Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant Markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels

Round 1

Reviewer 1 Report

The manuscript titled: Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant Markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels by A. Atey et al. is of great interest. It deals with genes that may interact with susceptibility to mastitis in dromedary camels. This issue is currently very important on a global scale. Mastitis infections, one of the most complicated and expensive endemic diseases of dairy animals, cause inflammation of the mammary gland and udder tissue. This causes a reduction in milk production, culling of animals, high veterinary costs and results in huge economic losses for breeders. The manuscript meets the requirements of the research work, i.e. is divided into individual chapters. The material and methodology are written clearly and comprehensibly. However, it is necessary to add the name of the company (eg. line 156 - missing company). Statistical analyzes are correctly presented. The interpretation of the results is concise, clearly formulated and correct. The results are interesting and new. The given links are correct.

 Therefore, I recommend the mentioned manuscript with minor modifications for acceptance into the journal: Agriculture (ISSN 2077-0472).

 Questions:

-          on what basis did you choose the mentioned genes?

-          you did gene expression from milk or from tissue

-     you used only one house keeping - reference gene - why this gene GAPDH, and it is possible to use another gene for this purpose,

-  for better reliability it is possible to use more genes (2-3)

- did you sequence both sides? you used both F and R primer

- what percent agarose gel did you use for control? (line 165)

 - why do you have such a large volume for the PCR reaction? - usually max. 50 microliters

 - table 2 (line 206), I don't see how long the annealing step took

 - how do you explain that for individual SNPs you did not find any numbers of animals (eg OTUD3 T120C - healty 28, but mastitits - no numbers, etc.), then which nucleotide was present for the given SNP?

  - were all genes up-regulated, or were some of them also down-regulated?

  - do you think that based on your study, cheap could be designed and applied

- I mean analyze animals and use them in breeding based on their genotype

 

Comments for author File: Comments.pdf

English is good, you can understand, but there are places in the article where sentences need to be reformulated. I am sending a proposal for modification in the attachment.

Author Response

Dear Reviewer,

Thank you very much for your interest and consideration. It is of great pleasure to receive your decision on our paper "Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels"

Please find our  response file to the reviewer comments. All comments were considered while revising this paper.

We hope that, our response is satisfactory.

Thank you very much for your cooperation and waiting for your response

Kind regards

Sincerely yours,

Corresponding author

Author Response File: Author Response.docx

Reviewer 2 Report

1. The number of the samples of camels should be presented in the figure legends.

2. The color and form of the figure should be modified to make them clear to read.

3. The control group in the RT-qPCR results would be around 1. The method for data analysis is 2-ΔΔCt, but it seems that the results were not obtained using this method. 

 

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

Comment

1. The number of the samples of camels should be presented in the figure legends.

Response

We are grateful to the reviewer for drawing it to our consideration. The number of the samples of camels is presented in the figure legends.

Comment

2. The color and form of the figure should be modified to make them clear to read.

Response

We are grateful to the reviewer for drawing it to our consideration. Better quality figures are added.

Comment

3. The control group in the RT-qPCR results would be around 1. The method for data analysis is 2-ΔΔCt, but it seems that the results were not obtained using this method. 

Response

We thank reviewer for this.  According to Livak and Schmittgen, 2001 and Pfaffl, 2001, the gene expression profile could be calculated in different ways as follows:

• The first way, you could make 1 for the control group calculations and in this case you can write the other groups by fold change using 2^-ΔΔCt method (for example 1.5 fold change etc…..). This method is preferred when there are different treatments in case of investigation of effect of different concentrations of certain treatment on different groups.
• The second way (as we made in our manuscript), you could calculate the mean for the control group (for all investigated animals) and then subtract each sample in the control group from this mean for the house keeping gene. In this way the control group is not necessary to be around or equal 1. The gene expression in this case could be also calculated also by using 2^-ΔΔCt. Actually we prefer this method especially when the number of animals is large enough and it is necessary in this way to calculate the overall mean for control group. We have published more than one paper by this way as follows:
• Ateya A, Al-Sharif M, Abdo M, Fericean L, Essa B. Individual Genomic Loci and mRNA Levels of Immune Biomarkers Associated with Pneumonia Susceptibility in Baladi Goats. Vet Sci. 2023 Mar 1;10(3):185. doi: 10.3390/vetsci10030185. PMID: 36977224; PMCID: PMC10051579.
• Essa B, Al-Sharif M, Abdo M, Fericean L, Ateya A. New Insights on Nucleotide Sequence Variants and mRNA Levels of Candidate Genes Assessing Resistance/Susceptibility to Mastitis in Holstein and Montbéliarde Dairy Cows. Vet Sci. 2023 Jan 3;10(1):35. doi: 10.3390/vetsci10010035. PMID: 36669036; PMCID: PMC9861242.
• Darwish, A.; Ebissy, E.; Ateya, A.; El-Sayed, A. Single nucleotide polymorphisms, gene expression and serum profile of immune and antioxidant markers associated with postpartum disorders susceptibility in Barki sheep. Anim. Biotechnol. 2023, 34, 327-339. 
• Al-Sharif, M.; Ateya, A. New Insights on Coding Mutations and mRNA Levels of Candidate Genes Associated with Diarrhea Susceptibility in Baladi Goat. Agriculture 2023, 13, 143. https://doi.org/10.3390/ agriculture13010143.
• The third way when there is no control group. In this case we depend on ΔCt by calculating Ct of each sample for investigated gene and subtracting it from Ct of the sample for housekeeping gene. In this situation we depend on the fact that ΔCt is inversely related to gene expression profile. Listed below are published papers depend on this way:
• Ateya AI, Hendam BM, Radwan HA, Abo Elfadl EA, Al-Sharif MM. Using Linear Discriminant Analysis to Characterize Novel Single Nucleotide Polymorphisms and Expression Profile Changes in Genes of Three Breeds of Rabbit (Oryctolagus cuniculus). Comp Med. 2021 Jun 1;71(3):222-234. doi: 10.30802/AALAS-CM-20-000103. Epub 2021 May 25. PMID: 34034856; PMCID: PMC8223864.
• Fouda, A. Ateya, I. EL Araby, and A. Elzeer. GENETIC POLYMORPHISMS AND EXPRESSION PATTERN OF IGF-I GENE IN COMMERCIAL BROILER STRAINS. Journal of Animal & Plant Sciences, 31(6): 2021, Page: 1542-1547.

Reviewer 3 Report

The manuscript titled “Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant Markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels ” by Ateyaet al., submitted to “Agriculture,” investigates the changes in immunological and antioxidant genes that may interact with mastitis susceptibility in dromedary camels. After carefully reviewing the manuscript, I commend the authors for their efforts in elucidating the potential benefits of expanding the knowledge of genes whose upregulation may influence mastitis susceptibility. There are certain areas in the paper that could be enhanced to improve its clarity and impact. Below, I provide my specific comments and suggestions: 

 –       The title accurately conveys the essence of the study, which is commendable.  

–       The abstract is comprehensive but might be more concise by adding the p-value in the brackets. 

–       In the introduction, when discussing the economic influence of mastitis prevention as well as losses connected with its treatment, it might be helpful to briefly touch upon why this is of particular importance in the context of camelid health, giving the reader a clearer picture of the study’s significance. 

–       The segment discussing the consequences of antibiotic resistance could be added.  

  

–       The objective statement at the end is clear, but it might benefit from an added emphasis on its novelty or what gap in the literature this study aims to address. This could further highlight the study’s significance. 

–       In the methods, specify the location or facility where the study took place as well as which clinical parameters were examined for health assessment.  

–       The paragraph on ROS production and oxidative stress seems quite dense. Breaking it into smaller paragraphs, each focusing on a distinct point or aspect of ROS production, might make it more digestible for readers. 

–       Throughout the discussion, references to previous studies and your own study’s results are intermixed. Consider reorganizing for clearer flow, possibly by presenting established knowledge first, followed by how your findings align or differ. 

–     How does oxidative stress lead to the modulation of immune system response? Delving deeper into this can provide readers with a more comprehensive understanding.  

–       The conclusion succinctly recaps the main findings. However, it might be helpful to add a sentence or two about the implications of these findings for future research or practical applications. 

–       The conclusion might benefit from a mention of any limitations in the study or areas where further research is recommended. This can provide context and direction for future studies in this area. 

–       The study, while comprehensive in its findings, may have some potential limitations. The specifics regarding the diversity of the camels, and different pathogens which may cause the mastitis were not detailed. Also, some other environmental factors are influencing the immune responses and oxidative stress could also impact the results. To fortify the study, it might be beneficial to add paragraph about potential study limitations. 

 

Minor corrections are needed.

Author Response

Reviewer 3

Comments and Suggestions for Authors

The manuscript titled “Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant Markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels ” by Ateyaet al., submitted to “Agriculture,” investigates the changes in immunological and antioxidant genes that may interact with mastitis susceptibility in dromedary camels. After carefully reviewing the manuscript, I commend the authors for their efforts in elucidating the potential benefits of expanding the knowledge of genes whose upregulation may influence mastitis susceptibility. There are certain areas in the paper that could be enhanced to improve its clarity and impact. Below, I provide my specific comments and suggestions: 

Comment

 –       The title accurately conveys the essence of the study, which is commendable.  

Response

We thank reviewer for this positive comment.

Comment

–       The abstract is comprehensive but might be more concise by adding the p-value in the brackets. 

Response

We thank reviewer for this comment. We have added p-value.

–       In the introduction, when discussing the economic influence of mastitis prevention as well as losses connected with its treatment, it might be helpful to briefly touch upon why this is of particular importance in the context of camelid health, giving the reader a clearer picture of the study’s significance. 

Response

We are grateful to the reviewer for drawing it to our consideration. A paragraph on influence of mastitis prevention on camelid health is added with reference.

Comment

–       The segment discussing the consequences of antibiotic resistance could be added.   

Response

We are grateful to the reviewer for drawing it to our consideration. The segment discussing the consequences of antibiotic resistance is added with reference.

Comment

–       The objective statement at the end is clear, but it might benefit from an added emphasis on its novelty or what gap in the literature this study aims to address. This could further highlight the study’s significance. 

We are grateful to the reviewer for drawing it to our consideration. The novelty statement is added at the end of introduction section.

Comment

–       In the methods, specify the location or facility where the study took place as well as which clinical parameters were examined for health assessment.  

Response

We are grateful to the reviewer for drawing it to our consideration. The information of health inspection and time of doing experiment is added.

Comment

–       The paragraph on ROS production and oxidative stress seems quite dense. Breaking it into smaller paragraphs, each focusing on a distinct point or aspect of ROS production, might make it more digestible for readers. 

Response

We are grateful to the reviewer for drawing it to our consideration. Generally paragraphs of the discussion section are broken it into smaller paragraphs, make it more digestible for readers. 

Comment

–       Throughout the discussion, references to previous studies and your own study’s results are intermixed. Consider reorganizing for clearer flow, possibly by presenting established knowledge first, followed by how your findings align or differ. 

Response

• We thank reviewer for this. Actually our study is novel to study these markers in dromedary camels. The investigated markers was proven to be candidates for mastitis susceptibility in other livestock. We mean that we have some share points with previous studies in proving the efficiency of these markers in other livestock. Therefore we put the listed below statements in the discussion section
• There needs to be more information on polymorphisms and expression profile of immune antioxidant biomarkers for mastitis incidence in dromedary camels. The initial evidence for this connection can be found in the gene sequences from the Camelus dromedarius that were used in our work and were published in PubMed. The variance of the immune (OTUD3, TLR2, TLR4, STAB2, MBL2, TRAPPC9, and C4A) and antioxidant (CAT, SOD3, PRDX6, OXSR1, NDUFS6, SERP2, and ST1P1) markers and how they relate to mastitis incidence in dromedary camels have not, to our knowledge, been the subject of any prior research.
• The limitations of earlier studies were overcome by our investigation of gene polymorphism using SNP genetic markers and gene expression. In order to address this issue, real-time PCR was used for the first time to measure the mRNA levels of key immunological and antioxidant markers in mastitic and healthy she-camels. As a result, the mechanisms of the investigated gene regulation are well understood in both healthy and mastitic she-camels.
• In both healthy and mastitis-affected dromedary she-camels, our study is the first to show the expression profile of immunological (OTUD3, TLR2, TLR4, STAB2, MBL2, TRAPPC9, and C4A) and antioxidant (CAT, SOD3, PRDX6, OXSR1, NDUFS6, SERP2, and ST1P1) However, the transcript analysis of immune and antioxidant markers was investigated to monitor mastitis incidence in other livestock; where gene expression rate of immune and antioxidant markers was proven to be a reference guide for tracking animal health in case of mastitis.
• We have divided the discussion sections into three sections to make it more digestible for readers.
• In appreciation to the reviewer efforts, we wrote some sentences that illustrate the coincidence of proving the efficiency of using candidate gene approach and expression profile of immune and antioxidant markers for monitoring of mastitis in livestock.

Comment

–     How does oxidative stress lead to the modulation of immune system response? Delving deeper into this can provide readers with a more comprehensive understanding. 

Response

We are grateful to the reviewer for drawing it to our consideration. Two paragraphs with references are added to illustrate how oxidative stress leads to the modulation of immune system response.

Comment

–       The conclusion succinctly recaps the main findings. However, it might be helpful to add a sentence or two about the implications of these findings for future research or practical applications. 

Response

• We are grateful to the reviewer for drawing it to our consideration. We have added implications of study as follows:
• These distinct functional variations present a significant opportunity to decrease the occurrence of mastitis by using genetic markers coupled with normal welfare during camel selection.
• Genetic selection, utilizing gene polymorphisms for enhanced innate immunity, is therefore a sustainable method of reducing the prevalence of disease and enhancing health in dromedary camels.
• Future treatments and selection for mastitis resistant animals may be made easier by the gene targets discovered here; therefore decrease economic losses afforded by animal breeders.

Comment

–       The conclusion might benefit from a mention of any limitations in the study or areas where further research is recommended. This can provide context and direction for future studies in this area. 

Response

We are grateful to the reviewer for drawing it to our consideration. Limitations of the study are mentioned in the conclusion.

Comment

–       The study, while comprehensive in its findings, may have some potential limitations. The specifics regarding the diversity of the camels, and different pathogens which may cause the mastitis were not detailed. Also, some other environmental factors are influencing the immune responses and oxidative stress could also impact the results. To fortify the study, it might be beneficial to add paragraph about potential study limitations. 

Response

We are grateful to the reviewer for drawing it to our consideration. A paragraph for the limitations of the study is added at the end of the discussion section.