Vaccination of Calves with the Mycobacterium bovis BCG Strain Induces Protection against Bovine Tuberculosis in Dairy Herds under a Natural Transmission Setting

Round 1

Reviewer 1 Report

In "Moreover, BCG has been shown to impart a variable protective efficacy, possibly due to the application of different BCG sub-strains and vaccination policies across the world [9,10]."

I understand that this is not the main goal of this work, but there are additional potential explanations to this phenomenon, such as host genetics and age of vaccination.  No current study supports any given BCG protects differently against human TB.

Introduction: it would be helpful to define what´s the cutoff value for “high prevalence” as their herds ranged from 15 to 76%. Also, it may be of help to define what cities or counties are considered in the “metropolitan region”.

Table 1. It would be helpful to include incidence rate per herd, so that one can see whether a potential variability in EV comes from this factor.

Table 1. Are the % shown at different time points the result of new positives/total animals at a particular time point? If so, please state this in the results or methods sections. Also, please mention why does the “total” decrease over time.  

In "What we could observe as possible factors associated to such contrasting results between herds, were the mortality rates of calves, and the incidences of tuberculous and NTM."

The authors did not really determine actual incidence of NTM, as they did not isolate NTM in their work, therefore this must be acknowledged.

In "The significant and negative correlation between these variables (r= -0.71, p=0.04) 280 suggests that the general health status of the animals around the vaccination period, and consequently the integrity of their immune system when exposed to BCG antigens, is essential for achieving a vaccine-associated protection."

There are,  in my opinion, at least 2 other potential explanations: (1) incidence rate in the herds with higher lethality and (2) virulence of the particular M. bovis strain circulating in that specific herd. I suggest the authors including these and discussing them in the proper section.

Further to this, as authors wrote “A high proportion of neonatal calf diarrhea and respiratory disease were recorded by producers in these mortality events”, then they must acknowledge the fact that they did not determine what other pathogens where circulating within the herds.

In "Institutional Review Board Statement: The animal study protocol followed the University of Chile guidelines on the care and use of animals.". The permit number must be included.

Author Response

Reviewer 1

Comment 1:

In "Moreover, BCG has been shown to impart a variable protective efficacy, possibly due to the application of different BCG sub-strains and vaccination policies across the world [9,10]."

I understand that this is not the main goal of this work, but there are additional potential explanations to this phenomenon, such as host genetics and age of vaccination.  No current study supports any given BCG protects differently against human TB.

Response:

In relation to potential explanations for the variable protective efficacy of BCG across the world, we agree with the reviewer about the existence of several other factors involved more than sub-strains and policies.

In order to consider the suggestion, we have changed the paragraph:

“Moreover, BCG has been shown to impart a variable protective efficacy, possibly due to the application of different BCG sub-strains, the genetic background of immunized individuals, the burden of disease and the vaccination policies across the world [9,10]”

 Comment 2

Introduction: it would be helpful to define what´s the cutoff value for “high prevalence” as their herds ranged from 15 to 76%. Also, it may be of help to define what cities or counties are considered in the “metropolitan region”.

Response

We have added those cutoff values in the text and mentioned that Santiago, the capital city, is located in the metropolitan region.

Comment 3

Table 1. It would be helpful to include incidence rate per herd, so that one can see whether a potential variability in EV comes from this factor.

 Response

There is a lot of information in this table, so we prefer not including this variable, which would not have some correlation with EV.

 Comment 4

Table 1. Are the % shown at different time points the result of new positives/total animals at a particular time point? If so, please state this in the results or methods sections. Also, please mention why does the “total” decrease over time.  

Response

The reviewer is right. Just new positives are informed in each time point.  In the second paragraph of results, we have added this phrase: “For these analyses, the DIVA IGRA positive animals were excluded from subsequent sampling activities and counted only at the time point of detection. Due to this and other causes of on-farm losses, the total number of animals under study decreased over time.”

Comment 5

In "What we could observe as possible factors associated to such contrasting results between herds, were the mortality rates of calves, and the incidences of tuberculous and NTM."

The authors did not really determine actual incidence of NTM, as they did not isolate NTM in their work, therefore this must be acknowledged.

Response

We agree with the reviewer. To clarify this issue, we made changes in two paragraphs.

In the cited text (4^th paragraph of discussion): “What we could observe as possible factors associated to such contrasting results between herds, were the mortality rates of calves, and the incidences of DIVA and PPDA reactors”.

In the 7^th paragraph of discussion: “In this work, the incidence of NTM, which is assumed from PPDA reactors in the control unvaccinated group (Table S1), was positively correlated with EV% results”

Comment 6

In "The significant and negative correlation between these variables (r= -0.71, p=0.04) suggests that the general health status of the animals around the vaccination period, and consequently the integrity of their immune system when exposed to BCG antigens, is essential for achieving a vaccine-associated protection."

There are,  in my opinion, at least 2 other potential explanations: (1) incidence rate in the herds with higher lethality and (2) virulence of the particular M. bovis strain circulating in that specific herd. I suggest the authors including these and discussing them in the proper section.

Response

We agree with the reviewer about several other factors that could be associated to the efficacy of the vaccine. For this reason, in the first part of the discussion’s 4^th paragraph, you can read “ What determines the variation in the vaccine protection between different reported field trials, could be related to several environmental, host, pathogen and vaccine related factors”.

The specific explanation (1), was effectively analyzed through the correlation between the incidence rate of bTB and the EV%, resulting in r= -0.22 with p=0.64. For this calculation we considered the incidence in the non-vaccinated group from each herd, as an expression of the exposition to the pathogen. In the discussion section we already have included this analysis, just after the text cited by the reviewer.

The second explanation we assume is contained in the aforementioned 4th paragraph. Since in this work we do not have any evidence on this source of variation, we do not consider relevant any additional mention of other possible drivers of EV% variability.

Comment 7

Further to this, as authors wrote “A high proportion of neonatal calf diarrhea and respiratory disease were recorded by producers in these mortality events”, then they must acknowledge the fact that they did not determine what other pathogens where circulating within the herds.

Response

We have modified this paragraph:

“A high proportion of neonatal calf diarrhea and respiratory disease were recorded by producers in these mortality events, although no etiological diagnosis was made”

Comment 8

In "Institutional Review Board Statement: The animal study protocol followed the University of Chile guidelines on the care and use of animals.". The permit number must be included.

Response:

We have added the number.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper is a field study of BCG vaccine efficacy against natural M bovis infection. 

The study design is sound and the discussion and conclusions are very valid. 

The major limitation of the study is the lack of correlation with post mortem data to demonstrate protection from the development of lesions or carcass defects. However, it is clear that this was not possible to obtain these data under the study design and real-time experimental conditions but this limitation is well covered in the Discussion section by the authors and their findings are not overstated in this context. 

Aside from minor typographical errors I would not recommend any changes to the manuscript prior to publicaton. 

Author Response

Reviewer 2

This paper is a field study of BCG vaccine efficacy against natural M bovis infection. 

The study design is sound and the discussion and conclusions are very valid. 

The major limitation of the study is the lack of correlation with post mortem data to demonstrate protection from the development of lesions or carcass defects. However, it is clear that this was not possible to obtain these data under the study design and real-time experimental conditions but this limitation is well covered in the Discussion section by the authors and their findings are not overstated in this context. 

Aside from minor typographical errors I would not recommend any changes to the manuscript prior to publicaton. 

Response

Thank you.

Author Response File: Author Response.docx

Reviewer 3 Report

This is a study on the efficacy of vaccinating calves against bTB using the BCG vaccine. Generally, the study was nicely done, however, there are two issues for considerations:

1. The conclusion, especially the last sentence in the Abstract, needs to be carefully re-structured. Results of the study clearly demonstrate low efficacy or protection rate (22.4%), which is generally similar with results of previous studies. Therefore, l felt that the 'low efficacy' must be included in the conclusion of Abstract for the readers to fairly judge the possible benefit of using BCG vaccination
2. The results appear to be similar with other previous study thus, the manuscript is not contributing new findings except for the confirmation of previous findings. 

Based on these issues, I could not accept the paper for publication in the current form. However, we can re-consider the acceptance status should the authors agree to modify the conclusion as suggested.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

This is a study on the efficacy of vaccinating calves against bTB using the BCG vaccine. Generally, the study was nicely done, however, there are two issues for considerations:

Comment 1

The conclusion, especially the last sentence in the Abstract, needs to be carefully re-structured. Results of the study clearly demonstrate low efficacy or protection rate (22.4%), which is generally similar with results of previous studies. Therefore, l felt that the 'low efficacy' must be included in the conclusion of Abstract for the readers to fairly judge the possible benefit of using BCG vaccination

Response

The concept of “low efficacy” was stated in the abstract, some lines before the conclusion. In addition and strictly speaking, the protection was temporally and geographically variable, with time points and herds with high, middle and low efficacies. So we believe that a more accurate conclusion is: “These results suggest that BCG vaccination of dairy calves in a natural transmission setting confers variable protection to animals against bTB in a high prevalence area”

Comment 2

The results appear to be similar with other previous study thus, the manuscript is not contributing new findings except for the confirmation of previous findings. 

Based on these issues, I could not accept the paper for publication in the current form. However, we can re-consider the acceptance status should the authors agree to modify the conclusion as suggested.

Response

We believe that this work resulted in similar or comparable results with other previous studies, although the experimental design of the study, and in particular the sample size, of both animals and dairy herds, is quite unique. And this is an important contribution to the knowledge of the protection conferred by BCG in a natural transmission setting, a condition that has been scarcely addressed in scientific literature (Srinivasan et al., 2021. https://doi.org/10.3389/fvets.2021.637580)

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

No further comment expect a slight change in the title: 'Vaccination of calves with the Mycobacterium bovis BCG strain induces variable protection against bovine tuberculosis in dairy herds under a natural transmission setting'