Non-Specific Cross Protection of BCG Vaccination in Dairy Calves

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents important results regarding the disease of concern, with implications not only for cattle but also for human health. The authors provided a very interesting introduction that highlights the importance of BCG vaccination and its potential to generate beneficial impacts against other infectious diseases. The methodology, however, is somewhat unclear, mainly due to the lack of a timeline, although the authors did apply relevant methods to address their objectives.

 

My main concern lies in the results section, which is not well presented and remains confusing. Some points discussed refer to results that were not provided (particularly concerning RNA and gene expression). On the other hand, the supplementary materials are well described, which suggests that the authors have sufficient data to build a clearer and stronger results section.

 

Overall, I recommend that the authors restructure the results before this manuscript can be considered for publication.

Line 83–85: Are you referring to the identification or to the isolation itself (including viral isolation of rotavirus)? Are those pathogens more frequently identified in which region (central Chile, whole Chile, Latin America, worldwide)?

 

Line 94: Which type of confinement? How was this farm characterized as positive for tuberculosis?

 

Line 107: I did not understand the timing of those vaccinations. Did they occur at a single time point in all 96 male calves, or were the vaccinations performed gradually throughout the year?

 

Line 176: The results section is somewhat confusing. I suggest that the authors start by presenting the number of animals that tested PCR-positive for the genetic targets, and then proceed to each evaluation, associating it with the mortality. In summary, it would be better if the authors begin the results with some descriptive data, then introduce the analytical ones. The results of DNA RT-PCR and RNA rt-RTPCR should be disclosed separately. 

 

Line 177: Again, I did not understand the timeline of your study. I think it would be clearer to the reader if you provided a timeline. 

 

Line 241 - 242: Please standardize the scientific names in italic.

 

Line 255-257:  In this sentence, the authors hypothesized a different host-pathogen interaction regarding the expression of stx1 gene, but the results of expression (RNA) are not provided in the Results section. 

 

Line 278: At the beginning of the methodology, the authors reported that the herd was characterized as positive for tuberculosis. However, in the discussion, it is stated that the infection status of the calves was not evaluated prior to vaccination. Is there any information available regarding the infection status of the dams of these animals? Moreover, is there any evidence that the vaccine fulfilled its main purpose, i.e., preventing tuberculosis in a high-prevalence herd?

Author Response

Comments and Suggestions for Authors

This manuscript presents important results regarding the disease of concern, with implications not only for cattle but also for human health. The authors provided a very interesting introduction that highlights the importance of BCG vaccination and its potential to generate beneficial impacts against other infectious diseases. The methodology, however, is somewhat unclear, mainly due to the lack of a timeline, although the authors did apply relevant methods to address their objectives.

 

Comments 1: My main concern lies in the results section, which is not well presented and remains confusing.

Response 1: In order to improve the clarity of this section, we have added three supplementary tables, describing results for the explanatory variables (vaccination group, season of birth and breed), and replaced the table 2 for showing the detection frequencies of target genes at 3 and 6 months of age. Additionally, we have changed the writing of main paragraphs in this section, including also OR values to facilitate the interpretation of results.

 

Comments 2: Some points discussed refer to results that were not provided (particularly concerning RNA and gene expression).

Response 2: In this work we did not analyze the gene expression. In methodology we described the RNA isolation process for the detection of the rotaviral nsp5 sequence through reverse transcription real-time PCR (indicated in line 135).

 

Comments 3: On the other hand, the supplementary materials are well described, which suggests that the authors have sufficient data to build a clearer and stronger results section.

Response 3: As indicated previously, we added three additional supplementary tables for a better description of variables.

Comments 4: Overall, I recommend that the authors restructure the results before this manuscript can be considered for publication.

Response 4: As indicated in the response of comment N°1, changes were made for improving the section of results.

Comments 5: Line 83–85: Are you referring to the identification or to the isolation itself (including viral isolation of rotavirus)?

Response 5: We were referring to the identification. We replaced “isolated” by “identified”

Comments 6: Are those pathogens more frequently identified in which region (central Chile, whole Chile, Latin America, worldwide)?

Response 6: Unfortunately, in Chile we do not have recent literature about main infectious causes of diarrhea and pneumonia. Our statement is based on the reference 23, which now has been indicated in the corrected version for clarity.

Comments 7:Line 94: Which type of confinement? How was this farm characterized as positive for tuberculosis?

Response 7: Animals were maintained in permanent confinement, which consisted on cows being housed indoors year-round, with restricted movement between pens and the milking parlor. And the sanitary condition about tuberculosis is defined by the official veterinary service, in this farm by detecting suspicious lesions in the slaughterhouse that were later confirmed with bacteriological isolation. Once the herd is classified in this category, it is expected that the owner implements the classical skin testing and elimination scheme for control. However, this farm made the last skin testing several years ago, without efforts for controlling the disease and, consequently, maintaining its “positive” condition for tuberculosis. Unfortunately the national control and eradication program is still working under voluntary adscription. 

Comments 8: Line 107: I did not understand the timing of those vaccinations. Did they occur at a single time point in all 96 male calves, or were the vaccinations performed gradually throughout the year?

Response 8: It was the second alternative. We were doing monthly visits to the farm, each time finding between 20-30 neonatal calves which were enrolled into one group (BCG or control), so all animals were gradually vaccinated during a period of 7 months.

Comments 9: Line 176: The results section is somewhat confusing. I suggest that the authors start by presenting the number of animals that tested PCR-positive for the genetic targets, and then proceed to each evaluation, associating it with the mortality. In summary, it would be better if the authors begin the results with some descriptive data, then introduce the analytical ones. The results of DNA RT-PCR and RNA rt-RTPCR should be disclosed separately. 

Response 9: As recommended, we have included a new table 2 for showing detection frequencies of target genes. Their distribution about categories of variables under study (vaccination group, season of birth and breed of animals) were included in three supplementary tables (4, 5 and 6, respectively). Additionally we made substantial changes in the writing of paragraphs, so we believe the text is clearer now than the original version.

Comments 10: Line 177: Again, I did not understand the timeline of your study. I think it would be clearer to the reader if you provided a timeline. 

Response 10: We have made changes in this paragraph for clarification. In methodology, between lines 100 and 112, we do believe that the description of timeline of the study is clear.  However, in case the reviewer still considers the need for more changes, we will be willing to work on that.

Comments 11: Line 241 - 242: Please standardize the scientific names in italic.

Response 11: These and other scientific names were corrected and written in italics.  

Comments 12: Line 255-257:  In this sentence, the authors hypothesized a different host-pathogen interaction regarding the expression of stx1 gene, but the results of expression (RNA) are not provided in the Results section. 

Response 12: As mentioned in the comment N°2, we did not analyze the gene expression in this work

Comments 13: Line 278: At the beginning of the methodology, the authors reported that the herd was characterized as positive for tuberculosis. However, in the discussion, it is stated that the infection status of the calves was not evaluated prior to vaccination. Is there any information available regarding the infection status of the dams of these animals? Moreover, is there any evidence that the vaccine fulfilled its main purpose, i.e., preventing tuberculosis in a high-prevalence herd?

Response 13: Because the farmer was not implementing skin test for the diagnosis of bovine tuberculosis in the last years, we could not know the infectious status of dams.

In relation to the BCG efficacy, we included this herd in a previous analysis vaccinating female calves in the first month after birth, and then making a follow up through blood testing with a DIVA-IGRA test during 18 months to evaluate their infection status. The efficacy of BCG was calculated in 42.2%. The description of this study appears in a previous publication (https://pubmed.ncbi.nlm.nih.gov/35565515/), in which this herd was identified as H1.

Reviewer 2 Report

Comments and Suggestions for Authors

Well written manuscript, study design is scientifically sound to answer the study questions.

See further comments on the attachment for authors.

Comments for author File: Comments.pdf

Author Response

Comments 1: Well written manuscript, study design is scientifically sound to answer the study questions.

Further comments on the attachment for authors:

The study is novel, justifiable and should contribute to a better understanding of the non-specific cross protection from vaccinating dairy calves. My congratulations to the authors for a well written manuscript.

The introduction engages the readers and sets the stage of the study from a broad understanding of the research field to the specific focus of your study. The materials and methods is sound and appropriate to answer the stated study objectives. The results section summarizes and presents the findings of the study in context with study research question(s). The data is presented in a logical sequence without bias or interpretation based on the study methodology. In the discussion the authors did a good job interpreting the result drawn from the study design,

Response 1: Thank you for the comments.

Reviewer 3 Report

Comments and Suggestions for Authors

Review Report

 Non-specific cross protection of BCG vaccination in dairy calves

 

Brief summary:

In the manuscript, the authors evaluate the non-specific protection of BCG vaccination in calves by monitoring pathogen-associated genes. A total of 186 calves were enrolled, with 96 vaccinated subcutaneously (BCG Russia strain) within 30 days of birth and 90 left unvaccinated. Fecal samples collected at 3 and 6 months were tested for Salmonella enterica (invA), E. coli Shiga toxins (stx1, stx2), and bovine rotavirus (nsp5). Logistic regression revealed higher detection of the stx1 gene at 3 months in unvaccinated calves and in those born during the cold season. Kaplan–Meier analysis showed reduced mortality from diarrhea and pneumonia in vaccinated calves (p=0.018). The authors conclude that BCG may provide non-specific protection during the first months of life under field conditions.

 

Broad comments:

The study focuses on a limited number of pathogen-associated genes detected in fecal samples. While this provides useful information, the interpretation of the results remains restricted, as no data on the overall microbial community composition or diversity are included.

The information on calf mortality is rather limited, as the authors only state that some deaths were attributed to pneumonia and others to diarrhea. However, no further details are provided on the etiological agents involved. For example, in calves that died of diarrhea, it would be highly relevant to know whether E. coli, rotavirus, or Salmonella were identified, as this information could directly support or contradict the molecular findings reported in the study. Including such data would greatly enhance the robustness and interpretability of the results.

 

Specific comments:

Abstract

The abbreviation “BCG” should be written in full (Bacillus Calmette Guérin) the first time it appears in the manuscript.

Lines 23-25: In the last sentence of the abstract, please consider rephrasing as “….in vaccinated calves compared with non-vaccinated animals (p=0.018)” to make the comparison explicit.

 

 Introduction

Bovine tuberculosis (bTB) eradication programs in many countries rely on the test-and-cull strategy, which involves the mandatory culling of animals that test positive, although this approach faces specific challenges in central Chile, as the authors state later in the manuscript.

Therefore, in the Introduction (Lines 30-33), please consider adding “mandatory culling of animals” in the sentence, between “export restrictions” and “condemnations”, to better describe the economic impact of eradication programs and provide an international perspective on the main aspect of bTB eradication programs worldwide.

 

In the Introduction the authors describe several beneficial effects of BCG in humans. For completeness, a brief mention of the use of BCG in cancer treatment would be advisable, for example its well-established role in non-muscle-invasive bladder cancer, where BCG remains the gold standard of intravesical immunotherapy (Pettenati, C., and Ingersoll, M.A. 2018. “Mechanisms of BCG immunotherapy and its outlook for bladder cancer”. Nature Reviews Urology 15: 615–625. https://doi.org/10.1038/s41585-018-0055-4 .

Lines 71–74, the sentence reads:

“This area is characterized by dairy herds with intensive production systems, which owners are unwilling to cull reactor animals due to lack of compensation, and consequently the Plan faces difficulties for cleaning up infected farms.”

Please consider rephrasing the sentence by adding “despite legal requirements” after “which owners” and ending with “ultimately hindering the eradication of the disease”, in order to better emphasize both the lack of compliance and its negative consequences.

For completeness, since the study involves BCG vaccination in cattle, it would be important to mention the well-known difficulty of differentiating vaccinated from infected animals in vivo. This issue has major implications for bovine tuberculosis control and eradication programs, where reliable diagnostic tools are essential. A brief reference to the concept of DIVA (Differentiating Infected from Vaccinated Animals) tests, and their potential use to overcome this limitation, would add valuable context to the Introduction.

 

Material and methods

In the Materials and Methods section, please consider adding sub-sections (e.g., study population, experimental design, biomolecular methods, statistical analysis) to improve clarity.

In Lines 167–171, calf mortality is described. As mentioned earlier, it would be important to provide more detailed information on the causes of death. In particular, the authors should explain why “the etiological diagnosis was not performed” and, if possible, indicate whether any post-mortem findings, or laboratory data were available to support the attribution of deaths to diarrhea or pneumonia. This would considerably strengthen the reliability of the results.

Please clarify whether diseased calves received any treatment; if so, specify the type, and if not, state this explicitly.

 

Results

In the Results section, particularly in the tables, the presentation of statistically significant results should be made clearer. For example, significant differences could be highlighted with an asterisk or another appropriate marker directly in the tables.

 

Discussion

Lines 258-261

The reference to changes in the “gut microbiota” may not be fully appropriate in the context of this study, as no microbiota analysis was performed. You may consider rephrasing or qualifying this statement.

If not already addressed in the Introduction, a brief mention in the Discussion of the difficulty in differentiating vaccinated from infected animals and the possible use of DIVA tests would also be appropriate.

 

 

 

 

Author Response

Brief summary:

In the manuscript, the authors evaluate the non-specific protection of BCG vaccination in calves by monitoring pathogen-associated genes. A total of 186 calves were enrolled, with 96 vaccinated subcutaneously (BCG Russia strain) within 30 days of birth and 90 left unvaccinated. Fecal samples collected at 3 and 6 months were tested for Salmonella enterica (invA), E. coli Shiga toxins (stx1, stx2), and bovine rotavirus (nsp5). Logistic regression revealed higher detection of the stx1 gene at 3 months in unvaccinated calves and in those born during the cold season. Kaplan–Meier analysis showed reduced mortality from diarrhea and pneumonia in vaccinated calves (p=0.018). The authors conclude that BCG may provide non-specific protection during the first months of life under field conditions.

 

Broad comments:

Comments 1: The study focuses on a limited number of pathogen-associated genes detected in fecal samples. While this provides useful information, the interpretation of the results remains restricted, as no data on the overall microbial community composition or diversity are included.

Response 1: we agree with the reviewer and because of that we were interpreting and concluding only about the genes analyzed. An analysis of microbial community composition exceeds the aim of this work.

 

Comments 2: The information on calf mortality is rather limited, as the authors only state that some deaths were attributed to pneumonia and others to diarrhea. However, no further details are provided on the etiological agents involved. For example, in calves that died of diarrhea, it would be highly relevant to know whether E. coli, rotavirus, or Salmonella were identified, as this information could directly support or contradict the molecular findings reported in the study. Including such data would greatly enhance the robustness and interpretability of the results.

Response 2: We agree with the reviewer. This information could be very useful to suggest or hypothesize about underlying non-specific immune mechanisms attributed to BCG, protecting animals against deadly pathogens in calves. However, the etiological diagnosis of death was not implemented in the farm, due to economic issues. Unfortunately the routinary procedure was the application of certain wide-spectrum antimicrobials, without any specific analysis. This weakness of the work was indicated in line 170-171.

  

Specific comments:

Abstract

Comments 3: The abbreviation “BCG” should be written in full (Bacillus Calmette Guérin) the first time it appears in the manuscript.

Response 3: It was corrected

Comments 4: Lines 23-25: In the last sentence of the abstract, please consider rephrasing as “….in vaccinated calves compared with non-vaccinated animals (p=0.018)” to make the comparison explicit.

Response 4: The phrase was modified for clarification.

 

 Introduction

Comments 5: Bovine tuberculosis (bTB) eradication programs in many countries rely on the test-and-cull strategy, which involves the mandatory culling of animals that test positive, although this approach faces specific challenges in central Chile, as the authors state later in the manuscript.

Therefore, in the Introduction (Lines 30-33), please consider adding “mandatory culling of animals” in the sentence, between “export restrictions” and “condemnations”, to better describe the economic impact of eradication programs and provide an international perspective on the main aspect of bTB eradication programs worldwide.

Response 5: We agree with the reviewer. The suggested phrase was incorporated.

Comments 6: In the Introduction the authors describe several beneficial effects of BCG in humans. For completeness, a brief mention of the use of BCG in cancer treatment would be advisable, for example its well-established role in non-muscle-invasive bladder cancer, where BCG remains the gold standard of intravesical immunotherapy (Pettenati, C., and Ingersoll, M.A. 2018. “Mechanisms of BCG immunotherapy and its outlook for bladder cancer”. Nature Reviews Urology 15: 615–625. https://doi.org/10.1038/s41585-018-0055-4 .

Response 6: We agree with the reviewer because the BCG effect on cancer treatment implies a very notorious non-specific benefit from this vaccine. We have added a brief mention in line 45. However, we preferred to maintain the reference of Covian et al., 2019 to support this phrase, because in this review the authors not only mention but also describe the underlying mechanisms of BCG protection against tumor cells in the specific type of cancer indicated by the reviewer.

 

Comments 7: Lines 71–74, the sentence reads:

“This area is characterized by dairy herds with intensive production systems, which owners are unwilling to cull reactor animals due to lack of compensation, and consequently the Plan faces difficulties for cleaning up infected farms.”

Please consider rephrasing the sentence by adding “despite legal requirements” after “which owners” and ending with “ultimately hindering the eradication of the disease”, in order to better emphasize both the lack of compliance and its negative consequences.

Response 7: We partially agree with the reviewer. In Chile, the control plan is voluntary, so owners decide whether to follow its requirements and cull reactor animals. On the farm in this study (as in many others in central Chile), the plan was not adopted, and animals were neither periodically tested nor culled for tuberculosis, without any legal requirement. The second suggestion was incorporated at the end of the phrase (line 76).

Comments 8: For completeness, since the study involves BCG vaccination in cattle, it would be important to mention the well-known difficulty of differentiating vaccinated from infected animals in vivo. This issue has major implications for bovine tuberculosis control and eradication programs, where reliable diagnostic tools are essential. A brief reference to the concept of DIVA (Differentiating Infected from Vaccinated Animals) tests, and their potential use to overcome this limitation, would add valuable context to the Introduction.

Response 8: We agree with the reviewer. We have now included some lines to describe the use of IGRA-DIVA for diagnosing vaccinated animals (lines 82-85)

 

Material and methods

Comments 9: In the Materials and Methods section, please consider adding sub-sections (e.g., study population, experimental design, biomolecular methods, statistical analysis) to improve clarity.

Response 9: We have added sub-sections.

Comments 10: In Lines 167–171, calf mortality is described. As mentioned earlier, it would be important to provide more detailed information on the causes of death. In particular, the authors should explain why “the etiological diagnosis was not performed” and, if possible, indicate whether any post-mortem findings, or laboratory data were available to support the attribution of deaths to diarrhea or pneumonia. This would considerably strengthen the reliability of the results.

Response 10: We have indicated that the etiological diagnosis was not performed because of economic issues. The attribution was performed by clinical symptoms of animals, which was mentioned between lines 177-181.

Comments 11: Please clarify whether diseased calves received any treatment; if so, specify the type, and if not, state this explicitly.

Response 11: Yes, diseased animals received broad-spectrum antibiotics (oxytetracycline and amoxicillin). In addition for diarrheic animals, parenteral fluids for hydration was included in the treatment. This was indicated in the text.

 

Results

12: Comments In the Results section, particularly in the tables, the presentation of statistically significant results should be made clearer. For example, significant differences could be highlighted with an asterisk or another appropriate marker directly in the tables.

Response 12: We have made substantial changes to the Results section, particularly in the description of the molecular detection of target genes, as recommended by two of the three reviewers. The detections according to vaccination groups, season of birth, and breeds are now presented in Supplementary Tables 4, 5, and 6, respectively. The new Table 2 includes only the results obtained at 3 and 6 months for all target sequences and deaths. Additional changes were incorporated into the text to improve the clarity of the statistically significant findings. We hope that this revised version is easier to understand.   

Discussion

Comments 13: Lines 258-261

The reference to changes in the “gut microbiota” may not be fully appropriate in the context of this study, as no microbiota analysis was performed. You may consider rephrasing or qualifying this statement.

Response 13: We agree with the reviewer and immediately after this phrase we have stated that “this study did not give clues in this issue” (line 279)

Comments 14: If not already addressed in the Introduction, a brief mention in the Discussion of the difficulty in differentiating vaccinated from infected animals and the possible use of DIVA tests would also be appropriate.

Response 14: As mentioned before, we have added information about IGRA-DIVA in lines 82-85.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In this last version, the authors improved the presentation of results, provided more data in the supplementary files, and the timeline of the study is now clearer to the readers. Therefore, I suggest the acceptance of the manuscript to be published.