Assessment of the Impact of Humic Acids on Intestinal Microbiota, Gut Integrity, Ileum Morphometry, and Cellular Immunity of Turkey Poults Fed an Aflatoxin B₁-Contaminated Diet

Round 1

Reviewer 1 Report

In the manuscript titled, “Assessment of the impact of humic acids on intestinal microbiota, gut integrity, ileum morphometry, and cellular immunity of turkey poults fed an aflatoxin B1-contaminated diet”, the authors wanted to determine if humic acid-fed diet (0.25% w/w) can ameliorate the toxic effect of aflatoxin B1 (AFB1). Indeed, the results suggested beneficial effects of HA on gut integrity. More importantly, this study also explores the identity of the microbes involved. The used of HA as feed supplement was also postulated. I found the manuscript to be interesting and scientifically sound. However, I’m disappointed with the one and only supplementary figure. There is no accompanying figure legend -- how are we as a reader can discriminate between one figure from another? Also, since this manuscript investigated the effects of HA, the source/preparation of the HA should be included herein and not let the readers search for the previously published sources.

One minor point:

Line 389, I’m pretty sure that the city of Wilmington is not in China.

Author Response

In the manuscript titled, “Assessment of the impact of humic acids on intestinal microbiota, gut integrity, ileum morphometry, and cellular immunity of turkey poults fed an aflatoxin B1-contaminated diet”, the authors wanted to determine if humic acid-fed diet (0.25% w/w) can ameliorate the toxic effect of aflatoxin B1 (AFB1). Indeed, the results suggested beneficial effects of HA on gut integrity. More importantly, this study also explores the identity of the microbes involved. The used of HA as feed supplement was also postulated. I found the manuscript to be interesting and scientifically sound. However, I’m disappointed with the one and only supplementary figure. There is no accompanying figure legend -- how are we as a reader can discriminate between one figure from another? Also, since this manuscript investigated the effects of HA, the source/preparation of the HA should be included herein and not let the readers search for the previously published sources.

A: Dear Reviewer, thank you very much for the time you have spent reviewing our manuscript. Your comments are very valuable and helpful. We have studied those comments carefully and have made corrections, which we hope to meet with the approval. The following is our point-by-point response.

• Figure legends were added.
• A brief description of the AFB1, HA, and Zeolite was added.

Reviewer 2 Report

The manuscript investigated the effect of dietary HA on intestinal microbiota, gut integrity, ileum morphometry, and immunity in turkey poults fed diet containing 250ng/g of AFB1. The study designed well, discussed comprehensively and obtained interesting information. Some comments for the manuscript need to be addressed before it can be accepted.

Lines 53-55 introduced the mitigating effects of humic acids (HA) on AFB1 toxicity. To make the “Introduction” more comprehensive and attractive, it is necessary to briefly introduce other detoxification methods of AFB1 for poultry such as curcumin and adsorbents, and allow me to suggest the following publication to be cited herein, please.

Zhang, J.; Sun, X.; Chai, X.; Jiao, Y.; Sun, J.; Wang, S.; Yu, H.; Feng, X. Curcumin mitigates oxidative damage in broiler liver and ileum caused by AFB1-contaminated feed through Nrf2 signaling pathway. Animals 2024, 14, 409. doi: 10.3390/ani14030409

Mucignat, G.; Bassan, I.; Giantin, M.; Pauletto, M.; Bardhi, A.; Iori, S.; Lopparelli, R.M.; Barbarossa, A.; Zaghini, A.; Novelli, E.; et al. Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure? Toxins 2022, 14, 435. doi: 10.3390/toxins14070435

In the section of “2. Results”, it is necessary to make several subsections according to the result content and add second or third level titles.

As elucidated in lines 98-100, PC and ZEO+AFB1 treatments showed a significant reduction in the presence of Anaerosipes compared to poults that received HA and HA+AFB1, but there was no significant difference of Lachnospiraceae among the HA, HA+AFB1 and ZEO+AFB1 treatments, only that of CP was significantly lower than that of HA, HA+AFB1 and ZEO+AFB1 as shown in Table 2. Rectify it please.

Line 23, the word “bar” is redundant, it can be left out.

Line 154, in the sentence “Poults in the NC and PC treatments……..”, the “PC” should be “HA+AFB1”.

Lines 160-162, the sentence “A comprehensive description of the observed results in morphometric analysis, serum levels of FITC-d, and CBH response is provided in Table 3” is redundant, it can be left out

Line 217，the word “added” should be “ fed”.

Line 231, the comma “,” in this line should be “.”.

Line 421, “………………………Baxter et al. (2017) [46]”, “(2017)” should be deleted.

Minor editing of English language required

Author Response

The manuscript investigated the effect of dietary HA on intestinal microbiota, gut integrity, ileum morphometry, and immunity in turkey poults fed diet containing 250ng/g of AFB1. The study designed well, discussed comprehensively and obtained interesting information. Some comments for the manuscript need to be addressed before it can be accepted.

A: Dear Reviewer, thank you very much for the time you have spent reviewing our manuscript. Your comments are very valuable and helpful. We have studied those comments carefully and have made corrections, which we hope to meet with the approval.

Lines 53-55 introduced the mitigating effects of humic acids (HA) on AFB1 toxicity. To make the “Introduction” more comprehensive and attractive, it is necessary to briefly introduce other detoxification methods of AFB1 for poultry such as curcumin and adsorbents, and allow me to suggest the following publication to be cited herein, please.

Zhang, J.; Sun, X.; Chai, X.; Jiao, Y.; Sun, J.; Wang, S.; Yu, H.; Feng, X. Curcumin mitigates oxidative damage in broiler liver and ileum caused by AFB1-contaminated feed through Nrf2 signaling pathway. Animals 2024, 14, 409. doi: 10.3390/ani14030409

Mucignat, G.; Bassan, I.; Giantin, M.; Pauletto, M.; Bardhi, A.; Iori, S.; Lopparelli, R.M.; Barbarossa, A.; Zaghini, A.; Novelli, E.; et al. Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure? Toxins 2022, 14, 435. doi: 10.3390/toxins14070435

In the section of “2. Results”, it is necessary to make several subsections according to the result content and add second or third level titles.

As elucidated in lines 98-100, PC and ZEO+AFB1 treatments showed a significant reduction in the presence of Anaerosipes compared to poults that received HA and HA+AFB1, but there was no significant difference of Lachnospiraceae among the HA, HA+AFB1 and ZEO+AFB1 treatments, only that of CP was significantly lower than that of HA, HA+AFB1 and ZEO+AFB1 as shown in Table 2. Rectify it please.

Line 23, the word “bar” is redundant, it can be left out.

Line 154, in the sentence “Poults in the NC and PC treatments……..”, the “PC” should be “HA+AFB1”.

Lines 160-162, the sentence “A comprehensive description of the observed results in morphometric analysis, serum levels of FITC-d, and CBH response is provided in Table 3” is redundant, it can be left out

Line 217，the word “added” should be “ fed”.

Line 231, the comma “,” in this line should be “.”.

Line 421, “………………………Baxter et al. (2017) [46]”, “(2017)” should be deleted.

A: Thank you very much for the observations, the changes have been made.

• References were added.

Reviewer 3 Report

This manuscript is a study that examines the effects of humic acid on turkey poults fed aflatoxin B1, and it is a topic that is of great interest to readers of Toxins due to its comprehensive exploration of various aspects of the impact. The experimental design is well-conceived, and the results are well-represented. The main findings of the paper are highly significant, so I recommend minor revision. However, the following points have to be considered.

1. (All tables in the manuscript) It is important for all tables and figures in the manuscript to convey their meaning independently. Therefore, I think all tables in the manuscript need to be revised. Please expand the abbreviation SEM and specify the meanings of superscripts a,b,ab, and abc. Even though those superscripts are widely recognized symbols in statistics, please clarify their significance and differences in every table.

2. Please check if there are any mistakes in the following sentences (and in other comparison sentences throughout the manuscript)

(1) Line 103-105: The PC and ZEO+AFB1 treatments had a numerical trend of decreased Peilou’s Evenness and Shannon index compared to the PC treatment, - Please check the second ‘PC’ is correct.

(2) Line 154-155: Poults in the NC and PC treatments showed the highest total area, - Please verify if PC should be replaced with HA+AFB1.

3. (Table 1 and Table 2) For readers who are not familiar with intestinal microbiota, it would be helpful to differentiate which taxa are beneficial and non-beneficial in tables. Additionally, is it possible to know the total count of intestinal microbiota besides the relative abundance presented in the tables?

4. (Line 123-125) Authors mentioned that no bacterial species were found to be enriched in the ZEO+AFB1 treatment. What is the implication of this result? Related to this question, what is the purpose of using zeolite in this study?

5. (Figure 3) Focusing on the Anaerostipes_F65 and Acutalibacter_F67, the relative abundance of NC is significantly lower that that of PC. What can be the possible reasons for that?

6. (Line 375-377) Even though the details of AFB1, HA, and Zeolite are described in the previous article, it is highly recommended to provide a brief explanation of those materials. How and where did you obtain (or culture) AFB1, what type of HA did you use, and are there any significant characteristics of HA and Zeolite used in this study?

7. (Figure 1S, supplementary) What are a,b,c,d,e stands for?

Comments for author File: Comments.pdf

Author Response

This manuscript is a study that examines the effects of humic acid on turkey poults fed aflatoxin B1, and it is a topic that is of great interest to readers of Toxins due to its comprehensive exploration of various aspects of the impact. The experimental design is well-conceived, and the results are well-represented. The main findings of the paper are highly significant, so I recommend minor revision. However, the following points have to be considered.

A: Dear Reviewer, thank you very much for the time you have spent reviewing our manuscript. Your comments are very valuable and helpful. We have studied those comments carefully and have made corrections, which we hope to meet with the approval. The following is our point-by-point response.

1. (All tables in the manuscript) It is important for all tables and figures in the manuscript to convey their meaning independently. Therefore, I think all tables in the manuscript need to be revised. Please expand the abbreviation SEM and specify the meanings of superscripts a,b,ab, and abc. Even though those superscripts are widely recognized symbols in statistics, please clarify their significance and differences in every table.
2. Please check if there are any mistakes in the following sentences (and in other comparison sentences throughout the manuscript)

(1) Line 103-105: The PC and ZEO+AFB1 treatments had a numerical trend of decreased Peilou’s Evenness and Shannon index compared to the PC treatment, - Please check the second ‘PC’ is correct.

(2) Line 154-155: Poults in the NC and PC treatments showed the highest total area, - Please verify if PC should be replaced with HA+AFB1.

3. (Table 1 and Table 2) For readers who are not familiar with intestinal microbiota, it would be helpful to differentiate which taxa are beneficial and non-beneficial in tables. Additionally, is it possible to know the total count of intestinal microbiota besides the relative abundance presented in the tables?

A: Thank you for your comment.  For readers who are unfamiliar with intestinal microbiota, we have provided a concise overview of the main findings of the microbiome analysis in the discussion, which are highlighted in green.  The major differentiation occurs not just at the phylum level, but also across the microbiome analysis.  We believe there is no need for more discussion with the material in the text; nevertheless, we could expand it if you believe it is necessary.  Unfortunately, in this investigation, we did not obtain a total bacterial count using typical microbiology protocols.  Thank you. 

4. (Line 123-125) Authors mentioned that no bacterial species were found to be enriched in the ZEO+AFB1 treatment. What is the implication of this result? Related to this question, what is the purpose of using zeolite in this study?

A: LEfSe, which stands for Linear discriminant analysis Effect Size, is a statistical method used in microbiome research to identify biomarkers that are differentially abundant between different groups of samples. This analysis is particularly applied to high-throughput sequencing data, such as 16S rRNA gene sequencing, which is commonly used to characterize microbial communities.  The analysis employs linear discriminant analysis (LDA), a statistical method that aims to find the linear combinations of variables (in this case, microbial taxa abundances) that best discriminate between two or more groups (e.g., healthy vs. diseased samples). The "Effect Size" in LEfSe refers to the biological relevance or magnitude of differences between groups. It helps identify not only statistically significant differences but also those that are biologically meaningful. It is specifically designed for identifying microbial biomarkers associated with different sample classes or conditions. These biomarkers are taxa (e.g., species or genera) that contribute significantly to the differences between groups.  LEfSe assesses statistical significance using non-parametric tests, which are well-suited for microbiome data due to its often non-normal distribution. The method considers both the presence/absence and the abundance of microbial taxa. Hence, the results are often visualized in the form of cladograms or bar plots, allowing researchers to easily interpret and communicate the identified biomarkers and their respective effect sizes.  By identifying biomarkers, LEfSe aids in characterizing the microbial signatures associated with different conditions, such as health and disease, or before and after a treatment intervention.  Researchers can use LEfSe results to generate hypotheses about the potential roles of specific microbial taxa in the context of the studied conditions. This can guide further investigations into the functional roles of these microbes.  LEfSe analysis is a valuable tool in microbiome research for identifying and characterizing microbial biomarkers associated with different sample groups, contributing to a better understanding of the microbial community dynamics in various biological contexts.

LEfSe is a statistical method used to identify features (in this case, bacterial species) that are significantly different in abundance between different classes or conditions. It incorporates both statistical significance and biological relevance in its analysis. The ZEO+AFB1 treatment is a specific experimental condition or group that involves a combination of factors represented by "ZEO" and "AFB1."

The absence of any species from the ZEO+AFB1 treatment in the LEfSe analysis suggests that, according to the statistical analysis, there were no bacterial species in the ZEO+AFB1 treatment that showed a significant difference in abundance compared to other treatments.  The absence of any species indicating that no bacterial species in the ZEO+AFB1 treatment were significantly more abundant implies that, in the context of this analysis, the microbial composition in the ZEO+AFB1 treatment did not differ significantly from other treatments in terms of the identified bacterial species. The relevance of this result is that, based on the LEfSe analysis, the ZEO+AFB1 treatment does not have specific bacterial species that stand out in terms of abundance compared to other treatments. This information can be crucial for understanding the impact of the ZEO+AFB1 treatment on the microbial community and may have implications for the study's objectives or the experimental conditions being investigated.  The absence of any species from the ZEO+AFB1 treatment in the LEfSe analysis indicates that, according to the statistical methodology employed, there were no bacterial species significantly more abundant in this treatment compared to others, highlighting a potential lack of distinct microbial composition under these experimental conditions.

Regarding your question about the purpose of using zeolite in this study we can mention that zeolites are crystalline aluminosilicate minerals with a three-dimensional porous structure. They have a high surface area and cation exchange capacity, making them suitable for various applications, including adsorption of mycotoxins in poultry feed. Because zeolites have a highly porous structure with a network of channels and cages of varying sizes, this porous nature provides a large surface area for adsorption.  Moreover, zeolites have ion exchange sites within their structure, allowing them to exchange cations. This property is useful in adsorbing mycotoxins, as the toxins often carry positive charges. Zeolite can attract and bind these positively charged mycotoxins.  In addition, zeolites have a chemical affinity for certain molecules, including mycotoxins. The surface properties of zeolites can interact with mycotoxins through van der Waals forces, hydrogen bonding, and electrostatic interactions, leading to adsorption.  For this characteristic, zeolites can exhibit selectivity in adsorbing different mycotoxins based on their chemical structure and properties and selectivity allows for targeted removal of specific mycotoxins, making them a cost-effective solution for mycotoxin management in poultry feed.  For this reason, we included it as an additional control group in the present study.  Thank you.

5. (Figure 3) Focusing on the Anaerostipes_F65 and Acutalibacter_F67, the relative abundance of NC is significantly lower that that of PC. What can be the possible reasons for that?

A:  Anaerostipes is a Gram-positive and anaerobic bacterial genus from the family of Lachnospiraceae. Anaerostipes may protect against colon cancer in humans by producing butyric acid.

Acutalibacter is a genus of the Oscillospiraceae family. Currently, little is known about Acutalibacter. On the other side, the abundance of Oscillospira in humans is positively correlated with the diversity of bacteria in the gut and can be a predictor of low body mass index (BMI) (https://doi.org/10.1016/B978-0-12-819265-8.00030-9).

6. (Line 375-377) Even though the details of AFB1, HA, and Zeolite are described in the previous article, it is highly recommended to provide a brief explanation of those materials. How and where did you obtain (or culture) AFB1, what type of HA did you use, and are there any significant characteristics of HA and Zeolite used in this study?
7. (Figure 1S, supplementary) What are a,b,c,d,e stands for?

A: Thank you very much for the observations, the changes have been made.

• Figure legends were added.
• A brief description of the AFB1, HA, and Zeolite was added.

Round 2

Reviewer 2 Report

All my comments have been addressed, and I consider that the revised manuscript can be accepted. 

Author Response

Thank you for your time