Molecular Epidemiology of Helminths at the Wildlife–Livestock Interface in Kazakhstan: Evidence from Sheep and Saiga

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS entitled "Molecular Diversity and Transmission Dynamics of Helminth Communities in Sheep and Saiga Antelope in West Kazakhstan" focuses on helminth co‑infections and cross‑species transmission between domestic sheep and wild saiga antelope, addressing pathogen spillover risks at the livestock‑wildlife interface. This is regionally relevant and of veterinary public health importance. The following issues can be applied to addressed to improve the quarlity of the MS.

1. The number of investigated animals is too small. Only 35 animals (20 sheep + 15 saiga) were examined, with no sample size calculation provided. Therefore, there may exist challenges in the interpretation of results. It will be more interesting to include more samples.
2. Lack of statistical analysis, which is also likely caused by sample size.
3. It will be more interesting to include morphological data in the MS, to intuitively display results.
4. Sequences identified in this study need to be submitted to public database, and include access number in the MS.
5. As to the Figure 1 and 2, support values less than 75 should not be indicated, and an outgroup is needed for the tree.
6. As to the discussion, no information is given on anthelmintic use in the region, nor its potential impact on parasite species distribution. It will be more interesting to discuss it. Meanwhile, explicitly state the limitations of the study (small sample size, single sampling time point, lack of seasonal dynamics.
7. There are quite a few language and formatting problems, such as repeated sentences and reference formatting is inconsistent. Please check reference formatting to comply with journal guidelines.

Comments on the Quality of English Language

Consider professional English editing or a native‑speaker colleague for language polishing

Author Response

Please refer to the attached

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting manuscript which details potential transmission of parasites between Saiga antelope and sheep in Kazakhstan. Using molecular methods the authors show several different species

The manuscript has some major issues which need to be addressed prior to publication. Not least the publication of the sequences in the manuscript is not appropriate, nor useful. These need to be uploaded to Genbank following scientific convention.

Also some inclusion of the techniques used will also be useful to the readers. The numbers of samples used are only small, and only a single gene is sequenced, and as such, some of the concluding comments need to be taken with this in mind. 

Further comments are detailed below

 

Line 93- you may want to say how the animals were euthanised. Is there any further details on these animals? Age? Sex? Breed etc

Line 97- perhaps needs some more data in here- was it Mcmasters technique or similar or was Baermanns technique employed. You do cite these but worth some more detail in here. Also if you use PCR from faecal samples, how can you be sure that cross reactivity in dual infected animals didn’t occur?

Line 100- ‘identificatied’- maybe identified is more appropriate here instead

Line 113- what were the reagents for the PCR ?

Line 119- what were the positive and negative controls used?

It would be useful to start the results with the simple data- prevalence in both animal species, counts, different species found etc – you have some of this- 137-141 which is worth moving up

Line 133- helminths and hosts doesn’t need capital letters

Line 142-148- here more specific would be worthwhile- so you say the majority of animals, how many? What %? how common was it?

Table 1- this is not worth including. These need to be uploaded to Genbank and the appropriate accession number reported here. The sequences are impossible to analyse in that format

You also use the sequences for the trees so having the raw data is not needed. You could include some discussion on any specific mutations seen within the genes if appropriate

Figure 1- worth stating the algorithm used to produce this and if it is a maximum likelihood or neighbour joining tree. You could also simplify it by removing the lower bootstrap values if you so wanted

Figure 2- similar comments to figure one regarding algorithm and tree production method and bootstraps. Also maybe worth including the colours as you do for figure 1

Line 199- could it be that recombination plays a part too? You only look at a single gene of many so there could be huge variations seen in other genes

Line 220- this is the first mention of the failed sequences, and that needs to be in the results

Author Response

Please refer to the attached

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most issues in the previous version. The following aspects need to be addressed.

1. As to the phylogenetic trees in Figure 1 and 2, more sequences from divergent species and same species in this study should be included for analysis to make the tree more interesting and meaningful.
2. There are still formatting issues, especially with inconsistent reference formats in the reference list. Please carefully check and verify.

Comments on the Quality of English Language

Consider professional English editing or a native‑speaker colleague for language polishing

Author Response

1. As to the phylogenetic trees in Figure 1 and 2, more sequences from divergent species and same species in this study should be included for analysis to make the tree more interesting and meaningful.

AU response: Thank you for this helpful suggestion. We agree that including a broader set of reference sequences could, in principle, provide additional phylogenetic context. However, in the present study, our analysis was deliberately restricted to a curated subset of well-characterized reference sequences to ensure reliable alignment and avoid introducing bias associated with highly heterogeneous or unevenly represented datasets, particularly given the limited number of sequences generated in this work. Expanding the dataset to include a larger number of divergent sequences would substantially alter the analytical framework and is beyond the scope of the current study. Therefore, we respectfully prefer to retain the current phylogenetic design, which is sufficient to support species-level identification and the main conclusions. We will, however, yield to the decision of the editor in this regard.

2. There are still formatting issues, especially with inconsistent reference formats in the reference list. Please carefully check and verify.

AU response: It is now revised as requested.

Reviewer 2 Report

Comments and Suggestions for Authors

I wish to thank the authors for addressing my comments. The manuscript is much improved and I have no further comments 

I wish you every success with your future research 

Author Response

Thank you for your valuable comments and wishes!