Reviewer ’s report  
 
Title: Impact of nutrition and rotavirus infection on the infant gut microbiota in a humanized pig 
model  
 
Version: 0 Date: 23 Nov 2017  
 
Reviewer: Julie Bines  
 
Reviewer's report:  
 
This area of research is topical and may provide important insights into the challenges that oral 
rotavirus vaccines face in generating a protective immune response in the infant gut.  The 
approach using a neonatal ge rm free piglet model has novelty and is an appropriate model for the 
investigation of human gastrointestinal studies such as this.  The study is ambitious, seeking to 
assess microbial diversity, diet and HRV infection in animals that had undergone a faecal  
transplant with 2 -month old infant faeces. However it is the complexity of interactions and the 
presentation of these results that is somewhat confusing to the reader.  
The study design describes 6 independent study groups with 3 to 5 animals in each grou ps.  Were 
investigators blinded to the group allocation? Considering the small sample size and the potential 
for individual diversity between animals within groups, it would be relevant to provide comment 
in the discussion on the potential for this to impa ct of interpretation of the results.  it would also 
be helpful to present the rationale for the sample size for key comparisons. Also to present the 
results in a table format with ranges and for comparisons using 95%CI in the table and text for 
comparisons  where appropriate. The results in text and figure 3 are only presented for 4 of the 6 
groups with the control groups for the sufficient and insufficient diets not presented. As a result 
the changes across domains for diets, HIFM and HRV challenge difficul t to interpret.  For 
example - a key stated outcome was " a sufficient diet combined with HRV infection increased 
the microbiota diversity in the gut." However no data is presented for the control groups with 
sufficient and deficient diet with no HRV infec tion so it is unclear if it is the diet alone or the diet 
plus the HRV infection that alters the microbiota although the potential impact of diet on the 
microbiome is noted in the discussion.  
A key differential between groups is the diet - which is defined  as deficient or sufficient. There is 
no definition of this - is this diet protein deficient, energy deficient, deficient of a specific 
nutrient or all of the above.  As the diet is based on mature cow milk with no mineral or 
micronutrient supplementation how does this relate to the normal nutritional requirement of 4 -
day old growing piglets. The diet content in terms of protein, energy, fat and micronutrient intake 
per kilogram of piglets for all groups and relative to normal neonatal piglet requirement to  
confirm that the diet that piglets received was in fact sufficient or deficient with respect to 
normal requirements and consistent within and between groups.  Body weight should be 
presented as kg in addition to % change and as there is no results present ed for body length or 
other anthropometric measure, results and discussion should be restricted to comments on weight 
and not stunting. The addition of the control groups to Figure 3A to understand the impact of 
HIFM and HRV on body weight.   
Considering t he development of the microbiome in the first months of life what was the rationale 
for using 2 month old infant stool for transplantation and not 4 day old for the 4 day old piglets?  
Stool score was based on stool consistency alone - was there any indica tor of stool volume? The 
results displayed in Figure 3 B appear to be at odds with the presentation of p values - further 
explanation is required to explain how this was calculated using n=3 -5, a table would be more 
helpful than a figure. It would be helpf ul to be consistent with terminology for the groups and 
times in text and figures as variably presented as GF, Groups 1 -6, sufficient etc and "days post 
challenge" and PTD/PCDs.  A major highlighted result "malnourished pigs exhibited severe 
diarrhea and e nhanced virus shedding following HRV infection" does not accurately reflect that 
the stool score was lower in the sufficient challenged group compared to the deficient HFIM 
challenge group and had an equally low stool score at day 3 post challenge when com pared to 
the deficient group. Also the highest level of stool HRV shedding in the study occurred in the 
sufficient challenge group at day 1 post -infection and shedding in this group was equal to the 
deficient challenge group on day 2.  Based on the small s ample size and the data presented the 
conclusion highlighted in text that "increased microbial dissemination was observed in systemic 
tissues in HRV infected deficient piglets" seems an overstretch and there is no presentation of 
statistical comparisons th at suggest that this is statistical significant or evidence to support 
clinical significance.  
Are the methods appropriate and well described?  
If not, please specify what is required in  your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explai n in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessme nt in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing int erests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financia l competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details be low. 
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including an y attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comm ents 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Impact of nutrition and rotavirus infection on the infant gut microbiota in a humanized pig 
model  
 
Version: 0 Date: 23 Nov 2017  
 
Reviewer: Julie Bines  
 
Reviewer's report:  
 
This area of research is topical and may provide important insights into the challenges that oral 
rotavirus vaccines face in generating a protective immune response in the infant gut.  The 
approach using a neonatal ge rm free piglet model has novelty and is an appropriate model for the 
investigation of human gastrointestinal studies such as this.  The study is ambitious, seeking to 
assess microbial diversity, diet and HRV infection in animals that had undergone a faecal  
transplant with 2 -month old infant faeces. However it is the complexity of interactions and the 
presentation of these results that is somewhat confusing to the reader.  
The study design describes 6 independent study groups with 3 to 5 animals in each grou ps.  Were 
investigators blinded to the group allocation? Considering the small sample size and the potential 
for individual diversity between animals within groups, it would be relevant to provide comment 
in the discussion on the potential for this to impa ct of interpretation of the results.  it would also 
be helpful to present the rationale for the sample size for key comparisons. Also to present the 
results in a table format with ranges and for comparisons using 95%CI in the table and text for 
comparisons  where appropriate. The results in text and figure 3 are only presented for 4 of the 6 
groups with the control groups for the sufficient and insufficient diets not presented. As a result 
the changes across domains for diets, HIFM and HRV challenge difficul t to interpret.  For 
example - a key stated outcome was " a sufficient diet combined with HRV infection increased 
the microbiota diversity in the gut." However no data is presented for the control groups with 
sufficient and deficient diet with no HRV infec tion so it is unclear if it is the diet alone or the diet 
plus the HRV infection that alters the microbiota although the potential impact of diet on the 
microbiome is noted in the discussion.  
A key differential between groups is the diet - which is defined  as deficient or sufficient. There is 
no definition of this - is this diet protein deficient, energy deficient, deficient of a specific 
nutrient or all of the above.  As the diet is based on mature cow milk with no mineral or 
micronutrient supplementation how does this relate to the normal nutritional requirement of 4 -
day old growing piglets. The diet content in terms of protein, energy, fat and micronutrient intake 
per kilogram of piglets for all groups and relative to normal neonatal piglet requirement to  
confirm that the diet that piglets received was in fact sufficient or deficient with respect to 
normal requirements and consistent within and between groups.  Body weight should be 
presented as kg in addition to % change and as there is no results present ed for body length or 
other anthropometric measure, results and discussion should be restricted to comments on weight 
and not stunting. The addition of the control groups to Figure 3A to understand the impact of 
HIFM and HRV on body weight.   
Considering t he development of the microbiome in the first months of life what was the rationale 
for using 2 month old infant stool for transplantation and not 4 day old for the 4 day old piglets?  
Stool score was based on stool consistency alone - was there any indica tor of stool volume? The 
results displayed in Figure 3 B appear to be at odds with the presentation of p values - further 
explanation is required to explain how this was calculated using n=3 -5, a table would be more 
helpful than a figure. It would be helpf ul to be consistent with terminology for the groups and 
times in text and figures as variably presented as GF, Groups 1 -6, sufficient etc and "days post 
challenge" and PTD/PCDs.  A major highlighted result "malnourished pigs exhibited severe 
diarrhea and e nhanced virus shedding following HRV infection" does not accurately reflect that 
the stool score was lower in the sufficient challenged group compared to the deficient HFIM 
challenge group and had an equally low stool score at day 3 post challenge when com pared to 
the deficient group. Also the highest level of stool HRV shedding in the study occurred in the 
sufficient challenge group at day 1 post -infection and shedding in this group was equal to the 
deficient challenge group on day 2.  Based on the small s ample size and the data presented the 
conclusion highlighted in text that "increased microbial dissemination was observed in systemic 
tissues in HRV infected deficient piglets" seems an overstretch and there is no presentation of 
statistical comparisons th at suggest that this is statistical significant or evidence to support 
clinical significance.  
Are the methods appropriate and well described?  
If not, please specify what is required in  your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explai n in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessme nt in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing int erests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financia l competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details be low. 
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including an y attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comm ents 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Impact of nutrition and rotavirus infection on the infant gut microbiota in a humanized pig 
model  
 
Version: 0 Date: 23 Nov 2017  
 
Reviewer: Julie Bines  
 
Reviewer's report:  
 
This area of research is topical and may provide important insights into the challenges that oral 
rotavirus vaccines face in generating a protective immune response in the infant gut.  The 
approach using a neonatal ge rm free piglet model has novelty and is an appropriate model for the 
investigation of human gastrointestinal studies such as this.  The study is ambitious, seeking to 
assess microbial diversity, diet and HRV infection in animals that had undergone a faecal  
transplant with 2 -month old infant faeces. However it is the complexity of interactions and the 
presentation of these results that is somewhat confusing to the reader.  
The study design describes 6 independent study groups with 3 to 5 animals in each grou ps.  Were 
investigators blinded to the group allocation? Considering the small sample size and the potential 
for individual diversity between animals within groups, it would be relevant to provide comment 
in the discussion on the potential for this to impa ct of interpretation of the results.  it would also 
be helpful to present the rationale for the sample size for key comparisons. Also to present the 
results in a table format with ranges and for comparisons using 95%CI in the table and text for 
comparisons  where appropriate. The results in text and figure 3 are only presented for 4 of the 6 
groups with the control groups for the sufficient and insufficient diets not presented. As a result 
the changes across domains for diets, HIFM and HRV challenge difficul t to interpret.  For 
example - a key stated outcome was " a sufficient diet combined with HRV infection increased 
the microbiota diversity in the gut." However no data is presented for the control groups with 
sufficient and deficient diet with no HRV infec tion so it is unclear if it is the diet alone or the diet 
plus the HRV infection that alters the microbiota although the potential impact of diet on the 
microbiome is noted in the discussion.  
A key differential between groups is the diet - which is defined  as deficient or sufficient. There is 
no definition of this - is this diet protein deficient, energy deficient, deficient of a specific 
nutrient or all of the above.  As the diet is based on mature cow milk with no mineral or 
micronutrient supplementation how does this relate to the normal nutritional requirement of 4 -
day old growing piglets. The diet content in terms of protein, energy, fat and micronutrient intake 
per kilogram of piglets for all groups and relative to normal neonatal piglet requirement to  
confirm that the diet that piglets received was in fact sufficient or deficient with respect to 
normal requirements and consistent within and between groups.  Body weight should be 
presented as kg in addition to % change and as there is no results present ed for body length or 
other anthropometric measure, results and discussion should be restricted to comments on weight 
and not stunting. The addition of the control groups to Figure 3A to understand the impact of 
HIFM and HRV on body weight.   
Considering t he development of the microbiome in the first months of life what was the rationale 
for using 2 month old infant stool for transplantation and not 4 day old for the 4 day old piglets?  
Stool score was based on stool consistency alone - was there any indica tor of stool volume? The 
results displayed in Figure 3 B appear to be at odds with the presentation of p values - further 
explanation is required to explain how this was calculated using n=3 -5, a table would be more 
helpful than a figure. It would be helpf ul to be consistent with terminology for the groups and 
times in text and figures as variably presented as GF, Groups 1 -6, sufficient etc and "days post 
challenge" and PTD/PCDs.  A major highlighted result "malnourished pigs exhibited severe 
diarrhea and e nhanced virus shedding following HRV infection" does not accurately reflect that 
the stool score was lower in the sufficient challenged group compared to the deficient HFIM 
challenge group and had an equally low stool score at day 3 post challenge when com pared to 
the deficient group. Also the highest level of stool HRV shedding in the study occurred in the 
sufficient challenge group at day 1 post -infection and shedding in this group was equal to the 
deficient challenge group on day 2.  Based on the small s ample size and the data presented the 
conclusion highlighted in text that "increased microbial dissemination was observed in systemic 
tissues in HRV infected deficient piglets" seems an overstretch and there is no presentation of 
statistical comparisons th at suggest that this is statistical significant or evidence to support 
clinical significance.  
Are the methods appropriate and well described?  
If not, please specify what is required in  your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explai n in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessme nt in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing int erests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financia l competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details be low. 
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including an y attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comm ents 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant