Molecular Detection of a Bartonella sp. in Fleas and Mites Collected from Wild Rodents (Cricetidae: Sigmodontinae) in the Northeast of Argentine Patagonia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal: Zoonotic Diseases

Title: Molecular detection of Bartonella quintana in fleas and mites collected from wild rodents (Cricetidae: Sigmodontinae) in the extreme northeast of Argentine Patagonia

General Overview:

            This study investigated Bartonella species in ectoparasites collected from rodents in selected regions of South America. Genetic characterization using the gltA and rpoB loci identified Bartonella quintana, an important zoonotic species, in fleas and mites collected from rodents. This finding raises concern regarding the potential circulation of zoonotic Bartonella in areas where wildlife, domestic animals, and humans coexist. PCR amplification of the gltA and rpoB genes was followed by DNA sequencing of selected positive samples, and the nucleotide sequences generated in this study were deposited in the GenBank database. The authors further performed concatenated phylogenetic analysis of the two loci and concluded that the detected Bartonella genotypes were identical to B. quintana, a well-recognized human pathogen and an important cause of infectious endocarditis. Overall, this study highlights the importance of molecular surveillance of vector-borne zoonotic bacteria in this geographical region. However, I have a major concern regarding the molecular methods used for species identification. Accurate species identification within the genus Bartonella relies on well-established genetic characterization using specific housekeeping genes, as many Bartonella species share high sequence similarity. Consequently, minor limitations or inappropriate interpretation of the genetic analyses may lead to incorrect species assignment and substantially affect the conclusions of this study.

Abstract

-The gene target(s) used for the molecular identification of Rickettsia spp. should be specified in the abstract.

-Lines 34–35: The housekeeping genes used for Bartonella species identification should be clearly stated. Since species identification was based on the gltA and rpoB loci, these genes should be explicitly mentioned rather than using the phrase "such as."

-Lines 35–37: The sequence identity of each locus (gltA and rpoB) should be reported in the abstract to support the species identification. According to the proposed sequence-based criteria for Bartonella species identification, the sequence identity should be ≥96.0% for the gltA locus and ≥95.4% for the rpoB locus for assignment to the same species (La Scola et al., 2003). Including these values would strengthen the molecular evidence supporting the identification of Bartonella quintana.

 

Materials and Method

-Table 1: Please correct the target gene name for Bartonella from "rpO" to "rpoB."

-The primer pairs used for amplification of the gltA and rpoB loci generated relatively short PCR products. The authors should discuss the limitations of using these short fragments for Bartonella species identification. The sequence-based criteria proposed by La Scola were established using partial sequences of 327 bp for gltA and 825 bp for rpoB, with proposed species-level thresholds of ≥96.0% and ≥95.4% sequence identity for the gltA and rpoB loci, respectively. The much shorter sequences analyzed in the present study may provide insufficient phylogenetic resolution for confident species identification.

-The GenBank accession numbers reported in the manuscript are currently unavailable. The authors should verify that the submitted sequences have been released or provide appropriate evidence that the submissions have been accepted and will become publicly accessible.

-According to Table 2, Bartonella DNA was detected in all ectoparasite groups (approximately 19 positive samples). However, only nine GenBank accession numbers are reported, and it is unclear whether these correspond to the gltA locus, the rpoB locus, or both. The authors should clarify how many sequences were generated for each gene and explain the discrepancy between the number of positive samples and the number of deposited sequences.

-The authors should clarify whether MEGA version 4 was actually used for the phylogenetic analysis. If so, please justify the use of this considerably outdated version instead of a more recent release.

-A major concern relates to the concatenated phylogenetic analysis. Figure 2 indicates that the concatenated gltA and rpoB alignment comprised only 309 bp, but the manuscript does not report the individual sequence lengths of each locus included in the analysis. This sequence length is substantially shorter than that used to establish the sequence-based criteria for Bartonella species identification (327 bp for gltA and 825 bp for rpoB). Consequently, the phylogenetic resolution of the concatenated dataset may be limited, and the original species delineation criteria cannot be directly applied to such a short concatenated fragment. The authors should justify the use of this dataset and discuss its limitations for accurate species identification.

Results and Discussion

Based on the major methodological concerns outlined above, the genetic evidence presented in this study is insufficient to confidently identify the detected Bartonella as B. quintana. The short gltA and rpoB fragments and the 309-bp concatenated phylogenetic analysis provide limited resolution for species-level identification. Therefore, the conclusion that B. quintana was detected in the studied ectoparasites should be interpreted with caution.

To strengthen the molecular evidence, the authors should consider reanalyzing the DNA samples using primer sets that generate longer gltA and rpoB fragments consistent with the published sequence-based criteria for Bartonella species identification. Sequencing of the longer amplicons would provide more robust evidence for species assignment.

If the authors choose to retain the current dataset, I do not believe that the available genetic evidence is sufficient to conclusively identify the detected organism as B. quintana. A more appropriate interpretation would be to describe the detected organism as a Bartonella sp. closely related to B. quintana rather than as confirmed B. quintana.

 

 

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please find my comments in the attached file.

Comments for author File: Comments.pdf

Author Response

Please see the attachement. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript reports the detection of Bartonella quintana in fleas and one mite species collected from wild rodents captured in Patagonia, Argentina. The study addresses a relevant topic, as B. quintana  is a vector-borne bacteria of veterinary and public health interest, and information on their occurrence in wildlife-associated ectoparasites from this region remains limited.

Unfortunately, the results are not suitable for publication. My major comments are outlined below:

1. The biological material (ectoparasites) analysed in this study is very limited. According to lines 197–200, only 26 fleas and 6 mites were collected from 126 trapped rodents. Ectoparasite sample is very small.
2. Moreover, the numbers reported in lines 197–200 are inconsistent with those presented in Table 2, where only 15 flea specimens and 3 mites are listed, with different numbers assigned to the reported ectoparasite species.
3. The authors state that all collected ectoparasites were positive for Bartonella quintana DNA. From a biological perspective, this result appears highly unlikely, particularly given the very small number of ectoparasites tested, their collection across different years, and their origin from different individuals and even different host species. Such a 100% positivity rate is not consistent with patterns reported in comparable studies and raises serious concerns regarding the reliability of the results, including the possibility of contamination. Although the authors report on the use of controls, the presence of controls alone is not sufficient to resolve these concerns, especially in the absence of independent confirmation of such an unexpected result. Therefore, these findings should be interpreted with great caution and, in their current form, cannot be considered sufficiently robust for publication.
4. The authors do not provide sufficient information on the sequence variability of the gltA and rpoB gene fragments detected in this study. It is unclear whether all positive ectoparasites yielded identical sequences or whether any nucleotide differences were observed among samples. From a biological perspective, it seems unlikely that ectoparasites collected from different host species would all harbour identical bacterial populations or strains.
5. The mite species name is misspelled in the manuscript. The authors use “Gigantolelpas”, whereas the correct spelling is Gigantolaelaps.
6. Several incorrect references in the Introduction raise concerns about the overall reliability of the manuscript. For example, reference 7 does not concern Bartonella, while the DOI provided for reference 10 is incorrect and in fact corresponds to a different article. Similarly, the DOIs listed for references 14 and 16 do not lead to the cited sources. I have not checked all remaining references; however, the errors identified above are sufficient to raise serious concerns from a reviewer’s perspective.
7. The description of the methods contains both factual and language errors. First, mites are not anatomically comparable to insects and do not possess clearly segmented abdominal tergites; therefore, the statement that they were cut “between the third and fourth abdominal tergites” is inappropriate for them. Second, the authors state that 25–100 ng of template DNA was used in the PCR reactions. This is highly questionable, as such amounts of DNA are unlikely to be obtained from a single ectoparasite specimen, particularly from an individual mesostigmatid mite, which would typically yield no more than a few to several nanograms of DNA per microliter. Third, the statement regarding ethidium bromide concentration also appears unreliable. While ethidium bromide may have been available as a stock solution at 10 µg/µL, gel staining would certainly not be performed in a 10 mg/µL solution. These issues indicate that the methodological section was prepared carelessly and does not provide a reliable account of the procedures actually performed.
8. The authors do not provide clear information on how many Bartonella sequences were included in the analysis, which species or strains they represented, or what criteria were used for their selection.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The research is inconsistent with the conclusions because the captures date back to 2017-2018, while we are in 2026.

The Materials and Methods section is not described exhaustively:
- Which portion of the sample was used for DNA extraction and which for morphological identification?
- How the fleas and mites were washed is not indicated.
- Indicate how many analyzed samples were positive for Bartonella compared to the total analyzed.

Finally, I suggest  aligning each target separately and reporting two phylogenetic trees , one for each target.
The number of bootstraps used is not indicated.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All of my comments have been satisfactorily addressed by the authors, and I have no further comments on this manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

All comments and suggestions have been addressed.

Reviewer 4 Report

Comments and Suggestions for Authors

In new version the abstract and conclusions are clear and appropriate to scope.
The section “Material and methods” have been described in a complete and exhaustive way and the results are accurately reported.
The supplementary material provided is useful for a better understanding of the study.
In conclusion, I believe that this article is suitable for publication.