Review Reports - A Porcine-Isolated <i>Mycobacterium bovis</i> Strain Exhibits Hypervirulence in a Murine Pulmonary Tuberculosis Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript measures the pathogenicity and virulence of M. bovis strain (MB894), isolated for TB-like lesions of pigs, in a mouse model, and its virulence was compared with the highly virulent strain MB303 isolated from wild boar. The higher bacterial loads and pathologic findings were observed in the lungs, spleens, and livers of mice infected with both strains. Higher levels of cytokines were also produced in splenocytes from mice infected with both strains when stimulated with MB lysate. The TLC pattern of both strains showed the characteristics of virulent strains. There is no significant difference in the virulence between the two strains. Finally, whole-genome sequencing and phylogenetic analysis of the MB894 strain were performed.

1. The abstract was written like an introduction. Please add the key results and the final conclusion. The abstract should include the following: state the principal objectives and scope of the investigation, describe the methods employed, summarize the results, and state the principal conclusion

2. In Fig. 2, please determine the bacterial load in the bone marrow and blood.

3. In Fig. 3A, please directly label the lung and liver in the figure to easily understand the figure and give a high-power field in the lung histopathology.

4. In Fig. 4, the high-virulence Mtb strain typically produces lower cytokine levels than the avirulent strain when antigen-presenting cells are stimulated with Mtb. Please perform these experiments in vitro using MB894 and the lower-virulent M. bovis strain. How about determining the survival curve in the macrophages of MB894 strain and the lower-virulent M. bovis strain.

5. In fig.6, to get more solid data, please include TLC analysis of lower-virulent M. bovis strain.

6. Too long discussion section.

7. Minor suggestion:

- In line 40, M. tuberculosis complex --> M. tuberculosis complex

- In line 47, Mycobacterium avium Complex --> Mycobacterium avium complex-

- In line 48, a M. bovis strain from tuberculosis-like lesions in pigs at slaughterhouse (MB894) and reconfirmed ~~ --> a M. bovis strain (MB894) from TB-like lesions in pigs at slaughterhouse and reconfirmed

- In line 82, tuberculosis --> TB

Author Response

Reviewer 1

Comments and Suggestions for Authors

The abstract was written like an introduction. Please add the key results and the final conclusion. The abstract should include the following: state the principal objectives and scope of the investigation, describe the methods employed, summarize the results, and state the principal conclusion

We appreciate the reviewer suggestion and have rewritten the abstract, also considering the reviewer 2 suggestions.

In Fig. 2, please determine the bacterial load in the bone marrow and blood.

We thank the reviewer for this suggestion, but as we work with intrathraqueal inoculation we usually perform the bacterial load testing in lungs, spleen and liver. We do not study this parameter in bone marrow so we didn´t keep this tissue for further analysis. The same occurs with blood as we understand that mycobacteria are rapidly cleared from blood particularly for this inoculation via and during the time frame used in our analysis.

In Fig. 3A, please directly label the lung and liver in the figure to easily understand the figure and give a high-power field in the lung histopathology.

We appreciate the reviewer’s comment. The histopathology shown in Figure 3A was acquired using a 40× objective with a 10× ocular on an Olympus CX31 microscope, corresponding to a total magnification of 400×, which represents a high-power field. To avoid confusion, we have clarified the magnification and directly labeled lung and liver within the figure, and the figure legend has been revised accordingly.

We also added Supplementary Figures 1, 2 and 3 to make more clear the histopathology results

In Fig. 4, the high-virulence Mtb strain typically produces lower cytokine levels than the avirulent strain when antigen-presenting cells are stimulated with Mtb. Please perform these experiments in vitro using MB894 and the lower-virulent M. bovis strain. How about determining the survival curve in the macrophages of MB894 strain and the lower-virulent M. bovis strain.

We appreciate the reviewer’s comments. However, we chose not to include macrophage infection data in the manuscript due to variations in the results. We conducted three independent experiments using porcine BMDMs, adapting the protocol from Vazquez et at 2014. We observed significant variability in CFU counts from the inoculum, both between strains and across different experiments, which made it challenging to interpret the results reliably. As a result, we opted to focus on the in vivo experiments using the Murine Pulmonary Tuberculosis Model, which are included in the manuscript. We believe this approach provides more consistent and interpretable data regarding the infection dynamics.

In fig.6, to get more solid data, please include TLC analysis of lower-virulent M. bovis strain.

We have now included in the TLC the analysis of lower-virulent M. bovis strain. However, we couldn´t include this strain in the quantification analysis, since PDIMs were almost undetectable by TLC in this strain.

Too long discussion section.

We have shortened the discussion accordingly to the reviewer’s suggestion

Minor suggestion:

- In line 40, M. tuberculosis complex --> M. tuberculosis complex

- In line 47, Mycobacterium avium Complex --> Mycobacterium avium complex-

- In line 82, tuberculosis --> TB

Reviewer 2 Report

Comments and Suggestions for Authors

This study systematically evaluated the pathogenicity of Mycobacterium bovis (MB894) isolated from pigs in a mouse model of pulmonary tuberculosis, and explored its possible molecular mechanisms through whole genome comparative analysis. The experimental design is reasonable, the methods are detailed, and the data is comprehensive, especially the combination of phenotype and genome analysis, providing valuable data for the toxicity study of porcine derived Mycobacterium bovis. The overall quality of the article is high, but there are still some important contents that need to be revised and improved. The paper has multiple areas for improvement in terms of data analysis depth, result interpretation, chart presentation, and language expression, as follows:

It is suggested to modify the title to: A Porcine-Isolated Mycobacterium bovis Strain Exhibits Hypervirulence in a Murine Pulmonary Tuberculosis Model

Abstract: It is possible to start from a global perspective and then focus on the special situation of Argentina and pigs, which will make the logic more coherent; The research objective should be clearly defined as “evaluating and comparing”, and the ultimate goal should be directly stated as "exploring the genomic basis".

Materials and Methods: Real time fluorescence quantitative PCR did not mention the validation information of internal reference genes (such as stability analysis); Lipid extraction and TLC methods were cited in earlier literature (2005), and it is recommended to supplement recent methodological references; Some statistical descriptions are unclear, such as' no significant difference ', but the statistical testing method and p-value are not labeled in the figure (e.g. Figure 3C).

Results: The lipid analysis section is not in-depth enough, and the PDIM content is semi quantified by TLC and ImageJ, which is a rough method. Suggest adding quantitative methods such as HPLC-MS, or at least providing repeated experimental data and statistical analysis. The results overlap with the discussion section.

Discussion: The discussion content covers too much background information, and the content directly related to this study is not prominent enough. Suggest simplifying the background and focusing on discussing the findings of this study, such as the differences, key mutations, and immune response characteristics between MB894 and MB303; The association between genome and phenotype is weak, and although multiple SNPs/INDELs were detected, the authors did not validate the impact of these mutations on virulence through functional experiments such as gene knockout/complementation and expression analysis. Suggest clearly stating in the discussion that this is a preliminary correlation analysis that requires further experimental verification; The data already described in the results, such as CFU and pathological score, should be elaborated again in the discussion. It is recommended to simplify the repetitive descriptions in the discussion and focus on interpretation and inference; Each section (such as 3.1, 3.2) lacks a brief descriptive title. It is recommended to supplement it to enhance readability (such as “3.1 Clinical signs and organic weights”).

Figures and Table: The coordinate axis labels in Figures 1 and 2 are unclear (such as “Coefficient” not indicating the organ weight/body weight ratio), and there is a lack of complete legend explanations, such as “triangles, boxes, circles” representing which groups are not clearly stated in the titles; The resolution of the pathological image in Figure 3 is low. It is recommended to provide higher definition images or annotate key lesion areas; The format of Table 1 (Genomic Mutation List) is confusing. It is recommended to organize it into a clear three line table and supplement necessary columns such as mutation location and gene function.

References: Some of the references have inconsistent page formats in their issue numbers. It is recommended to adjust them according to the requirements of the journal; Part of the references in the article are cited as superscripts (such as ¹), while others are numbers in parentheses (such as (1)), which should be consistent with the format required by the journal; Some entries in the reference list lack DOI numbers or issue information (such as references 5 and 6).

Supplementary materials: Supplementary Tables 1-3 are mentioned in the text, but their contents are not briefly explained in the main text. It is recommended to provide additional explanations in the methods or results section.

Language expression and format: There are multiple grammar errors and unclear expressions in the manuscript, for example, “The observed data was supported by...” should be changed to “The observed data was supported by...”; It is suggested to change the title from “pulmonary TB mouse model” to “pulmonary tuberculosis mouse model” to maintain consistency in terminology throughout the text; Abbreviations that are not defined or not fully written for the first time appear multiple times in the text, such as “MB894”. When it first appears in the abstract, it should be clearly stated as “Mycobacterium bovis strain 894”; Some abbreviations were not defined when they first appeared (such as BALT, PE-PGRS).

Author Response

REVIEWER 2

Comments and Suggestions for Authors

It is suggested to modify the title to: A Porcine-Isolated Mycobacterium bovis Strain Exhibits Hypervirulence in a Murine Pulmonary Tuberculosis Model

We accepted the reviewer suggestion, and have changed the title accordingly

Abstract: It is possible to start from a global perspective and then focus on the special situation of Argentina and pigs, which will make the logic more coherent; The research objective should be clearly defined as “evaluating and comparing”, and the ultimate goal should be directly stated as "exploring the genomic basis".

We have changed the abstract, considering the suggestions of both reviewers

Materials and Methods: Real time fluorescence quantitative PCR did not mention the validation information of internal reference genes (such as stability analysis); Lipid extraction and TLC methods were cited in earlier literature (2005), and it is recommended to supplement recent methodological references; Some statistical descriptions are unclear, such as' no significant difference ', but the statistical testing method and p-value are not labeled in the figure (e.g. Figure 3C).

We thank the reviewer for the comments. Regarding quantification of cytokines, we did not use RT-PCR for cytokines-genes expression.

Conversely, we have used the BD Cytometric Bead Arrays system that is based on protein quantification. This method allows the identification of the cytokines present in the sample that can be measured using flow cytometry. The intensity of the fluorescence of the sample, reveals the concentration of a particular protein when compared with the standards for each cytokine.

We added another more recent methodological references (The Cell Wall Lipid PDIM Contributes to

Phagosomal Escape and Host Cell Exit of

Mycobacterium tuberculosis

Jeff Quigley,a V. Keith Hughitt,a,b Carlos A. Velikovsky,a,c Roy A. Mariuzza,a,c

Najib M. El-Sayed,a,b Volker Brikena)

The statistical analysis has now been clarified by adding the statistical testing method and corresponding p-values to the figure legend (e.g., Figure 3C). This information has been included to clearly support statements such as “no significant differences”

Results: The lipid analysis section is not in-depth enough, and the PDIM content is semi quantified by TLC and ImageJ, which is a rough method. Suggest adding quantitative methods such as HPLC-MS, or at least providing repeated experimental data and statistical analysis. The results overlap with the discussion section.

We appreciate the reviewer suggestion. We understand that the Image J method is not very accurate. However, in our current scenario it is not possible to perform a quantitative method such as HPLC-MS. Moreover, we just want to show that both virulent strains behave similarly in terms of PDIM content, and we don’t want to know the exact amount of PDMIs in each sample. The results shown in the figure is one representative experiment of three independent lipid extractions. And thus, we do not do statistical analysis

Discussion: The discussion content covers too much background information, and the content directly related to this study is not prominent enough. Suggest simplifying the background and focusing on discussing the findings of this study, such as the differences, key mutations, and immune response characteristics between MB894 and MB303;

The association between genome and phenotype is weak, and although multiple SNPs/INDELs were detected, the authors did not validate the impact of these mutations on virulence through functional experiments such as gene knockout/complementation and expression analysis. Suggest clearly stating in the discussion that this is a preliminary correlation analysis that requires further experimental verification;

We have shortened the discussion accordingly to the reviewer’s suggestion

The data already described in the results, such as CFU and pathological score, should be elaborated again in the discussion. It is recommended to simplify the repetitive descriptions in the discussion and focus on interpretation and inference; Each section (such as 3.1, 3.2) lacks a brief descriptive title. It is recommended to supplement it to enhance readability (such as “3.1 Clinical signs and organic weights”).

We understand that the reviewer suggests simplifying the discussion and don´t repeat what is already described in the results section. We have shortened the discussion accordingly.

We have also added a brief descriptive title in the results section

Figures and Table: The coordinate axis labels in Figures 1 and 2 are unclear (such as “Coefficient” not indicating the organ weight/body weight ratio), and there is a lack of complete legend explanations, such as “triangles, boxes, circles” representing which groups are not clearly stated in the titles;

We have changed the figures accordingly

The resolution of the pathological image in Figure 3 is low. It is recommended to provide higher definition images or annotate key lesion areas;

We improved the resolution of the figure and provides additional Supplementary Figures 1,2 and 3 to make more clear the histopathology results

The format of Table 1 (Genomic Mutation List) is confusing. It is recommended to organize it into a clear three line table and supplement necessary columns such as mutation location and gene function.

Table1 was edited, eliminating columns, but keeping the most relevant information

References: Some of the references have inconsistent page formats in their issue numbers. It is recommended to adjust them according to the requirements of the journal; Part of the references in the article are cited as superscripts (such as ¹), while others are numbers in parentheses (such as (1)), which should be consistent with the format required by the journal; Some entries in the reference list lack DOI numbers or issue information (such as references 5 and 6).

References were managed using Mendeley Reference Manager, according to the journal´s format

Supplementary materials: Supplementary Tables 1-3 are mentioned in the text, but their contents are not briefly explained in the main text. It is recommended to provide additional explanations in the methods or results section.

The Supplementary Table 1 (assembly summary statistics) is described in M&M section 2.8 Genome sequencing, assembly, and annotation

The content of Supplementary Table 2 is explained in M&M section 2.10 Phylogenetic analysis

The content of Supplementary Table 3 is described in the Results section 3.5 Comparative genomics analysis between MB894 and MB303 strains

Language expression and format: There are multiple grammar errors and unclear expressions in the manuscript, for example, “The observed data was supported by...” should be changed to “The observed data was supported by...”;

It is not clear what the reviewer wants to change, since the sentence is the same

It is suggested to change the title from “pulmonary TB mouse model” to “pulmonary tuberculosis mouse model” to maintain consistency in terminology throughout the text;

This change was done in the text

Abbreviations that are not defined or not fully written for the first time appear multiple times in the text, such as “MB894”. When it first appears in the abstract, it should be clearly stated as “Mycobacterium bovis strain 894”; Some abbreviations were not defined when they first appeared (such as BALT, PE-PGRS).

We thank the reviewer for the observation. All the abbreviations where described the first time they appear in the text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did not provide appropriate responses to my key questions with the additional experiments.

Author Response

Reviewer 1

The authors did not provide appropriate responses to my key questions with the additional experiments.

Reviewer 1 requested additional experiments, to determine the bacterial load in the bone marrow and blood.

According to the infection model, Mycobacterium sp. is measured in lungs, spleen and sometimes in liver. Accordingly, we have modified the discussion, with a paragraph from lane 481-503, providing some references for the presence of M. bovis in lungs, spleen and liver in the mouse model.

They also suggested performing in vitro experiments to determine the survival in the macrophages.

We have previously performed in vitro macrophage experiments derived from porcine and the results were inconclusive, so we first decided not to include them in the manuscript. We have now included this information in the manuscript and provide the data as supplementary material (Supplementary Figure S4).

Reviewer 2 Report

Comments and Suggestions for Authors The manuscript has been sufficiently improved to warrant publication in Biology.

Author Response

The manuscript has been sufficiently improved to warrant publication in Biology