Impact of Stocking Densities on the Microbiota of the Cloaca, Eggshell, and Egg Content of White Egg Layers in Colony Cages

Round 1

Reviewer 1 Report

The study reported in this manuscript is scientifically sound and appears to have been conducted with due care. The manuscript is well written and, presents new information or interpretation in such a way to be of interest to a wider audience.

This referee has two minor queries.

No production data are presented. Why? – not recorded. it is acknowledged that the focus of the study is on ‘food safety’. However, there must be some balancing with ‘productivity’ and profitability. Please comment.

ALSO note that the appropriate statistical method for this design, where the responses may be density-dependent, is orthogonal polynomials to examine the linear and quadratic effects of increasing stocking densities. As such, differences between individual treatments and the use of superscripts to separate significance between treatment means are not relevant. Using linear or quadratic effects would enable a much focussed presentation. The data should be presented accordingly in the Tables and, the text may need to be revised. Please consult a statistician.

Well written paper

Author Response

Hello Reviewer 1,

Thank you for your comments in regard to my manuscripts. Below are answers to the queries that you left for me:

“No production data are presented. Why? – not recorded. it is acknowledged that the focus of the study is on ‘food safety’. However, there must be some balancing with ‘productivity’ and profitability. Please comment.”

We are currently preparing a manuscript that addresses the production, egg quality, welfare, and other parameters. The hens involved in this study were also a subset of those hens. Therefore, we felt it appropriate to focus this manuscript on microbial contamination and submit a separate paper focusing on production and welfare.

“ALSO note that the appropriate statistical method for this design, where the responses may be density-dependent, is orthogonal polynomials to examine the linear and quadratic effects of increasing stocking densities. As such, differences between individual treatments and the use of superscripts to separate significance between treatment means are not relevant. Using linear or quadratic effects would enable a much focussed presentation. The data should be presented accordingly in the Tables and, the text may need to be revised. Please consult a statistician.”

While orthogonal polynomials may be another way to analyze the data, we believe that the general linear model and Tukeys HSD is still an appropriate method to contrast the data. The density values we utilized are analyzed as separate categorical treatments and we are not treating them as continuous variables. We are interested in how these densities perform in comparison to each other and for this we believe that the Tukeys HSD is the most appropriate for the multiple comparisons of this data. Further, when we consulted a statistician, the statistician agreed with the methodology in the paper.

Reviewer 2 Report

The manuscript entitled "Impact of stocking densities on the natural microflora of cloaca, eggshell, and egg content of white egg layers in colony cages" determines the presence of pathogenic bacteria and enterobacteria associated with poultry production, in particular industrial egg production. In addition, it correlates their presence among the different matrices and analyzes possible causes of what was observed.

Abstract: Previous works are mentioned in which the results differ in some parameters with those of the current work, and in the discussion it is suggested that more studies should continue to be carried out, for example, controlled studies of infection with pathogens. I think it would be desirable that the abstract of the paper reflects and emphasizes that the results are for this particular study, instead of generalizing.

The methodology is very clear and the results are well presented.

Discussion: although it is correctly written, the Enterobacteriaceae/coliforms/E. coli section lacks a minimal introduction, something that the Salmonella section does have. I suggest unifying the criteria.

I suggest including and discussing that no animal health-status variables were measured, nor was the composition of the microbiota studied. Determining the presence of pathogens is important in terms of cross-contamination and risk to human health. Additionally, recent work shows that overcrowding can have an effect on animal stress, which can affect the gut microbiota of poultry, and consequently the production of meat and eggs.

Finally, I have some minor comments to detail below:

-Line 110: the assays are not collected, the samples are. I suggest to change to "Biological samples were collected..."

-Line 231 and abstract: I suggest changing microflora to microbiota. The term microlofra refers to an ancient association of microroganisms to the plant kingdom. Although this word can still be seen in some studies, it would be desirable to start correcting it.

Author Response

Hello Reviewer 2,

Thank you for taking your time to provide comments to my manuscript. Below I hope to address your main concerns:

Abstract:  Previous works are mentioned in which the results differ in some parameters with those of the current work, and in the discussion it is suggested that more studies should continue to be carried out, for example, controlled studies of infection with pathogens. I think it would be desirable that the abstract of the paper reflects and emphasizes that the results are for this particular study, instead of generalizing.

I am unsure what you are asking. I do not see any mention of previous studies in the abstract. I have included comments about other research within the introduction however, I did this to help build the need for the research we performed. I also wanted to avoid including any results of the current study within the introduction because I do not believe that is the place for them. Could you please clarify what you are asking?

Discussion: although it is correctly written, the Enterobacteriaceae/coliforms/E. coli section lacks a minimal introduction, something that the Salmonella section does have. I suggest unifying the criteria.

We have added a few sentences to address this.

I suggest including and discussing that no animal health-status variables were measured, nor was the composition of the microbiota studied. Determining the presence of pathogens is important in terms of cross-contamination and risk to human health. Additionally, recent work shows that overcrowding can have an effect on animal stress, which can affect the gut microbiota of poultry, and consequently the production of meat and eggs.

Thank you for the suggestion, I have added a few sentences about this in the conclusion.

-Line 110: the assays are not collected, the samples are. I suggest to change to "Biological samples were collected..."

Added a few words to help clear this up.

-Line 231 and abstract: I suggest changing microflora to microbiota. The term microlofra refers to an ancient association of microroganisms to the plant kingdom. Although this word can still be seen in some studies, it would be desirable to start correcting it.’

Thank you for the suggestion. I have made the corrections.

Reviewer 3 Report

The present study investigated the impact of stocking densities on the natural microflora of cloaca, eggshell, and egg content of white egg layers in colony cages. The study is very interesting and readers focusing on poultry production will benefit from the findings. I have only several minor concerns about this manuscript.

1. Apart from microflora load  in cloaca and egg, it would be better if the authors could provide data on egg production. If so, the sounds of science will be geatly improved. 

2. The introduction is so long and some part is not highly relavant to this study. P51-P62 could be deleted.

3. In table 1, the compositions of Mineral Premix, Vatamin Premix and Selenium Premix should be annotated.

4, In table 3, the meaning of letters like a, b and c should also be annotated.

5, P217, we theorize that this may be due to 

6, P267-P269, The sentence  should be re-written. 

7, P275-278, These senteces describing Salmonella should be combined with 4.3.

Author Response

Hello reviewer 3,

Thank you for taking the time to edit my manuscript. Below I hope I can answer your comments and concerns.

1. Apart from microflora load in cloaca and egg, it would be better if the authors could provide data on egg production. If so, the sounds of science will be geatly improved.

We are currently preparing a manuscript that addresses the production, egg quality, welfare, and other parameters. As this study involved a small subset of hens within the production, quality, and welfare study, we felt it appropriate to focus this paper on the microbial profile exclusively.

2. The introduction is so long and some part is not highly relevant to this study. P51-P62 could be deleted

We have removed this paragraph to shorten the introduction.

3. In table 1, the compositions of Mineral Premix, Vatamin Premix and Selenium Premix should be annotated.

We have added this information.

4, In table 3, the meaning of letters like a, b and c should also be annotated

Thanks for the catch! I have added it.

5, P217, we theorize that this may be due to

Added

6, P267-P269, The sentence  should be re-written.

Broke up the sentence so it is hopefully less confusing.

7, P275-278, These senteces describing Salmonella should be combined with 4.3.

I removed the final sentence in this paragraph because it is discussed in the Salmonella section

Reviewer 4 Report

This study presents a compelling analysis of the influence of stocking densities on the microbiological aspects within the poultry industry. However, a notable concern arises from the approach employed for the isolation of Salmonella spp. In the manuscript, the authors described the plating of samples onto selective media after pre-enrichment in BPW, omitting the typically recommended enrichment steps (eg: MKKTTN/RV broth) as outlined in recognized methodologies such as ISO 6579 or BAM Chapter 5 for Salmonella species isolation. The absence of an enrichment method increases the risk of potential oversight in Salmonella detection.

Furthermore, the reliance solely on the observation of Salmonella growth on selective media lacks comprehensive conclusiveness. Definitive confirmation of Salmonella identity necessitates supplementary measures, such as a series of biochemical tests or PCR-based assays. The current methodology's potential pitfalls are twofold: firstly, false negatives may arise due to the absence of enrichment, and secondly, incomplete identification of cultures could lead to false positives. These limitations hold significant implications, particularly considering the study's concluding assertion that altering space allocation per hen does not impact eggshell microbiological profiles or Salmonella prevalence in cloaca or eggshell.

Given the potential ramifications associated with the aforementioned concerns, I recommend a substantial revision of the manuscript. Addressing these methodological issues is crucial to uphold the accuracy and reliability of the study's findings.

Author Response

Hello Reviewer 4.

Thank you for reviewing my manuscript. Below you will find your comments and my response to them.

In the manuscript, the authors described the plating of samples onto selective media after pre-enrichment in BPW, omitting the typically recommended enrichment steps (eg: MKKTTN/RV broth) as outlined in recognized methodologies such as ISO 6579 or BAM Chapter 5 for Salmonella species isolation. The absence of an enrichment method increases the risk of potential oversight in Salmonella detection.

Thank you for pointing it out. We did use RV but somehow didn’t include it in the manuscript. We have made corrections in the methodology session.

Furthermore, the reliance solely on the observation of Salmonella growth on selective media lacks comprehensive conclusiveness. Definitive confirmation of Salmonella identity necessitates supplementary measures, such as a series of biochemical tests or PCR-based assays. The current methodology's potential pitfalls are twofold: firstly, false negatives may arise due to the absence of enrichment, and secondly, incomplete identification of cultures could lead to false positives. These limitations hold significant implications, particularly considering the study's concluding assertion that altering space allocation per hen does not impact eggshell microbiological profiles or Salmonella prevalence in cloaca or eggshell.

Totally agree. We randomly picked typical colonies to confirm using BAX PCR. However, we didn’t include it in the manuscript and we have made the corrections in the methodology.