Follow-Up of the Immune Response and the Possible Presence of Brucella melitensis Strains in Peripheral Blood in Hoggets Vaccinated by Rev1 in Greece

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Dear Authors,

I sincerely appreciate all the revisions made to the article, and I’m happy with the final version. I have no further comments at this stage.I have no further comments.

Author Response

Comment 1: I sincerely appreciate all the revisions made to the article, and I’m happy with the final version. I have no further comments at this stage. I have no further comments.

Response 1: We would like to thank Reviewer for his sincere appreciation for our effort.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

• Several grammatical and syntactic errors throughout. For example:
  
  • "seemes" (Conclusion) → should be "seems"
    
  • "immunoreaction" is used repeatedly—more appropriate terms could be "immune response" or "serological response."
  • coinciding with a high variability" could be better written as "correlating with variable immune responses."
• Control Group: The absence of a control group is acknowledged but weakens causal inference. Even using historical controls or statistical modeling could help infer comparative effectiveness.
• Sample Size: N=10 is very small. While field constraints are understandable, the statistical power is limited.
• Strain Tracking: Excellent use of WGS, but the results would benefit from clearer visualization—such as a heatmap showing SNP distances or a time-series graph per animal.
• Figure 2 (phylogenetic tree): Include more detail in the legend to help non-experts interpret clustering.
• Figure 3: Needs clarification. The four sub-panels (A–D) are not well explained. What distinguishes each group mechanistically?

• The link between field strain identity and immune response is interesting but remains speculative. The authors could:

  • Propose mechanistic hypotheses or cite immunological literature to support this.

  • Discuss whether Rev1-induced antibodies cross-react with field strains or if these immune responses are strain-specific.

  • The public health implications, especially considering human brucellosis risk with delayed vaccination, could be more explicitly addressed.

Comments on the Quality of English Language

Consider professional English editing or at least a thorough grammar and clarity review.

Author Response

Consider professional English editing or at least a thorough grammar and clarity review.

English editing has been further performed

Comment 1: "seemes" (Conclusion) → should be "seems"
Response 1: Authors agree with the comment of the reviewer and changed the word accordingly.

Comment 2: "immunoreaction" is used repeatedly—more appropriate terms could be "immune response" or "serological response."

Response 2: “immunoreaction” has been changed according to reviewer’s suggestion to “immune response” wherever it is needed.

Comment 3: coinciding with a high variability" could be better written as "correlating with variable immune responses."

Response 3: The sentences have been rephrased (lines 376-378). We did not want to give the impression of statistically significant correlations, so we rephrased it differently than proposed.

Comment 4: Control Group: The absence of a control group is acknowledged but weakens causal inference. Even using historical controls or statistical modeling could help infer comparative effectiveness.

Response 4: Authors agree with reviewer’s comment. However, to their knowledge, no similar study on delayed vaccinated hoggets has been reported. Therefore, there is not any source to retrieve historical control or statistical modeling to strengthen the conclusions, as very kindly the reviewer suggests. For this reason, the authors extended the discussion mainly to grounds of other relative studies (i.e. bibliography 13 and 52) and avoided reaching strong conclusions. The concept that further studies are required is added to lines 418-419.

In addition, at the end of the first paragraph of the Discussion, in lines 314-316, we have added the following sentences: Previous analyses performed in our lab (unpublished data) in blood samples from flocks located on Greek islands – which belong to “eradication zone” of brucellosis - did not result in the detection of Brucella..

Comment 5: Sample Size: N=10 is very small. While field constraints are understandable, the statistical power is limited.

Response 5: The authors do agree with the reviewer’s comment. Due to the small sample size we did not include statistical analysis, as they would not give reliable results. We would very much like to be able to analyze a greater number of cases from various farms. However, there is a significant limitation for extending such a study to various farms, since the environment and management between different farms may significantly vary changing unpredictably the state of infection in the animals.  However, is a future perspective, and effort is given in rising financial resources to extend this study further.

Comment 6: Strain Tracking: Excellent use of WGS, but the results would benefit from clearer visualization—such as a heatmap showing SNP distances or a time-series graph per animal.

Response 6: A phylogenetic tree is traditionally used as visualization. However, we understand the reviewers wish to depict SNP distances more clearly. Therefore, we added a distance matrix stating the cgSNP differences between each strain.

 Comment 7: Figure 2 (phylogenetic tree): Include more detail in the legend to help non-experts interpret clustering.

Response 7: The authors now have added some information in the figure and tried to explain the clustering better in the figure caption.

 Comment 8: Figure 3: Needs clarification. The four sub-panels (A–D) are not well explained. What distinguishes each group mechanistically?

Response 8: The authors appreciated the reviewers query and apologized for this: The four groups have been created based on the wild field strain detected in the hogget’s blood samples except for hogget H9 from which two different strains were recovered (F1 & F2) and it has been included in group A.

The title of Figure 3 is now changed to be clearer as follows: “: Graphs A-D represents the association between the group of B. melitensis field strains detected in hoggets and the strength and duration of the hoggets’ immune reaction during the experimental period (pre - vaccination and until 120d post – vaccination)”.

Comment 9: The link between field strain identity and immune response is interesting but remains speculative. The authors could:

Comment 9A: Propose mechanistic hypotheses or cite immunological literature to support this.

Response 9A: The authors agree with this suggestion of the reviewer.

During recent years, extended research targeting to creation of a successful recombinant vaccine towards Brucella, as an alternative to Rev1, has established that various genes, encoding specific antigens, induce different cytokines and provoke different types of inflammatory response. Genomic and phenotypic differences have been observed among B. melitensis field strains i.e. in lipopolysaccharide (LPS) composition, presence of outer membrane proteins (OMP), effectiveness of the Type IV secretion system (T4SS). These molecular features, affect the host’s pattern recognition receptor engagement, cytokine milieu, and subsequent T-cell responses, inducing different levels and profiles of immune responses following an infection (de Jong et al., 2010; Martirosyan et al., 2011). The authors have, now, added relevant information in lines 392-400.

Comment 9B: Discuss whether Rev1-induced antibodies cross-react with field strains or if these immune responses are strain-specific.

Response 9B: Although the authors appreciate the suggestion of the reviewer, they considered unnecessary the inclusion of additional statements in the discussion since it has been already referred in the Introduction (lines 55-58) “….. The diagnosis of brucellosis in cattle and small ruminants is usually performed by serological testing using the Rose Bengal (RBT) and the Complement Fixation tests (CFT). However, due to cross-reactivity of field strains with Rev1 vaccine strain these tests may not be reliable .…..”

The Rev1 vaccine strain, induces a robust humoral and cellular immune response which is not strictly specific to the Rev1 strain. Several studies have demonstrated that the antibodies and T-cell responses generated post-vaccination with Rev1, cross-react with multiple field strains of B. melitensis, primarily due to shared immunodominant antigens such as smooth lipopolysaccharide (S-LPS) and outer membrane proteins (OMPs) (Bowden et al., 1995; Banai 2005).

Comment 9C: The public health implications, especially considering human brucellosis risk with delayed vaccination, could be more explicitly addressed.

Response 9c: As mentioned in the manuscript according to Dougas et al.,2022 delayed vaccination of small ruminants past the optimal age of 3–6 months coincided with a higher rate of human brucellosis. To our knowledge there is no other relevant study supporting such a hypothesis.

 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Well written manuscript but needs improvement

Introduction

Add more review on relatedness of human brucellosis cases in Greece to animal contact, products if any. Cases of animal brucellosis if any.

Line 62 and Line 69-unclear what this means.

Materials and Methods (more detailed description needed)

Detailed description of animal husbandry(all animals) including the ewes, rams and hoggets-production system, feeding, management. Farm history-why did you choose this farm or area. How frequent is Brucella related diseases in the area/farm in both animals and human?

Breeding management of the test hoggets need to be described.

Line 144-145-describe fully-unclear what was done.

PCR description details of Brucella detection, primers, fragment size instead of citing references

Results(more details)

Results

PCR results-gels

With WGS probably more indepth analysis of the sequences data would add insight on strain differences apart from cgSNPs. Adds impact and new information.

Discussion

Need further discussion once more information is added to the materials and methods and farm/location history to relate findings with study location and husbandry practices.

Also section on the pregnancy outcome depends on description of breeding management of study animals in materials and methods.

Author Response

Comment 1: Add more review on relatedness of human brucellosis cases in Greece to animal contact, products if any. Cases of animal brucellosis if any.

Response 1: The authors include additional information in lines 74-79

Commet 2: Line 62 and Line 69-unclear what this means.

Response 2: The information written in lines 62-69 have been rearranged as follows “Since there is no vaccine for humans, the only way to prevent human brucellosis is by controlling the disease in livestock. The pathogen persists in flocks mainly through non-symptomatic carriers, the proportion of which has not yet been precisely estimated [22]. These latent carriers can transmit B. melitensis both horizontally and vertically within the herd [23,24], while vaccination with the live-attenuated vaccine strain B. melitensis Rev1 is considered the most effective way to control animal brucellosis.” (lines 47-53)

 

Materials and Methods (more detailed description needed)

Comment 3: Detailed description of animal husbandry (all animals) including the ewes, rams and hoggets-production system, feeding, management. Farm history-why did you choose this farm or area. How frequent is Brucella related diseases in the area/farm in both animals and human?

Response 3: the authors appreciate the reviewer’s comment. To address it, a Supplementary Table (Table S1) has been added in the revised manuscript, describing production, reproduction, and management traits, infrastructures and housing conditions, feeding and nutrition, incidence of health and welfare issues, and the vaccination program of hoggets in the studied farm.

A sentence to explain how the farm was selected has been added as follows: ‘The farm was selected on the basis of having applied late vaccination against B. melitensis. Brucella-related diseases in animals and humans in the farm or the region had not been reported in the past 15 years that the farm operates.’

Comment 4: Breeding management of the test hoggets need to be described.

Response 4:As described in the previous comment; Breeding management of the hoggets has been added in Supplementary Table S1.

Comment 5: Line 144-145-describe fully-unclear what was done.

Response 5: The authors hope that the added explanation in lines 157-160 will be considered as adequate by the reviewer.

Comment 6: PCR description details of Brucella detection, primers, fragment size instead of citing references

Response 6: The PCR description details have been added in the manuscript in lines 171-175 and 179-182.

Results (more details)

Comment 7: PCR results-gels

Response 7: PCR gels have been added as Supplementary Figures 1 and 2 as written in lines 229-230.

Comment 8: With WGS probably more in depth analysis of the sequences data would add insight on strain differences apart from cgSNPs. Adds impact and new information.

Response 8: The focus of this study was on the immune response in the hoggets. Thus, genomic differences of the isolates were not considered further, as little is known about genetic determinants that influence immune response to Brucella. Further, the Brucella genome is extremely stable and differences usually can only be detected on SNP level.

Discussion

Comment 9: Need further discussion once more information is added to the materials and methods and farm/location history to relate findings with study location and husbandry practices.

Also section on the pregnancy outcome depends on description of breeding management of study animals in materials and methods.

Response 9: All the information has been added and explained

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Well improved. In the discussion and conclusion section-comment on the public health implication of the study findings after late vaccination-refer to the literature review line 85-87.

Did you include negative controls for the PCR- not mentioned in the text and none in the gel.

Author Response

Comment 1: Well improved. In the discussion and conclusion section-comment on the public health implication of the study findings after late vaccination-refer to the literature review line 85-87.

Response 1: The authors would like to sincerely thank the reviewer for urging to comment on the public health implication of the study findings after late vaccination.  Following his advice, we have now added a few words to lines 412-415.  However, due to the limited number of available animals, we avoid drawing any conclusions on this subject.

Comment 2: Did you include negative controls for the PCR- not mentioned in the text and none in the gel.

Response 2: The authors appreciated the reviewer’s comment and apologized for the omission. Yes, to check the specificity of the PCRs we have included negative controls, such as non-template reaction as well as other relative bacterial species such as Y. enterocolitica, E. coli Ο:157, Shigella flexneri, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus. However, we did not include those gels but only the ones that contained our samples and a B. abortus sample.

We have now added this information in Materials and Methods and the Results in lines 182-184 and 234-235, respectively.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This research is interesting and valuable for brucellosis control and prevention. Generally, the experimental design is rational and the results and discussions are  organized well. However, my comments are listed as follow.

1. line 229: PCR showed that all the isolated strains belonged to the B. melitensis species. Please added the strain biotype. 

2. line 232-233: by applying a threshold of a maximum five cgSNPs difference, the isolates could be assigned to four groups (Figure 2). Supplement tabel 1 showed the detailed SNPs (>200), the results seemed to be contradictory, please explained.

3. Please check the contents between Figure 2  and texts. (isolates and Group)

4. Figure 2: the colors of the blocks ?

5. human isolates were included and what differences were showed and please discussed.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comment 1: line 229: PCR showed that all the isolated strains belonged to the B. melitensis species. Please add the strain biotype. 

Response 1: We thank the reviewer for his comment. However, the biotype is not required for this point as identification of Brucella species was the target.

Comment 2: line 232-233: by applying a threshold of a maximum five cgSNPs difference, the isolates could be assigned to four groups (Figure 2). Supplement tabel 1 showed the detailed SNPs (>200), the results seemed to be contradictory, please explained:

Response 2: In figure 2 the isolates mainly fall into two clusters at two different branches of the tree. Within these clusters, the SNP difference is >200 cgSNPs, but the differences within the clusters is low. A visualization of the similarities give the coloured blocks at the right of the tree.

Comment 3: Please check the contents between Figure 2  and texts. (isolates and Group)

Response 3: The reviewer’s comment is correct. There was a mix-up between the isolates H6A and H7B and the Groups of strains to which they belonged. The text now has been revised appropriately.

Comment 4: Figure 2: the colors of the blocks ?

Response 4: The colours indicate those isolates that belong to the same group at the given cgSNP threshold (indicated above the blocks). We added this in the figure caption.

Comment 5: human isolates were included and what differences were showed and please discussed.

Response 5: Human isolates were indeed included in Figure 2. These strains originated from a previous study  (Brangsch et al., 2023), where isolates from both, small ruminants and humans from Greece were analyzed. Explanatory notices have been added in the text and more specifically, in the section of Results (lines 227-228) and in Discussion (lines 337-340).

Comment 6: The English could be improved to more clearly express the research.

Response 6: English editing has been carried out

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors describe humoral responses and detection of B. melitensis in blood of sheep vaccinated at 9 months of age with Rev1 as compared to the recommended 3-6 months of age.  I have the following comments for the authors to consider:

1. My biggest concern is the lack of a control (unvaccinated group) to compare both immunologic responses post vaccination and pathogenic effects of B. melitensis during pregnancy.  Not all animals abort that are infected and early infection with a virulent field strain may induce cellular immunity that prevents pathogenic effects during pregnancy.  In the absence of an unvaccinated group, the authors conclusions that late vaccination induces protective effects are invalid.  The study animals were obviously from a herd endemically infected with B. melitensis and the immunogenic and pathogenic effects in unvaccinated animals were not assessed.
2.  For this reviewer, the numbers of animals included in each WGS analysis are not sufficient to make the interpretations as reported on lines 251-264 and in the discussion on lines 353-269.  The largest group contained 5 similar isolates from 3 animals and the other 2 groups were of 3 and 2 isolates from 3 and 2 animals, respectively.  The limited data acquired is not sufficient to support the conclusions stated in the manuscript.
3. This reviewer was surprised that only 1 serologic test was performed on the 10 animals in the study.  I would have expected to see a high sensitivity screening test such as the rose bengal test also employed.  This may have given different results than those of the ELISA used in the study which this reviewer would consider to be a confirmatory (high specificity) test. 
4. I would suggest that Figure 1 and Table1 presents the same data.  I prefer the table to the figure as it presents actually data rather than an interpretation.
5. In general, study design makes it impossible to segregate the effects of a high degree of latent infection from effects of Rev1 vaccination at 9 months of age. 

Minor

1. I did not find the citation for reference 38 which should have been after line 279. I did not find the citation for reference 43 which should have been after line 296.  I did not find the citation for reference 50 which should have been prior to line 315.   Reference 56 was cited on lines 339 whereas references 57 and 58 were cited on line 338.

 

Author Response

Comment 1: My biggest concern is the lack of a control (unvaccinated group) to compare both immunologic responses post vaccination and pathogenic effects of B. melitensis during pregnancy.  Not all animals abort that are infected and early infection with a virulent field strain may induce cellular immunity that prevents pathogenic effects during pregnancy.  In the absence of an unvaccinated group, the authors conclusions that late vaccination induces protective effects are invalid.  The study animals were obviously from a herd endemically infected with B. melitensis and the immunogenic and pathogenic effects in unvaccinated animals were not assessed.

Response 1: Unfortunatelly, no control (unvaccinated) with animals exposed to the same environmental conditions to the studied ones was available, since the study was performed in a farm with healthy regularly vaccinated herd, as mentioned in lines 23, 93, 99-106 and 281-283.

Comment 2: For this reviewer, the numbers of animals included in each WGS analysis are not sufficient to make the interpretations as reported on lines 251-264 and in the discussion on lines 353-269.  The largest group contained 5 similar isolates from 3 animals and the other 2 groups were of 3 and 2 isolates from 3 and 2 animals, respectively.  The limited data acquired is not sufficient to support the conclusions stated in the manuscript.

Response 2: We do agree with the comment of the reviewer. We only attempted to present our observation regarding the onset of the immune response. We changed the expression we used i.e. “it becomes evident” with “it is assumed”  (line 250) which we hope that the reviewer will find more appropriate.

Comment 3: This reviewer was surprised that only 1 serologic test was performed on the 10 animals in the study.  I would have expected to see a high sensitivity screening test such as the rose bengal test also employed.  This may have given different results than those of the ELISA used in the study which this reviewer would consider to be a confirmatory (high specificity) test.

Response 3: In lines 49-63 in our text, we offer a number of explanations on why we have chosen to use iELISA combined with molecular methods (PCR and WGS), as well as blood cell cultures. In addition, we have used only iELISA as a more sensitive and reliable method as supported by to other published studies too (i.e. Mathur et al 2022).

Comment 4: I would suggest that Figure 1 and Table1 presents the same data.  I prefer the table to the figure as it presents actually data rather than an interpretation.

Response 4: Although we find the reviewer’s comment useful, we included figure 1 because it shows clearly that in our experiments, the overall immune response followed the same pattern mentioned by other investigators also and we comment in the discussion section on that (line 306-309)

Comment 5: In general, study design makes it impossible to segregate the effects of a high degree of latent infection from effects of Rev1 vaccination at 9 months of age. 

Response 5: In Figure 3, graph D, it is presented the onset of immune response of an animal vaccinated with Rev1 and where no any natural infection was detected. It can be seen that the onset of immune response of this animal differs substantially from those observed in animals with latent infection(s).

Minor

1. I did not find the citation for reference 38 which should have been after line 279. I did not find the citation for reference 43 which should have been after line 296.  I did not find the citation for reference 50 which should have been prior to line 315.   Reference 56 was cited on lines 339 whereas references 57 and 58 were cited on line 338.

Response: According to the reviewer’s suggestions, the references have been revised appropriately in the manuscript

Reviewer 3 Report

Comments and Suggestions for Authors

The study by Maria Babetsa and colleagues presents a well-structured and detailed analysis of Brucella melitensis REV1 vaccination in Greece, covering epidemiological concerns, immune response variability, and the detection of field strains. It addresses a critical gap in knowledge regarding the effects of delayed vaccination, a practical concern in disease control programs. The study is well-supported by references and previous research, providing a strong foundation for your claims. It has a well-structured methodology with clear vaccination and sampling protocols, and a good combination of serology, culture, PCR, and WGS for comprehensive Brucella spp. detection. The manuscript is well-written and does not contain many grammatical errors. Overall, the findings presented are worthwhile and will be informative to the field. However, some points could be discussed, improving the quality of the article.

The small sample size (N=10) limits statistical power, and limits the generalizability of the findings. Including a larger cohort or conducting a multi-farm study would strengthen conclusions.

Other point is that the Authors clearly states the routine annual vaccination at 5 months as part of the national Brucellosis control program, ensuring regulatory compliance. However, there is no direct comparison with hoggets vaccinated at the standard 5-month age. Having an early-vaccinated control group would help quantify differences in immune response and infection rates between early and delayed vaccination. Also, there is no discussion on potential maternal antibody interference, which could affect vaccine efficacy. Blood samples at younger ages (e.g., 3–6 months) could help clarify whether maternal antibodies influenced immune response at 9 months.

Regarding serological analysis, using a commercially available and validated ELISA test ensures standardization, but diagnostic reliability could be increased if additional confirmatory serological tests (e.g., Rose Bengal Test, Complement Fixation Test) were performed. Did the Authors used any other serological test?

The discussion of fluctuating antibody responses is interesting but could benefit from a clearer takeaway message. Are these variations expected? What practical implications do they have for disease surveillance and control?

Author Response

Comment 1: The study by Maria Babetsa and colleagues presents a well-structured and detailed analysis of Brucella melitensis REV1 vaccination in Greece, covering epidemiological concerns, immune response variability, and the detection of field strains. It addresses a critical gap in knowledge regarding the effects of delayed vaccination, a practical concern in disease control programs. The study is well-supported by references and previous research, providing a strong foundation for your claims. It has a well-structured methodology with clear vaccination and sampling protocols, and a good combination of serology, culture, PCR, and WGS for comprehensive Brucella spp. detection. The manuscript is well-written and does not contain many grammatical errors. Overall, the findings presented are worthwhile and will be informative to the field. However, some points could be discussed, improving the quality of the article.

Response 1: We thank the reviewer for the positive feedback and we modified the manuscript based on the comments raised by reviewer

Comment 2: The small sample size (N=10) limits statistical power, and limits the generalizability of the findings. Including a larger cohort or conducting a multi-farm study would strengthen conclusions.

Response 2: We do agree with the reviewer’s comment, and we would very much like to be able to analyze a greater number of cases from various farms. However, there is a significant limitation for such a study, namely the fact that the environment and management between different farms may significantly vary creating a significant source of variation affecting the likelihood of infection in the studied animals.  However, such a study is among our future perspectives, and we are searching for the financial resources to support it.

Comment 3: Other point is that the Authors clearly states the routine annual vaccination at 5 months as part of the national Brucellosis control program, ensuring regulatory compliance. However, there is no direct comparison with hoggets vaccinated at the standard 5-month age. Having an early-vaccinated control group would help quantify differences in immune response and infection rates between early and delayed vaccination.

Response 3: We focused on the analysis of one farm, where the vaccination took place at the same time in all hoggets, at the age of 9 months. We agree, that if we had the opportunity to include more groups would help to get a wider picture. However, taking in consideration that vaccination is applied in the same day on all the animals of the farm, we did not have the capacity to use earlier (e.g., 3-5 months-old) vaccinated lambs as controls. In that case we had to use animals from other farm and maybe from other region facing all those factors described in the previous comment.

Comment 4: Also, there is no discussion on potential maternal antibody interference, which could affect vaccine efficacy. Blood samples at younger ages (e.g., 3–6 months) could help clarify whether maternal antibodies influenced immune response at 9 months.

Response 4: Yes, maternal antibodies are important in the first months of age, but they remain into the blood circulation of the offspring usually until the age of five months. However, to exclude such an effect, we tested for the occurrence of antibodies at the initiation of the study (day zero), and all of the studied animals were found negative. Thus, we expect that in this group of hoggets, no maternal antibodies influenced the immune response.

Comment 5: Regarding serological analysis, using a commercially available and validated ELISA test ensures standardization, but diagnostic reliability could be increased if additional confirmatory serological tests (e.g., Rose Bengal Test, Complement Fixation Test) were performed. Did the Authors used any other serological test?

Response 5: We have used only iELISA as a more sensitive and reliable method as supported by to other published studies too (i.e. Mathur et al 2022).

Comment 6: The discussion of fluctuating antibody responses is interesting but could benefit from a clearer takeaway message. Are these variations expected? What practical implications do they have for disease surveillance and control?

Response 6: As mentioned in the text, (lines 314-319 and 352-357), such fluctuations are frequently observed due to a variety of reasons, creating serious problems to surveillance and control of disease (as added in line 316-317). Since our sampling size does not allow for generalized conclusions, we avoided stating messages that are not statistically supported by our findings.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revisition looks great. 

Author Response

Comment 1:The revisition looks great.

Response 1: We thank the reviewer for his comment