Progression of Trypanosoma cruzi Dm28c Strain Infection in a BALB/c Mouse Experimental Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes infection progression in an experimental mouse model of low virulence T. cruzi infection, which the authors state could be useful for studying disease progression from acute to chronic phases. However, the analyses was limited only to blood and cardiac parasite levels, cardiac cellular infiltrate, and splenic levels of a limited panel of cytokines. While the data presented is informative, the manuscript could be strengthened by addressing the questions and comments listed below.

The introduction could be strengthened by addressing the following comments:

1. What is the original source of the Dm28c strain? Was it from a vector, animal reservoir, or human patient?
2. Why would the Dm28c strain as a representation of DTU TcI be useful as a model of disease, as opposed to a DTU TcVI strain that may cause more severe disease?
3. Why is the BALB/c mouse particularly beneficial as an animal model of T. cruzi infection? What immune profile is characteristic of this strain and why is it beneficial for experimental T. cruzi infection studies?

The discussion could be strengthened by addressing the following questions.

1. The results showed that parasitemia was not detectable by microscopy at 21 dpi and cardiac inflammation was mild by 56dpi. However, T. cruzi causes a systemic infection and persists in other tissues, such as adipose tissue and gastrointestinal tissue. Why was only heart tissue examined in these studies?
2. The differences in cardiac pathology between different experimental infection models, such as Sylvio X10/4 infection in C3H/He mice vs C57BL/6 mice, are mentioned, but there is no discussion of the significance of the genetic differences in these mouse models. Please discuss the specific immunologic difference in these mouse models and how that is likely affecting the clinical progression of experimental infection.
3. It is indicated in line 307 that by 56 dpi cardiac pathology in Dm28c infected BALB/c mice is largely resolved. However, cardiac fibrosis with low grade inflammation is a hallmark of chronic T. cruzi infection leading to cardiac dysfunction. Why was fibrosis not evaluated at the 56dpi timepoint to determine if the pathology had evolved to progressive fibrosis?
4. Please discuss the limitations of this study and answer the following questions:
   1. Why were only male mice used to characterize infection progression?
   2. Why was gene expression only performed on splenic tissue and not also cardiac tissue to determine tissue specific immune responses to parasites?
   3. Discuss the limitations of evaluating gene expression only and not complementing this with levels of cytokines using ELISA or similar assays.

Author Response

Response to Reviewer 1 Comments

1. Summary

 Thank you very much for taking the time to review this manuscript. We ´ve considered your valuable suggestions and comments, and incorporated modifications in the manuscript accordingly. We evaluated fibrosis in cardiac tissues as suggested. The micrographs of cardiac tissues stained with Picrosirius Red/Fast Green, which show that infection with Dm28c did not progress to fibrotic remodeling during the study period, have been included in Appendix 1 and references to these results are mentioned in the text in the revised version.

Please find the detailed responses below. The corresponding corrections in the re-submitted files are highlighted (Revised document with changes highlighted.pdf) and in track changes (Revised document with track changes highlighted.docx) respectively. We thank you for all suggestions since they clearly contributed to improve the paper.

3. Point-by-point response to Comments and Suggestions for Authors

(Authors´ Responses in red)

Comments 1:

This manuscript describes infection progression in an experimental mouse model of low virulence T. cruzi infection, which the authors state could be useful for studying disease progression from acute to chronic phases. However, the analyses were limited only to blood and cardiac parasite levels, cardiac cellular infiltrate, and splenic levels of a limited panel of cytokines. While the data presented is informative, the manuscript could be strengthened by addressing the questions and comments listed below.

Response 1:

Thank you for pointing this out. While our study focused on early infection dynamics using a low-virulence T. cruzi strain, we recognize the limitations of our approach and have expanded both the Introduction and Discussion to clarify the rationale, scope, and future directions of the model.

Comments 2:

The introduction could be strengthened by addressing the following comments:

1. What is the original source of the Dm28c strain? Was it from a vector, animal reservoir, or human patient?

The Dm28 strain has a sylvatic origin and was first isolated from the opossum Didelphis marsupialis. The Dm28c clone used in this study was selected based on key trypomastigote-stage biological traits, including the ability to infect triatomine vectors and vertebrate hosts, invade cultured cells, resist macrophage digestion, and complement lysis, and differentiate in vitro and has been extensively characterized in different laboratories, including ours.

Information regarding the strain origin has been added in the Introduction section (lines 87-90) and cited in Ref #12.

2. Why would the Dm28c strain as a representation of DTU TcI be useful as a model of disease, as opposed to a DTU TcVI strain that may cause more severe disease?

Considering the observed variability between animal models and the consequent limitations of generalizing results, our study does not intend to replace or oppose to other animal models, but to complement available information regarding Chagas disease. It is accepted that different factors, including the parasite strain, can influence pathogenesis, therapeutic response, and the final outcome of the infection. The different strains and DTUs also differ in organ tropism and geographical distribution. Although DTU TcVI strains are often associated with severe disease, TcI strains are widespread in the Americas, can be found in both domestic and wild transmission cycles and are frequently linked to cardiac forms. Moreover, since only aproximately 30% of infected people develop symptomatic chronic Chagas disease, mild or low virulence models are relevant from an epidemiological point of view. Also, the low-virulence Dm28c model offers a valuable system to investigate immune mechanisms driving disease progression in a controlled setting. Finally, as addressed in the Discussion section, we believe that this non-lethal model could contribute to a better understanding of the complex variability observed in T. cruzi infections. Characterizing different animal models individually should enhance our understanding of the huge variability observed in humans too.

The importance of this model was also addressed in the introduction: “Because of the low to moderate virulence, the Dm28c-infection model allows the study of the disease progression through the acute to chronic phase in contrast to more virulent highly mortal strains.” (lines 99-101)

3. Why is the BALB/c mouse particularly beneficial as an animal model of T. cruzi infection? What immune profile is characteristic of this strain and why is it beneficial for experimental T. cruzi infection studies?

The BALB/c and C57BL/6 mouse strains are both commonly used in our laboratory for T. cruzi infection studies. While the C57BL/6 strain is known for its relative resistance to infection, BALB/c mice typically develop marked parasitemia during the acute phase. Preliminary experiments in our hands showed that T. cruzi Dm28c infection in C57BL/6 mice resulted in undetectable parasitemia levels. In contrast, BALB/c mice consistently exhibited detectable parasitemia, enabling the monitoring of parasite burden and disease progression. Additionally, the BALB/c strain is known to mount a well-characterized Th2/Th1 immune response, making it a valuable and complementary model for studying host–pathogen interactions, particularly in infections with low-virulence T. cruzi strains such as Dm28c.

This clarifying information has been added in the Introduction and Reference #13 was included accordingly (lines 92-98).

Comments 3:

The discussion could be strengthened by addressing the following questions.

1. The results showed that parasitemia was not detectable by microscopy at 21 dpi and cardiac inflammation was mild by 56dpi. However, T. cruzi causes a systemic infection and persists in other tissues, such as adipose tissue and gastrointestinal tissue. Why was only heart tissue examined in these studies?

In this study, we focused our analysis on heart tissue because Chagas cardiomyopathy is the most clinically relevant manifestation of chronic T. cruzi infection. One of the main aims of the study was to evaluate early signs of myocardial damage in a low-virulence model. Although we acknowledge that the Dm28c strain exhibits tissue tropism beyond the heart, including gastrointestinal involvement in BALB/c mice, the present study was specifically designed to assess cardiac parasitism and general inflammatory response. Future studies will be necessary to evaluate parasite persistence and the associated inflammation in other tissues.

2. The differences in cardiac pathology between different experimental infection models, such as Sylvio X10/4 infection in C3H/He mice vs C57BL/6 mice, are mentioned, but there is no discussion of the significance of the genetic differences in these mouse models. Please discuss the specific immunologic difference in these mouse models and how that is likely affecting the clinical progression of experimental infection.

As recommended, we have expanded the Discussion section (lines 306-352) to include a detailed explanation of how the distinct immune responses of C3H/He and C57BL/6 mice may contribute to the differences in cardiac pathology. We now highlight that C3H/He mice tend to mount a more robust Th1-type response, which may promote parasite control but also exacerbate immunopathology, leading to severe myocarditis. In contrast, C57BL/6 mice show a more balanced response, with higher expression of IL-10 that in this context is associated with a protective effect, reducing tissue damage, and contributing to the milder cardiac phenotype observed in T. cruzi Sylvio X10/4.

3. It is indicated in line 307 that by 56 dpi cardiac pathology in Dm28c infected BALB/c mice is largely resolved. However, cardiac fibrosis with low grade inflammation is a hallmark of chronic T. cruzi infection leading to cardiac dysfunction. Why was fibrosis not evaluated at the 56dpi timepoint to determine if the pathology had evolved to progressive fibrosis?

We thank the reviewer for this observation. Indeed, cardiac fibrosis is one of the most relevant pathological features of the chronic phase of Chagas disease, both in humans and murine models. In mice, fibrosis is typically minimal or absent during the acute phase (0–30 dpi) and tends to increase during the chronic phase, depending on both the parasite strain and the host genetic background. For example, rrevious studies using strains such as Y, Colombiana, or CL Brener have demonstrated significant cardiac fibrosis, while infections with low-virulence strains, may show a slower or even self-limited progression toward fibrosis.

In agreement with the reviewer comments, we considered it important to evaluate the extent of fibrosis in cardiac tissue during both the acute and early chronic phases in our model, so we performed Picrosirius Red/Fast Green staining on cardiac slices corresponding to different days post-infection analyzed in our study, which was added to the manuscript as Appendix A. The staining revealed no evidence of fibrosis at any of the analyzed time points, suggesting that, as with other low-virulence strains, infection with Dm28c did not progress to fibrotic remodeling in BALB/c mice during the study period (Appendix, Fig. A1). References to this new assay and the observed lack of fibrosis, were included in the text:

Material and Methods (line 159): “Additionally, cardiac fibrosis was assessed using Picrosirius Red/Fast Green staining.”

Results (lines 233-236): “Additionally, Picrosirius Red/Fast Green staining revealed no evidence of fibrosis at any of the analyzed time points, indicating that infection with Dm28c did not progress to fibrotic remodeling during the study period (Fig. A1).”

Discussion (lines 355-358): “Previous studies using strains such as Y, Colombiana, or CL Brener have demonstrated significant cardiac fibrosis [25,26]. In contrast, infections with low-virulence strains may show a slower or even self-limited progression toward fibrosis. In our model, no cardiac fibrosis was detected during the acute phase or at 56 dpi.”. Also, References #25 and #26 have been added.

4. Please discuss the limitations of this study and answer the following questions:

1. Why were only male mice used to characterize infection progression?

We agree with the reviewer that using only male mice may be a limitation of our study. However, when designing the study, we deliberately decided to exclude female animals since  it is known that immune response may behave differently in male and female mammals. Previous assays form our lab, suggest that using both male and female mice with a small “n” (total numbero of animals) may lead to high dispersion of data due to the hormonal interference associated with female sex hormones, thus making results difficult to be interpreted. Therefore, by evaluating disease progression only in male mice we reduced the total number of animals, according to ARRIVE guidelines. Nevertheless, it would be interesting to assess the sexual dimorphism exhibited by Dm28c strain in BALB/c mice and to determine whether Dm28c strain exhibits sex-specific differences in pathogenesis. The inclusion of gender as another variable will be considered in future studies.

In the text, this limitation is addressed in lines 109-111: “Only male BALB/c mice were used to minimize variability associated with sex-related hormonal fluctuations, which can modulate immune responses and influence the course of T. cruzi infection.”

2. Why was gene expression only performed on splenic tissue and not also cardiac tissue to determine tissue specific immune responses to parasites?

We focused on the spleen because it is a central immune organ involved in systemic responses during acute infection. It plays a key role in both innate and adaptive immunity and is pivotal for parasite clearance during the acute phase. Evaluating splenic cytokine gene expression allowed us to infer the systemic immune response to infection. Although cardiac tissue is likely the primary target organ in Chagas disease, our main aim in this study was to provide an overview of immune activation rather than organ-specific responses. We agree that cardiac gene expression would offer additional insights and plan to include this in future studies.

In the manuscript, this rationale is summarized as: “Given the spleen’s central role in systemic immunity during acute T. cruzi infection, we analyzed cytokine expression at the mRNA level in this tissue to capture broader immune trends complementing heart histology.” (lines 367-370)

3. Discuss the limitations of evaluating gene expression only and not complementing this with levels of cytokines using ELISA or similar assays.

Our decision to focus on cytokines gene expression was guided by the scope and the available resources for this study, aiming to have an overview of the systemic inflammatory response during infection. We acknowledge that mRNA levels do not always directly correlate with protein abundance due to post-transcriptional and post-translational regulation, thus the method employed may be considered a limitation of our work. We agree that cytokines´ levels confirmation using ELISA, multiplex bead arrays, or similar methods would provide a more complete picture of cytokine activity and functional immune responses, however, we consider that our transcriptional data provide insights into cytokine-mediated inflammatory regulation. In fact, in previous studies from our lab, we analyzed cytokine gene expression by RT-qPCR to infer the inflammatory response both in vitro and in vivo (Cribb et al., 2017; https://doi.org/10.1371/journal.pntd.0005350).

This limitation was addressed in the text (lines 370-372): “Although protein-level quantification (e.g., by ELISA) was not performed, the gene expression data provide a reliable overview of the dynamics of immune activation in this low-virulence infection model.”

4. Response to Comments on the Quality of English Language

Both reviewers marked:

The English is fine and does not require any improvement.

5. Additional clarifications:

As mentioned in the cover letter, we included additional information regarding the use of animals in this study as suggested by the editor, according to ARRIVE guidelines.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The entry manuscript (MS) “Progression of Trypanosoma cruzi Dm28c strain infection in a 2 BALB/c mouse experimental model” ID parasitologia-3783343 fits very well in the in the scope of Pathogens MDPI. The main text comprises an excellent MS about a mice infection model to study T. cruzi Dm28c strain. You should be proud of the study submitted to this Journal. After a detailed evaluation of the manuscript, my major suggestion comprises the insertion of more references during the main. Additional experimental studies may be discussed, specially papers employing different strains not mentioned in this MS

Finally, please solve small points before the final acceptance:

• The keywords should provide an alternative way to find studies from similar topics. Therefore, the addition of distinct words from the title could help other researches to find your manuscript. Please, consider providing just keywords not presented in the title. As suggestion, you could be use words like “Inflammatory response”, “parasitemia” “non-lethal” “spleen”;
• Please, write what is CONICET and UNR in full at the Author´s description;
• In the first paragraph of the Introduction section, the references should be added in the final of the sentences, not just at the end of the paragraph. In addition, there are three different issues: epidemiology, clinical phases of Chagas disease and treatment. This suggests that three different references should be provided during this paragraph (lines 35-57). Please, don´t skimp on the references in the entire MS;
• In line 78, consider replace “As with” to “Similar to” or just “As”;
• The paragraph of line 79 should not be separated from the previous one. Please, consider not dividing the sentences into many paragraphs during the main text of the MS;
• The first subsection of the Methodology don´t need the word infections. Just “Mice and parasites” seems more adequate;
• In addition, this subsection describing the trypomastigotes’ acquisition should be referenced;
• There are so many paragraphs in the second Methodology subsection, “Experimental infection”. Please, consider merging the first and the second paragraphs;
• In line 179 from the third Methodology subsection, replace “sacrificed” to “"euthanized animals (or mice)";
• In the last Methodology subsection, insert at least one reference;
• During the Results section, the division in additional subsections (like "3.1.1"...) of the subsection 3.1 is unnecessary. Please, remove it.
• The paragraphs from lines 183 and 184 could be merged.
• The Figure 1 could have an elongated X axis and don´t need a number of 150 in the Y axis. Please, consider making a better figure;
• Table 2 should have a minor title. Information about the number of parasites, score, sections and days post-infection may be given as a table footnote. Please, consider a better formation of table 2;
• In the Discussion section, it´s not necessary to specify “(Figure 1)” in line 272, since the description of the result was already be presented in the Results section;
• The second reference, “WHO”, should have the date of the access the website.

Author Response

Response to Reviewer 2 Comments

1. Summary

We thank Reviewer 2 for their positive evaluation of our work and for the constructive suggestions that have helped improve the clarity, formatting, and completeness of our manuscript. We have addressed all points raised, as detailed below, and believe these changes have strengthened the final version. Additional experimental studies employing different strains have been considered to improve the Discussion, and new references have been in included both in Introduction and Discussion sections. The corrections in the re-submitted files are highlighted (Revised document with changes highlighted.pdf) and in track changes (Revised document with track changes highlighted.docx) respectively.

3. Point-by-point response to Comments and Suggestions for Authors

(Authors´ Responses in red)

Comments 1:

The keywords should provide an alternative way to find studies from similar topics. Therefore, the addition of distinct words from the title could help other researches to find your manuscript. Please, consider providing just keywords not presented in the title. As suggestion, you could be use words like “Inflammatory response”, “parasitemia” “non-lethal” “spleen”;

Response 1:

We have revised the keywords to remove those already present in the title and included additional terms suggested by the reviewer.

New Keywords: TRYPANOSOMA, INFLAMMATORY RESPONSE, PARASITEMIA, NON-LETHAL, SPLEEN

Comments 2:

Please, write what is CONICET and UNR in full at the Author´s description;

Response 2:

We have added the full name of each institution. Lines 6 to 13.

Comments 3:

In the first paragraph of the Introduction section, the references should be added in the final of the sentences, not just at the end of the paragraph. In addition, there are three different issues: epidemiology, clinical phases of Chagas disease and treatment. This suggests that three different references should be provided during this paragraph (lines 35-57). Please, don´t skimp on the references in the entire MS;

Response 3:

            Additional references have been incorporated in the revised manuscript (2, 4, 5, 12-14, 17, 24-26). New references provide relevant information about Chagas Disease epidemiology, clinical phases and treatment, parasite and mouse strains, related assays and different Chagas experimental models. Each reference was included at the end of the sentence.

Comments 4:

In line 78, consider replace “As with” to “Similar to” or just “As”;  

Response 4:

We have replaced “As with” to “Similar to”, line 80. 

Comments 5:

The paragraph of line 79 should not be separated from the previous one. Please, consider not dividing the sentences into many paragraphs during the main text of the MS;

Response 5:

This modification has been added, the number of paragraphs in the Introduction section were optimized.. 

Comments 6:

The first subsection of the Methodology don´t need the word infections. Just “Mice and parasites” seems more adequate;

Response 6:

We agree with the reviewer and this title has been modified to “Mice and parasites”.

Comments 7:

In addition, this subsection describing the trypomastigotes’ acquisition should be referenced;

Response 7:

A citation has been added to the subsection detailing the acquisition of trypomastigotes, to suport the methodology used.

Comments 8:

There are so many paragraphs in the second Methodology subsection, “Experimental infection”. Please, consider merging the first and the second paragraphs;

Response 8:

            The number of paragraphs in the “Experimental Infection” subsection, has been reduced.

Comments 9:

In line 179 from the third Methodology subsection, replace “sacrificed” to “"euthanized animals (or mice)";

Response 9:      

The term “sacrificed” animals has been replaced in the text, line 153.

Comments 10:

In the last Methodology subsection, insert at least one reference;

Response 10:    

The citation by Mishra et al. (2019) [17], which discusses the application of Student’s t-test, ANOVA, and covariance, has been added to the Statistical Analysis section to provide appropriate reference for the statistical methods employed in this study.

Comments 11:

During the Results section, the division in additional subsections (like "3.1.1"...) of the subsection 3.1 is unnecessary. Please, remove it. 

Response 11:    

In the revised version, Results have been reordered for more clarity. We removed the subsections and it is now divided only in Sections such as “3.1, 3.2, etc”.

Comments 12:

The paragraphs from lines 183 and 184 could be merged.

Response 12:

This modification has been included and additional information regarding to the use of animals was added.

Comments 13:

The Figure 1 could have an elongated X axis and don´t need a number of 150 in the Y axis. Please, consider making a better figure;

Response 13:

Figure 1 has been modified accordingly, with an elongation in X axis and the removal of the 150 value from the Y axis to improve clarity.

Comments 14:

Table 2 should have a minor title. Information about the number of parasites, score, sections and days post-infection may be given as a table footnote. Please, consider a better formation of table 2;

Response 14:

The table has been modified according to the suggestions provided. It has now a title and the information is explained in a footnote.

Comments 15:

In the Discussion section, it´s not necessary to specify “(Figure 1)” in line 272, since the description of the result was already be presented in the Results section;

Response 15:

We have removed all figure references from the Discussion section, including the mention of “(Figure 1)”, as the corresponding results are already described in the Results section.

Comments 16:

The second reference, “WHO”, should have the date of the access the website

Response 16:

The date of access has been added to the reference using the format suggested by the journal. The WHO citation is now numbered as Ref #1.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revisions have sufficiently addressed my comments and the manuscript is much improved.