Orally Fed Recombinant Lactococcus lactis Displaying Surface Anti-Fimbrial Nanobodies Protects Piglets against Escherichia coli Causing Post-Weaning Diarrhea
Round 1
Reviewer 1 Report
I don't have any comment in this version
Author Response
Reply to Review Report 1
Reviewer 1 does not have any comment.
Reviewer 2 Report
As most of my comments have been addressed in this new version, I would be inclined to accept the manuscript if few minor changes are taken into account:
Line 92: Nanobodies have
L88: Abbreviate (Nbs) just once, the authors already did it on L85.
L219-221: Were the piglets housed each one in individual pens so the authors knew the amount of feed they were eating or not? As it is read, it seems like 5 piglets were housed together in different pens, so the amount of L. lactis cells delivered to them could have been different. Was this solved through rationing the feed? Also, were the piglets housed in the same room or in different rooms to avoid any potential cross-contamination?
L332, L360, L398: Missing numeration, a fourth digit might be added.
Figure 5: As I already requested, add significance to the barplots when necessary. Although it is stated as “nearly complete inhibitory effect” and “partial inhibition”, comparison between groups for the number of E. coli cells adhered to villus requires a statistical analysis such as ANOVA to assess its significance.
L416-418: What does it mean “To control infection”? I guess it is referred to control the F4+ E. coli infection. Also, wasn’t the positive infection control the one infected and receiving the L. lactis MG1363 strain? Table 1 states that the negative control neither received E. coli nor L. lactis.
L433: “Supplementary” Table 5.
L436-437: “while a significant weight gain (p < 0.001) of 1.2 kg above initial weight was predicted 436 for experiment day 14” Doesn’t this just mean that the animals were growing and thus, gaining more weight? I don’t think it is relevant, as nothing is significant in the comparisons between groups on different days.
L455: I would say a tendency, as p = .071 is higher than the consensus p ≤ .05.
L502: Remove the strikethrough text.
L520: It is curious that the challenge experiment was performed with anti-FaeG L. lactis, after showing that the anti-FadF L. lactis had a greater inhibitory effect than anti-FaeG L. lactis. More argumentation is required.
L533: In addition, none of
L569: I guess the expression of the mucin4 gene should also be included when screening for the F4R status of piglets.
L582: I wouldn’t say complete protection, as F4-negative ETEC exist for example.
Author Response
Reply to Review Report 2
Line 92: Nanobodies have
Authors: correction is made.
L88: Abbreviate (Nbs) just once, the authors already did it on L85.
Authors: correction is made.
L219-221: Were the piglets housed each one in individual pens so the authors knew the amount of feed they were eating or not? As it is read, it seems like 5 piglets were housed together in different pens, so the amount of L. lactis cells delivered to them could have been different. Was this solved through rationing the feed? Also, were the piglets housed in the same room or in different rooms to avoid any potential cross-contamination?
Authors: The piglets were house in groups of 5 piglets per pen. All the pens were located within on large housing unit. The pens were separated by solid wall (1.25 m height) which avoided direct contact and cross contamination between piglets in different pens. The feed was rationed into 2 daily portions (morning and evening).
L332, L360, L398: Missing numeration, a fourth digit might be added.
Authors: Numeration is added to the text.
Figure 5: As I already requested, add significance to the barplots when necessary. Although it is stated as “nearly complete inhibitory effect” and “partial inhibition”, comparison between groups for the number of E. coli cells adhered to villus requires a statistical analysis such as ANOVA to assess its significance.
Authors: We added significance to the bar plots. The number E. coli cells adhered to the villi in each adhesion assay and the reversal assay were compared using paired t-test and the level of significance reported in the manuscript text.
L416-418: What does it mean “To control infection”? I guess it is referred to control the F4+ E. coli infection. Also, wasn’t the positive infection control the one infected and receiving the L. lactis MG1363 strain? Table 1 states that the negative control neither received E. coli nor L. lactis.
Authors: We meant “To control F4+ E. coli infection” and adapted the manuscript accordingly. The positive control group received L. lactis MG1363 in diet while the negative control group received feed not supplemented with L. lactis MG1363. The manuscript was revised for clarity.
L433: “Supplementary” Table 5.
Authors: Manuscript is adapted.
L436-437: “while a significant weight gain (p < 0.001) of 1.2 kg above initial weight was predicted 436 for experiment day 14” Doesn’t this just mean that the animals were growing and thus, gaining more weight? I don’t think it is relevant, as nothing is significant in the comparisons between groups on different days.
Authors: We agree with the above observation; it was an expected weight gain as the piglets were growing. We removed the unnecessary detail from the manuscript.
L455: I would say a tendency, as p = .071 is higher than the consensus p ≤ .05.
Authors: We agree with this observation. The manuscript was adapted to state that “..mean CFU was not significantly different for all groups.”
L502: Remove the strikethrough text.
Authors: Strikethrough is removed.
L520: It is curious that the challenge experiment was performed with anti-FaeG L. lactis, after showing that the anti-FadF L. lactis had a greater inhibitory effect than anti-FaeG L. lactis.
More argumentation is required.
Authors: There is not a reliable in vivo challenge model for F18-fimbriated E. coli (encoding the FedF adhesins) in piglets. For F4-fimbriated ETEC strains we used the challenge model that was published in Virdi et al. (Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic escherichia coli infection. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 11809–11814). Therefore the challenge experiment was done with a F4ac-producing ETEC strain. We adapted the text in the manuscript to clarify why only challenge in piglets was performed for F4.
L533: In addition, none of
Authors: Text is adapted.
L569: I guess the expression of the mucin4 gene should also be included when screening for the F4R status of piglets.
Authors: The presence of the mucin4 gene is only an indication for piglets carrying the F4ac receptor. The other fimbrial variants F4ab and F4ad recognize other intestinal receptors. The only way to be absolutely certain that the F4R receptor for F4ac is present, is to do the postmortem analysis using the in vitro adhesion test on microvilli.
L582: I wouldn’t say complete protection, as F4-negative ETEC exist for example.
Authors: We adapted the text in the manuscript.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Author exam the transgenic L. Lactis that express the nanobodies which prevent the E. Coli colonization by neutralizing fimbrial adhesins FaeG (F4 fimbriae) and FedF (F18 fimbriae). The study provided clear evident on in vitro and in vivo, and is well written. However, the statistical analysis was not done properly and need to be redone.
Line 21: What is VHHs stand for?
Line 166: Was section collected from more than one pig? (more than on donor?)
Were piglets received any medication treatment prior to kill for sampling?
Which stage of the piglets when they were sampled? (preweaning or post weaning).
Line 201 Were all piglets from one gender and from the same parity sow?
Line 204: Was pig housed individually?
Line 212: What was the concentration of zinc in the diet?
Line 239 (TMB, Invitrogen)
Line 242: what was experimental unit? Please provide detail information on statistical analysis
Line 247: change Nanobodies to nanobodies
Line 245 I would move 3.1 to material and methods
Line 386 Was the average weaning body weight comparable between treatment groups at weaning?
The different on weaning body weight can greatly impact the outcome of growth performance.
If there were different on initial BW, was initial BW being used as covariance while analyzing data?
Line 390: What was the results of type 3 test and multiple comparison?
Line 406: a repeat measurement should be considered using in time series data.
P value in F test didn’t tell you which group is significantly higher than others.
Why group 1 had increased CFU on d 3?
Line 420: Was there a standard for ELISA assay? What was inter and intra assay CV?
Line 424: There was no significant different on ELISA results.
Line 463: Do we know how much of L. lactis survive pepsin and bile acids? It would be great if in vitro test being carried out to show L. lactis is still viable when reach the target segment of GI tract.
Line 485: Again this is not significantly different.
Reviewer 2 Report
This study investigated the effects of using a recombinant L. lactis to protect weaners against post-weaning disease through the use of nanobodies to chelate F4 and F18 fimbriae expressing E. coli. The article is well written in most of its length. However, the results section needs to report clearly the differences between groups and the discussion section is should be expanded. In specific, here are some comments that need to be addressed before further considering the manuscript:
Lowercase Nanobodies in the title
L43-44: As they act as adjectives, epithelial and endothelial should be used rather than epithelium and endothelium plural noun forms.
L68-70: Sounds more like a discussion point, better use “were not validated”
The end of the introduction, should be moved up before the aim of the manuscript. In other words, introduce and expand a little bit further the use of nanobodies in animal production. Also, make it clearer the aim of the study as now it is diluted as part of the introduction. The last sentence is a result/conclusion and should not be reported in the introduction.
L227: I understand that we are talking about F4+ E. coli isolates that were shed within the faecal samples. Improve the readability of this section.
Figure 5: Add significance to the barplots when necessary. In line 334-335 is read that long protein A anchor had an increased inhibitory effect, but this effect is not measured.
L382: Was the absence of the F4R receptor expected in some pigs at this stage? A preliminary analysis should have been done in advance due to the scarcity of the numbers used in the trial.
Figure 7: The results are not comparable in the fashion that is reported in the text (L390-391). Please report the differences between groups with p-values.
L399: Specify how and which CFU were determined. Were they just ETEC? I haven’t found in the methods section how they were cultured. In addition, report the statistical differences between groups as stated before.
L420-424: How the Group 3 had a faster sero conversion rate than the other two groups if the difference in mean IgG between groups was not significant? Again, report the differences between groups.
L444-445: I don’t think that the sentence is correctly written, rewrite. Are not any other examples of the use of nanobodies in pigs rather than in mice?
The discussion is surprisingly short with the amount of results present and resembles a future work section. There is not any comparison with other probiotic treatment studies. How other groups have used antibodies strategies to inhibit ETEC cell adhesion? How long the recombinant Lactococcus remained in the host? Has the administration of these recombinant strains with nanobodies any potential drawback?
In the same manner, the conclusion is too general and does not summarizes all the results of the manuscript. Future work is not necessary in this section and the last sentence is redundant.
