not the reverse? Explain why.

Line 320: It is not clear to whom "their" refers in this context?

Line 320-322:  Lost of meaning.

• Conclusions are absent, highly desirable. They should summarize the results and clearly demonstrate new scientific information obtained during the study.
• Data Availability Statement: Here you are required to provide links to specific data, rather than sending readers to scour the text and the Internet.
• There are some recurring inaccuracies in the formatting of bibliography. Follow the MDPI Reference List and Citations Style Guide (https://www.mdpi.com/authors/references). For highlighted errors, see the attached PDF.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Check the English throughout the manuscript, paying particular attention to the Discussion.

Author Response

The manuscript “Genetic identification of Giardia isolates from understudied rodent hosts: Brazilian porcupine, Coendou prehensilis (Family Erethizontidae); crested porcupine, Hystrix cristata (Family Hystricidae); and naked mole rat, Heterocephalus glaber (Family Heterocephalidae)” submitted to the Special Issue

The Long-Standing Problem of Parasitic Diseases in Zoo Animals: Current Challenges and Searching for Solutions

of the journal Journal of Zoological and Botanical Gardens.

We would like to thank the reviewer for the time and effort dedicated to reviewing this manuscript. The comments have greatly improved the readability and significance of the work. We have implemented the requested changes in most cases, and in a few instances, we have provided a reasoned justification for maintaining an alternative perspective.

Comment 1: Basic remarks

The subject matter presented in the Manuscript corresponds to the subject areas of the JZBG and is particularly well fit to its special issue.

Title should be corrected by excluding unnecessary punctuation and the words "Family" (Latin names are self-explanatory for biologists), but adding the epithet "parasitic" to the Giardia isolates.

Suggested title:

Genetic identification of parasitic Giardia isolates from rodent hosts: Brazilian porcupine Coendou prehensilis (Erethizontidae), crested porcupine Hystrix cristata (Hystricidae), and naked mole rat Heterocephalus glaber (Heterocephalidae)

Response 1: In response to this comment, and also taking into consideration the suggestions made by reviewer #1, we have revised the title as follows:

Genetic identification of parasitic Giardia enterica in three wild rodent species from a zoological institution: first host records in Brazilian porcupine (Coendou prehensilis) and naked mole rat (Heterocephalus glaber), and detection in crested porcupine (Hystrix cristata).

Comment 2: Abstract: remove redundant information (see attached pdf for proposed changes).

Response 2: In response to this comment, as well as the suggestions provided by Reviewer 1, we have extensively revised the abstract and removed the sections marked in the PDF supplied by the reviewer.

Comment 3: Introduction provides some information about rodents, their biology and distribution, as well as possible contacts with humans, which is important for understanding their transmission of intestinal parasites; it also provides a description of the parasitic protozoa that affect rodents and details of their taxonomy, which explains for the need for additional research.

In zoological nomenclature, it is advisable to provide full Latin names of genera and species, including author and year, when they are first mentioned in the text. Subsequently, the generic name of the species should be abbreviated if the context makes it clear which genus is being referred to.

Response 3: The journal guidelines do not indicate that the use of the complete binomial with author and year is required in the main text. While we agree that this system is appropriate (and even necessary) in taxonomically focused papers, such as those dealing with the validity or reclassification of taxa or the description of new species or genera, we believe it is not essential for a study such as the present one, where the taxonomic authority and year of description are not relevant to the objectives.

Moreover, including the author and year would create space constraints in Table 1, potentially affecting readability. For practical reasons, we have followed the commonly accepted convention of using only the binomial genus and species name. We kindly ask the reviewer to accept the use of this format in the manuscript.

Comment 4: The minor changes I have suggested can be found in the attached pdf (lines 37, 50, 51, 62, 73, 74, 79, 97, and 107).

Response 4: We have taken into consideration the suggestions made by the reviewer (line numbers refer to those in the revised manuscript):

• In line 37, we have replaced “extant” in the original manuscript by “currently living”; the reviewer suggested “currently known”, but we think this could be interpreted as including extinct (fossil) known species.
• In lines 51, 57-58 and 63, the format suggested by the reviewer (e.g., “The Old World porcupines belonging to the family Hystricidae”) may, in our opinion, imply that only a subset of Old World porcupines belongs to that family. Although there is no other family that includes Old World porcupines, readers unfamiliar with porcupine taxonomy may interpret the phrase as referring to a subgroup of Old World (or, ahead, New World) porcupines. This ambiguity can be avoided by using commas (e.g., “The Old World porcupines, belonging to the family Hystricidae, are …”) or parentheses. We have chosen this structure to maintain consistency with how species names are presented elsewhere in the text (i.e., the common name is given in the main text, with the Latin binomial in parentheses, as in lines 52 or 85-87).
• We have revised lines 62-64 regarding mole rats as follows: “Respect to the mole rats, this term refers to two families: Heterocephalidae (which includes a single genus and species, the naked mole rat Heterocephalus graber) and Bathyergidae.”
• Regarding species names in the text, we use the full genus name when it appears at the beginning of a sentence (since a sentence should not begin with an abbreviation or a numeral), or when a species is mentioned for the first time, regardless of whether other species of the same genus have already been cited. For this reason, in lines 85-87 and 91, we have retained the full genus names for the species as they were first introduced.
• In line 110, we believe that reintroducing “parasitic flagellates” is unnecessary, as it has clearly stated in lines 83-84 that Giardia species are flagellate parasitic protozoa.
• In table 1, we have removed the abbreviations for order and suborders, as suggested. The table has also been revised to correct the taxonomic classification of rodents, which was used to organize the data but was previously inaccurately presented.

Comment 5: Materials and Methods are described in sufficient detail, perhaps even too much detail.

Response 5: We agree with the reviewer that this section could be shortened by omitting the detailed PCR protocols, as we have followed previously published methods and appropriate citations are provided. However, based on our recent experience with two other submissions, when methodological details are summarized too briefly, reviewers often request that the protocols be described “briefly” within the manuscript. Ultimately, including a moderate level of detail (along with the relevant references) offers a practical benefit to readers by allowing them to understand and replicate the methods without needing to consult additional sources or search through external databases. For this reason, we kindly ask that the current level of detail be retained, as it may facilitate reproducibility and accessibility for other researchers.

Comment 6: The first paragraph (lines 112-117) is somewhat confusing. It is unclear what samples are being discussed: those studied in this work or some others? Please explain this more clearly.

Response 6: We have modified the text (lines 131-134) to clarify that the samples used in this study were obtained from the Faunia zoological park in 2024: “The present study is based on Giardia-positive fecal samples collected in 2024 from crested porcupines, Brazilian porcupines, and naked mole rats housed at Faunia, a zoological park located in Madrid (Spain). These are the samples now analyzed to achieve the genetic identification of Giardia isolates in these host species.”

Comment 7: Line 126: there is a reference that is incorrectly formatted and is not in the list of references.

Response 7: The reviewer is correct. The reference was included in the bibliography as number 40, but for an unknown reason, the author’s name and date was retained in the text instead of the reference number. After incorporating additional references, it is now listed as reference number 43 and has been correctly cited in the text.

Comment 8: Lines 138, 141, 167, and 174: PCR is an abbreviation for polymerase chain reaction, so the additional word reaction is not needed.

Response 8: The reviewer is correct; although the expression “PCR reaction” is commonly used, it is indeed a redundant use of the word “reaction”. We have replaced “reaction” with “amplification” where appropriate throughout the manuscript.

Comment 9: Line 139: What did you mean by "Fragments of four genes were partially amplified"?

Response 9: We expressed this incorrectly. What we actually did was amplify a fragment of each of the genes. Including responses to other reviewer’s comments, the correct phrasing should be: “partial fragments of four genes commonly used for Giardia genotyping were amplified …”. The text has been corrected accordingly (lines 161-162).

Comment 10: Paragraphs 4-7 (147-176): Please explain to the readers why it was necessary to conduct PCR in two stages? Was there a second stage, the so-called re-PCR using the amplicons obtained in the first stage? There are many questions that require clarification.

Response 10: The paragraph has been slightly revised in response to comments from other reviewers, but the description of the protocol has been essentially maintained. As stated in the text (line 161) and demonstrated in the descriptions of the individual protocols for each marker (which are properly referenced: references 36, 45-48), we did not develop new protocols but rather followed those established by other authors, who have also discussed their problems and advantages. These two-step PCR approaches are commonly employed to increase the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low amounts of target DNA and potential PCR inhibitors. In each case, the second-round PCR (nested or semi-nested) uses a new set of internal primers and the product of the first round as a template, thereby reducing the likelihood of non-specific amplification. We have added the following text (lines 321-326) to explain the rationale for using a two-step PCR protocol: “Nested or semi-nested PCR approaches are commonly employed to enhance the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low concentrations of target DNA, and PCR inhibitors [44]. In each case, the second-round PCR (nested or semi-nested) used a new set of internal primers and the product from the first round as a template, thereby minimizing the likelihood of non-specific amplification [36, 45, 47, 48].”

Comment 11: Moreover, is it worth repeating the primer structures and PCR parameters if they are described in the relevant publications (references 33, 41-44) and you have not made any changes? In my opinion, the description of these procedures should be significantly shortened, but the general scheme of the experiment should be explained in more detail, as already stated above.

Response 11: As explained in Response 5, we believe it is worthwhile retaining the detailed protocols in the manuscript, and we kindly ask the reviewer to consider maintaining them.

In Response 10, we clarified that the overall analytical scheme follows the standard methodology commonly used by researchers investigating the genetic variability, species diversity, or molecular epidemiology of Giardia. There is nothing unusual in the general structure or in the protocols themselves (aside from the use of two-step PCRs), and in our view, the study design is clearly outlined in the Materials and Methods section: selection of positive samples following microscopic analysis, DNA extraction, PCR amplification and sequencing, sequence analysis and comparison, and phylogenetic analysis (the value of which is further addressed in our response to Comment 12). To improve clarity, we have now included subheadings within the Materials and Methods section.

Comment 12: Last paragraph (186-196): It is unclear for what purpose the phylogenetic trees were reconstructed. To determine evolutionary relationships or to visualize species affiliation, which was determined using genetic distances? This point should be specified in the text.

Response 12: The objective of our study is to identify the species to which the rodent isolates belong, and this can be reliably achieved by comparing our sequences with those available in GenBank. A Neighbor-Joining tree provides a useful way to summarize all pairwise comparisons between our sequences and those from other rodent hosts. We have added the following text to the manuscript (lines 210-212): “Phylogenetic trees were constructed separately for each gene to enable comparative analyses between the sequences obtained in this study and those previously published from Giardia isolates in rodents.“

Comment 13: Results

Figure 1: Scale bars would be better to better to place in the lower right corner of each drawing.

Response 13: We have corrected the position of the scale bars.

Comment 14: Tables 2-4: I think that these tables should be removed and their contents easily explained in words. Otherwise, they clutter up the manuscript too much.

Response 14: In our opinion, removing tables 2-4 and presenting the polymorphisms solely in the text would make the information more difficult to follow and interpret. For example, compare the clarity and visual accessibility of the tables in the manuscript with the equivalent text:

“The comparison between sequences obtained from the different host species revealed that they were not identical, and specific base differences were observed across all three molecular markers analyzed (bg, tpi, and gdh). In the bg alignment (Supplementary File S1), all three isolates differed from the reference G. enterica sequence (ACGJ01002392) at position 97 (G instead of R) and position 232 (C instead of T). In addition, the Brazilian porcupine and crested porcupine sequences showed a T at position 301, whereas the naked mole rat matched the reference with a C at this position.

The tpi marker showed more extensive divergence (Supplementary File S2). The Brazilian porcupine isolate differed from the reference sequences in two positions (76 and 387), while the crested porcupine exhibited notable divergence at eight positions (24, 30, 76, 153, 161, 195, 256, 265, and 282), suggesting it represents a more distinct variant. The naked mole rat sequence was identical to the reference in all positions except 519, where it differed by a single base.

In the gdh gene alignment (Supplementary File S3), the Brazilian porcupine isolate showed changes at positions 120 (T vs. C), 405 (T vs. C), and 429 (Y vs. T). The crested porcupine also exhibited differences at positions 309 (A vs. C) and 429 (Y vs. T), while the naked mole rat differed at positions 333 (T vs. C) and 429 (Y vs. T). Notably, the ambiguous base Y (C/T) at position 429 was shared by all three isolates and may indicate either a true polymorphism or the presence of mixed base calls at this site.”

We kindly ask the reviewer to accept the current presentation of tables 2-4 as a concise and visually clear summary of the polymorphisms identified in the different gene markers. We believe this format facilitates a more effective understanding of the sequence differences.

Comment 15: Tables 5-7 should be combined into one, which will make the material easier to understand.

	G. duodenalis	G. intestinalis	G. enterica-1	G. enterica-2
beta giardin
Coendou
crested porcupine
naked mole rat
triose phosphate isomare
…
…
glutamate dehydrogenase
…

Response 15: In response to the reviewer’s comment, we have combined the three tables into a single table. However, it is important to note that each gene marker requires comparison with different reference sequences, which necessitates including a specific header row for each marker subgroup within the table. In our view, the only real advantage of combining the tables is the consolidation of their captions into a single legend. For clarity and ease of interpretation, we believe it would be preferable to keep each table separate.

Comment 16: Discussion contains many inaccuracies.

Line 272: Despite it… - change “it” to name of a parasite to make clear meaning.

Line 276: … due to reduced enclosure sizes change to “due to the limited life space”

Response 16: We have corrected the use of the English language thorough the text.

Comment 17: Lines 278-279, 282: 50 % at the moment of positive samples – what did you mean?

Response 17: We sampled the animals several times throughout the year, but positive samples were detected only during certain sampling events. In those positive events, only 50% of the animals in the group were shedding cysts; that is, prevalence was 50% during the sampling events in which the parasite was detected. We acknowledge the expression “at the moment of positive samples” was unclear and have revised it to “The observed prevalence in the present study (50% in each positive sampling event) …” (lines 305-306) and “the prevalence observed (21.4% of samples testing positive over the year, and 50% of positive animals in the positive sampling events) …” (lines 309-311).

Comment 18: Line 294: comma instead of semicolon

Line 295: need square bracket [ instead of round bracket (

Lines: 296-300: The text needs some correction. The English may not be correct, so the meaning is lost.

Line 303: use word morphology.

Response 18: We have revised the use of the English throughout the manuscript, and numerous changes have been made to improve clarity and readability.

Comment 19: Line 304: organism? Did you mean parasite?

Response 19: Yes, we meant “parasite”. We have changed the text.

Comment 20: Lines 310-311: You should clarify whose unpublished data you are referring to.

Response 20: We conduct parasitological analyses of animals from several zoological institutions on a weekly basis. The results from a 10-year period (2013-2022) for Faunia (the zoo from which the present samples were obtained) and a second institution were published in 2024 (ref. 11). In that study, we reported that crested porcupines were first found to be infected in 2020, while Brazilian porcupines consistently tested negative during that period. The first positive findings in Brazilian porcupines occurred in 2023, and these are the unpublished results to which we refer. The text has been updated to (lines 381-382): “(Esteban-Sánchez, de la Riva-Fraga, Pérez de Quadros, García-Rodríguez and Ponce-Gordo, unpublished results)”

Comment 21: Line 311: mole rats can not be negative. Avoid scientific jargon.

Response 21: We have changed the text (lines 382-383) to “All samples from naked mole rats had consistently tested negative”.

Comment 22: Line 313: see in attached pdf.

Response 22: Our intention was to differentiate between mechanical carriers (such as insects or contaminated footwear) and biological hosts that can become infected and actively transmit the parasite (e.g., small rodents, zookeepers). To clarify this distinction, we have rephrased the sentence as follows (lines 383-387): “It could be hypothesized that crested porcupines were initially infected and that the parasite was subsequently transmitted to other species through mechanical carriers (e.g., insects or fomites such as zookeepers’ footwear), through infected local microfauna (e.g. small rodents), or potentially via infected zookeepers.”

Comment 23: Line 315-316: So, you allow the transfer from humans to rodents (line 313), but not the reverse? Explain why.

Response 23: We have changed the text (lines 390-396) to: “Transmission of the parasite from these animals to humans can be considered unlikely in the current zoo setting, as the animals are housed in isolated enclosures with minimal human contact. Only trained zookeepers enter the enclosures, and they do not directly interact with the animals except during veterinary check-ups. Enclosure cleaning is performed according to established protocols, and fecal material is disposed of under controlled conditions. Nevertheless, transmission risks cannot be completely excluded, and zoo animal-human transmission has been documented in other institutions [60].”

Comment 24: Line 320: It is not clear to whom "their" refers in this context?

Response 24: The text has been changed to (lines 396-400): “In the present case, further investigation is needed within this zoological facility to clarify the potential routes of introduction and transmission of the parasite. Additionally, studies in porcupines and naked mole rats from other zoological institutions and wild populations are needed to better assess their potential role as zoonotic reservoirs of Giardia spp.”

Comment 25: Line 320-322:  Lost of meaning.

Response 25: We have changed the text as follows (lines 401-403): “While further research and future discoveries may reveal additional Giardia genotypes or species in previously unreported hosts, there appears to be an association between G. muris, G. microti, and G. cricetidarum and rodents of the suborder Myomorphi.”

Comment 26: Conclusions are absent, highly desirable. They should summarize the results and clearly demonstrate new scientific information obtained during the study.

Response 26: We have added a conclusion section with the following text (lines 409-423): “This study provides the first genetic identification of G. enterica in the Brazilian porcupine and the naked mole rat, and confirms the presence of this parasite in captive individuals of the crested porcupine. Although all isolates were identified as G. enterica through three independent genetic markers (tpi, gdh, and bg), the sequences differed among host species and from sequences available in GenBank, suggesting independent infection events and previously undescribed variants. These findings support the hypothesis that captive environments may influence the circulation and selection of specific Giardia genotypes, which may differ from those found in wild populations.

No clinical signs of giardiasis were observed in infected individuals, and in both porcupine species, repeated positive results over time suggest persistent colonization. Given the zoonotic potential of G. enterica, ongoing surveillance in both captive and wild rodent populations is warranted, particularly to clarify transmission dynamics and evaluate the role of these hosts as reservoirs. The genetic data generated in this study provide a valuable reference for future epidemiological, comparative, and taxonomic research on Giardia in porcupines and mole rats.”

Comment 27: Data Availability Statement: Here you are required to provide links to specific data, rather than sending readers to scour the text and the Internet.

Response 27: The data generated in this study are presented in the manuscript, in the supplemental files, or as sequences submitted to GenBank. The supplemental files have been uploaded to the journal’s submission system; if the manuscript is accepted, these files will be available in the journal’s website, with a download link provided by the journal. Accordingly, we have indicated this in the manuscript as: “Supporting information can be downloaded at: www.mdpi.com/xxx/s1”, with the placeholder ‘xxx’ to be replaced by the journal upon publication.

Regarding the sequence data, it is a standard practice to submit sequences to GenBank and keep them in reserved status until the corresponding publication is released. In accordance with the journal’s guidelines, accession numbers were provided in the text and, where appropriate, in tables and figures. The sequences will become publicly available once the manuscript is accepted for publication. There is no requirement for sequences to be publicly accessible at the time of manuscript submission.

Comment 28: There are some recurring inaccuracies in the formatting of bibliography. Follow the MDPI Reference List and Citations Style Guide (https://www.mdpi.com/authors/references). For highlighted errors, see the attached PDF.

Response 28: Bibliography has been revised according to journal guidelines.

Comment 29: Comments on the Quality of English Language

Check the English throughout the manuscript, paying particular attention to the Discussion.

Response 29: The use of English throughout the manuscript has been carefully revised, and corrections have been made where appropriate to improve clarity and readability. In this regard, we would like to thank the reviewer for his/her comments on both the form and content of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes the characterization of Giardia in Brazilian porcupines, Coendou prehensile, crested porcupines, Hystrix cristata, and naked mole rats, Heterocephalus glaber, from captive areas, using a molecular method. The result of this study can be the baseline of Giardia circulation in rodent species and further transmission between other species of rodents and humans, which is consistent with the one health concept.
However, this manuscript needs to be revised on these points, including:  
Major revision: 
1)    Please clarify the four specific PCR fragments that were targeted in this study. Although, SSU-rRNA gene was not successful in sequencing. 
2)    Please clarify why only the β-giardin gene was constructed in the NJ tree, and sequences in this study were identified as G. enterica, despite the β-giardin locus of G. duodenalis among human and animal isolates showing genetic heterogeneity.
Minor revision:
1)    Please add the aim of the study in the abstract.
2)    Line 91 …. assignments as G. duodenalis assemblage ‘X’. Please add a citation.
3)    Line 92 …. are considered the ethiological agents of human giardiasis. Please add a citation.
4)    Line 209: Please specify the cysts observed in the Figure caption.
5)    The ambiguity R (G/A) in table 2 was possibly not different from “G” in other sequences
6)    Tables 2, 3, 4, and Tables 5, 6, 7 can be described in the text with significant information, and all tables can be represented in the supplement section.
7)    Figure 2 showed only one targeting fragment, which is not consistent with the materials and methods section. Please clarify why the phylogenetic tree of this specific target was constructed.
8)    Accession no. of Giardia enterica (strain GS) in Table 2 and Figure 2 are different.

Author Response

This manuscript describes the characterization of Giardia in Brazilian porcupines, Coendou prehensile, crested porcupines, Hystrix cristata, and naked mole rats, Heterocephalus glaber, from captive areas, using a molecular method. The result of this study can be the baseline of Giardia circulation in rodent species and further transmission between other species of rodents and humans, which is consistent with the one health concept.

We sincerely thank the reviewer for the time and effort dedicated to reviewing this manuscript. Their comments have significantly improved the readability and overall significance of the work. We have made the requested modifications in most cases, while in a few instances we have provided reasoned arguments in support of a different perspective.

Comment 1: However, this manuscript needs to be revised on these points, including:  
Major revision: 
1) Please clarify the four specific PCR fragments that were targeted in this study. Although, SSU-rRNA gene was not successful in sequencing. 

Response 1: The markers used in this study (bg, gdh and tpi) are those routinely applied for the characterization and identification of Giardia isolates (Capewell et al. Trends Parasitol. 2021, 37, 142-153; doi 10.1016/j.pt.2020.09.013). Other markers, such as elongation factor 1 alpha (el-1a) (Monis et al. Mol. Biol. Evol. 1999, 16, 1135-1144; doi 10.1093/oxfordjournals.molbev.a026204) and the ribosomal ITS region (Beck et al. Vet. Parasitol. 2011, 175, 40-46, doi 10.1016/j.vetpar.2010.09.026) have also been used occasionally. Additional markers such as microsatellites and repeatitive DNA sequences could also be valid, but they appear to be underrepresented in the genome (Capewell et al. 2021).

Based on the literature, we selected the commonly used bg, gdh and tpi genes and included the SSU-rRNA gene to complete the panel for characterizing the isolates analyzed in this study. Once sequencing results were obtained, we initially considered omitting the SSU-rRNA gene from the manuscript because sequencing was unsuccessful and the results from the other markers were clear and consistent. However, we ultimately chose to include it as the analysis was performed, and negative results (such as the failure to obtain usable sequences) are rarely published but can be helpful to other researchers in avoiding similar pitfalls. This gene is not mentioned in the abstract or in the keywords. Nonetheless, if the reviewer believes that the information related to this marker (in the material and methods, results, and discussion) should be removed, we are willing to do so and would instead include a brief statement in the discussion indicating that amplification and sequencing of this gene was attempted but unsuccessful, and therefore not included in the analysis.

We have added the following text (lines (161-163) to clarify the rationale for the selection of these gene markers: “Following Capewell et al. (2011) [44], partial fragments of four genes commonly used for Giardia genotyping were amplified by nested or semi-nested PCR and subsequently sequenced: “

During sequencing, we encountered no issues with the bg, gdh, and tpi amplicons, and the results allowed clear identification and differentiation of the isolates. However, the chromatograms corresponding to the SSU-rRNA amplicons showed weak signals and poor resolution, with overlapping peaks that rendered the sequences unreadable. We were unable to determine the exact cause of this failure, but since the single-copy genes were successfully sequenced, we ruled out sample-related issues such as low DNA concentration, poor DNA quality, or the presence of PCR inhibitors. It is possible that the problem was due to low DNA concentration in the purified PCR product, excess salt content, or suboptimal sequencing conditions. We have added the following text to the manuscript (lines 321-335) to explain these observations and to clarify why we did not attempt to repeat the SSU-rRNA analysis: “The reasons for the failure to obtain readable sequences of the SSU-rRNA gene remain unclear. Nested or semi-nested PCR approaches are commonly employed to enhance the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low concentrations of target DNA, and PCR inhibitors [44]. In each case, the second-round PCR (nested or semi-nested) used a new set of internal primers and the product from the first round as a template, thereby minimizing the likelihood of non-specific amplification [36, 45, 47, 48]. Given that high-quality sequences were obtained for the other markers (which are single-copy genes), it is unlikely that the SSU rRNA gene amplification failure was due to low DNA concentration, poor DNA quality, or the presence of PCR inhibitors. It is therefore more plausible that the issue was related to amplicon purification or suboptimal sequencing conditions. Since the bg, gdh, and tpi markers yielded clear and informative sequences for genotyping, additional attempts to sequence the SSU-rRNA gene were not pursued, particularly in light of its lower discriminatory power for differentiating assemblages or species within the G. duodenalis complex [44].”

Comment 2: 2) Please clarify why only the β-giardin gene was constructed in the NJ tree, and sequences in this study were identified as G. enterica, despite the β-giardin locus of G. duodenalis among human and animal isolates showing genetic heterogeneity.

Response 2: We constructed phylogenetic trees for all three genes (bg, tpi, and gdh), including all available sequences from rodent hosts deposited in GenBank. The resulting trees were highly similar, with the isolates from porcupines and naked mole rats consistently clustering within the G. enterica clade. To avoid excessive repetition of essentially the same results, we selected one of the trees (bg) as representative, while the trees for tpi and gdh are provided as supplementary material (files S4-S6).

The full trees are very large, and including the complete bg tree in the main text would significantly reduce readability due to space constraints. Therefore, we chose to include a condensed version of the bg tree in the manuscript. This reduced tree shows in detail the branch containing the sequences obtained in this study, while the remaining branches corresponding to other Giardia species are summarized for clarity. The complete bg tree is available as supplementary file S4.

To clarify this, we have added the following text to the manuscript (lines 258–260): “A condensed tree based on the bg gene is shown in Figure 2 as representative of the results obtained for each gene; full phylogenetic trees for the bg, tpi and gdh markers are provided in supplementary files S4-S6.”

Comment 3: Minor revision:
1)    Please add the aim of the study in the abstract.

Response 3: We have revised the abstract and clearly stated the aim of the study.

Comment 4: 2) Line 91 …. assignments as G. duodenalis assemblage ‘X’. Please add a citation.
3)    Line 92 …. are considered the ethiological agents of human giardiasis. Please add a citation.

Response 4: We have modified the text (lines 102-104) as follows: “In citing identifications from previous studies, we will adopt this taxonomic framework and refer to these species names, rather than by assemblage letter dessignations”. Please note that “we will adopt this taxonomic framework” refers to that by Wielinga et al. (2023) (reference 14), which is cited in the same paragraph.

Comment 5: 4) Line 209: Please specify the cysts observed in the Figure caption.

Response 5: We have modified the figure caption to indicate that “Giardia cysts observed on temporary slides …”

Comment 6: 5) The ambiguity R (G/A) in table 2 was possibly not different from “G” in other sequences

Response 6: The reviewer is correct. The ambiguity observed in the bg sequence is most likely due to the GS/M strain possessing two alleles that differ at that particular base position. We included this position in the table because, although the ambiguous base is compatible with the sequences from the porcupine and naked mole rat isolates, the sequences may not be strictly identical at that site.

Comment 7: 6) Tables 2, 3, 4, and Tables 5, 6, 7 can be described in the text with significant information, and all tables can be represented in the supplement section.

Response 7: In our opinion, removing Tables 2-4 and instead describing all polymorphisms solely in the main text would make the information harder to follow and less accessible. For comparison, consider the clarity of the tables versus the following text version:

“The comparison between sequences obtained from the different host species revealed that they were not identical, and specific base differences were observed across all three molecular markers analyzed (bg, tpi, and gdh). In the beta-giardin (bg) alignment (Supplementary File S1), all three isolates differed from the reference G. enterica sequence (ACGJ01002392) at position 97 (G instead of R) and position 232 (C instead of T). In addition, the Brazilian porcupine and crested porcupine sequences showed a T at position 301, whereas the naked mole rat matched the reference with a C at this position.
The tpi marker showed more extensive divergence (Supplementary File S2). The Brazilian porcupine isolate differed from the reference sequences at two positions (76 and 387), while the crested porcupine exhibited notable divergence at eight positions (24, 30, 76, 153, 161, 195, 256, 265, and 282), suggesting it represents a more distinct variant. The naked mole rat sequence differed by a single base at position 519.
In the gdh gene alignment (Supplementary File S3), the Brazilian porcupine isolate showed changes at positions 120 (T vs. C), 405 (T vs. C), and 429 (Y vs. T). The crested porcupine also exhibited differences at positions 309 (A vs. C) and 429 (Y vs. T), while the naked mole rat differed at positions 333 (T vs. C) and 429 (Y vs. T). Notably, the ambiguous base Y (C/T) at position 429 was shared by all three isolates and may indicate either a true polymorphism or the presence of mixed base calls at this site.”

We kindly ask the reviewer to accept the current presentation of Tables 2-4, which we believe provide a clearer and more accessible summary of the key polymorphisms across the different gene markers.

A similar situation may apply to Tables 5-7 (now merged into a single Table 5, following another reviewer’s suggestion). The advantage of presenting the data in table format is that it offers a clear and organized view, whereas describing the same information in the main text would result in a long sequence of numerical values, which could be harder to follow. We kindly ask the reviewer to accept the revised Table 5 (or the previous separate Tables 5-7) as a straightforward and effective way to present the data.

Comment 8: 7) Figure 2 showed only one targeting fragment, which is not consistent with the materials and methods section. Please clarify why the phylogenetic tree of this specific target was constructed.

Response 8: Please refer to the response to Comment 2. Phylogenetic trees were constructed for all three genes (bg, gdh, and tpi) and are presented as Supplementary Files S4-S6. Figure 2 shows a condensed version of the bg gene tree, included in the main manuscript as a representative example of the results obtained for all three genes, and formatted to fit within the page constraints.

Comment 9: 8) Accession no. of Giardia enterica (strain GS) in Table 2 and Figure 2 are different.

Response 9: Thanks very much for pointing out the typographic error. We have corrected the number in Table 2.

 

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

It would have been better to take samples from zookeepers as well. 

 

Author Response

Comment: It would have been better to take samples from zookeepers as well. 

Response: First, we would like to sincerely thank the reviewer for taking the time to read and comment on the manuscript.

We agree that analyzing samples from the zookeepers would have been valuable; however, this would have presented several challenges. First, such a study would require approval from an ethics committee, which would take longer than the time frame available for conducting prompt analyses of human samples following detection of infection in animals. A general, prospective, and undefined approval “just in case” would likely have been rejected. Second, participation would be voluntary, and there would be no guarantee that the appropriate individuals—specifically, those in charge of the animals—would provide samples.

In addition, we would like to emphasize that the focus of the manuscript is on the genetic characterization and identification of Giardia isolates from the animals, rather than on epidemiological aspects such as transmission routes, infection sources, or longitudinal molecular prevalence. For the specific objective of this study, the analysis of zookeeper samples was not essential.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript was revised to meet academic standards by clearly describing the scientific methods. The presentation and discussion of the study findings have been improved, with more precise descriptions of the identified Giardia species and their genetic characteristics. Therefore, it should be accepted to publish this manuscript version. This study will be interesting for researchers and veterinarians in the field of Parasitology, in particular the topic of protozoan diseases or wildlife diseases