Erysipelothrix spp. and other Erysipelotrichales detected by 16S rRNA microbial community profiling in samples from healthy conventionally reared chickens and their environment
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Outbreaks of erysipelas, a disease caused by infection with Erysipelothrix rhusiopathiae (ER), is a re-emerging problem in cage-free laying hen flocks. The source of ER infection in hens is usually unknown and serological evidence has also indicated the presence of ER or other antigenically related bacteria in healthy flocks. The aim of the present study was to evaluate sample collection, culture methods and DNA-based methodology to detect ER and other Erysipelotrichales in samples from healthy chickens and their environment. We used samples from a research facility with conventionally reared chickens with no history of erysipelas outbreaks where hens with high titres of IgY recognising ER previously have been observed. Microbial DNA was extracted from samples either directly or after pre-culture in nonselective or ER-selective medium. Real-time PCR was used for detection of Erysipelothrix spp. and high-throughput amplicon sequencing of 16S rRNA sequencing was used for detection of Erysipelotrichales. A pilot serological analysis of some Erysipelotrichales members with IgY from unvaccinated and ER-vaccinated high-biosecurity chickens, as well as conventionally reared chickens, was also performed. All samples were negative for ER, E. tonsillarum and E. piscisicarius by PCR analysis. However, 16S rRNA community profiling indicated the presence of several Erysipelotrichales genera in both environmental samples and chicken intestinal samples, including Erysipelothrix spp. that were detected in environmental samples. Sequences from Erysipelothrix spp. were most frequently detected in samples pre-cultured in ER-selective medium. At species level the presence of Erysipelothrix anatis and/or Erysipelothrix aquatica was indicated. Serological results indicated that IgY raised to ER showed some cross-reactivity with E. anatis . Hence, environmental samples pre-cultured in selective medium and analysis by 16S rRNA sequencing proved a useful method for detection of Erysipelotrichales, including Erysipelothrix spp., in chicken flocks. The observation of such bacteria in environmental samples offers a possible explanation for the observation of high antibody titres to ER in flocks without a history of clinical erysipelas.
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The work presented is clear and the arguments well formed. Thank you for you're considered revisions (and rebuttals), i'm pleased to say, following review, the manuscript has now been accepted. Best wishes, John.
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The reviewers raise concerns regarding the scientific rigour and experimental design of the work. The reviewers believe the results shown in the manuscript do not support the conclusions presented.
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Comments to Author
1) Introduction section needs a bit cohesion and has to be proofread for English language. Some sentences are weak and do not convey the proper meaning. 2) Sampling procedure can be better explained if a figure or a map can be added describing the structure and architect of the the poultry facility. Samples could be categorized for example, Cage and ground is related to structure, facility worker shoes are miscellaneous, and mites (as the vector). 3) Please add a figure or flow chart for the methods. 4) Supernatant from 3 protocols were mentioned were used for DNA extraction, however the sampling procedure 1 does not indicate any supernatant. 5) Line 294 does not convey the proper meaning. How the low virulence Erysipelothrix can be predicted? Have any samples from infected chickens were …
Comments to Author
1) Introduction section needs a bit cohesion and has to be proofread for English language. Some sentences are weak and do not convey the proper meaning. 2) Sampling procedure can be better explained if a figure or a map can be added describing the structure and architect of the the poultry facility. Samples could be categorized for example, Cage and ground is related to structure, facility worker shoes are miscellaneous, and mites (as the vector). 3) Please add a figure or flow chart for the methods. 4) Supernatant from 3 protocols were mentioned were used for DNA extraction, however the sampling procedure 1 does not indicate any supernatant. 5) Line 294 does not convey the proper meaning. How the low virulence Erysipelothrix can be predicted? Have any samples from infected chickens were tested for virulence? 6) Please add a conclusion to the discussion section which clearly justify the objective of the study.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Summary: The paper seeks to unravel why chickens exhibit positive antibody presence against ER despite no recorded outbreaks in their facility. It demonstrates that the qPCR with probe method lacks sensitivity for detecting Erysipelothrix rhusiopathiae (ER) or Erysipelothrix spp., while the proposed 16S sequencing method can identify species within Erysipelothrix spp. but not specifically ER. I've provided suggestions below, including adding experimental data in supplementary tables to bolster the paper. Line 126: Kindly specify in the method the supernatant was obtained after lysing bacteria Line 130: Section 8.3. Maybe it could be a good idea to add the graph of the qPCR with probe in supplementary data to show that positive controls amplified while tested DNA did not. Same for data for SYBR qPCR …
Comments to Author
Summary: The paper seeks to unravel why chickens exhibit positive antibody presence against ER despite no recorded outbreaks in their facility. It demonstrates that the qPCR with probe method lacks sensitivity for detecting Erysipelothrix rhusiopathiae (ER) or Erysipelothrix spp., while the proposed 16S sequencing method can identify species within Erysipelothrix spp. but not specifically ER. I've provided suggestions below, including adding experimental data in supplementary tables to bolster the paper. Line 126: Kindly specify in the method the supernatant was obtained after lysing bacteria Line 130: Section 8.3. Maybe it could be a good idea to add the graph of the qPCR with probe in supplementary data to show that positive controls amplified while tested DNA did not. Same for data for SYBR qPCR experiment with the identified sequenced the in a supplementary table. Line 132: Please specify the sequence of the probe Line 141: Kindly specify if you add any positive control for the 16S RNA sequencing Line 143: Please specify the sequence of used primers. Line 149: Please specify the version of phyloseq R package Line 151: Kindly specify the version of Mega X used. Line 152: Please specify the number of bootstraps and the model that were used for the ML analysis. Line 157: I believe that using "Genes encoding for proteins" is better has a blastn analysis has been done. Line 158: Please specify the parameters that have been used for the blast. Line 179: Please specify if the Elisa assay was performed on technical replicates as the data in figure on only show individual values. Line 183: Maybe it could be a good idea to add the data obtained for this assay and calculus in supp data. Line 203-207: Are the reads numbers after removing any contaminants? Line 221-224 and 232-234: Please add the sequence alignment sin the supp data? Line 238: Please clarify why you did a serological analysis on chicken kept in the conventional center at two different ages? Data showed that there is no clear different in antibody titration between both ages. Line 254 Table 1: Please explain/suggest why pathogens were not detected in all samples isolated from Ilieum Caecum and Cloaca? Maybe it will be easier to re-specify the ages of the chicken in the table (10-12 weeks) to make it easier for the reader. Line 258 Figure 1: I would suggest changing the plot for a heatmap with a scale going between the lowest and the highest value at 3800 while having the same color for samples above 3800. I think that it will make the figure easier to read. Line 300-302: Please review the sentence, I am not sure of the meaning. Line 315: The results on the figure 1 showed that levels of antibody are higher in the chicken from the conventional facility. Would it mean that ER and others are present in the facility (mentioned in line 42-43 but not in the discussion) and ER would not have been detected or misidentified during sequencing? Would it be possible that the presence of these pathogens is too low to induce any disease? I am wondering: if the positivity of the Elisa assay was only a question of cross-reactivity between similar immunogenic proteins within species then I would expect similar or higher levels of antibody against E. anatis compared to ER as E. anatis was detected during the sequencing. What is your opinion about it? Line 322: It would be interesting to discuss about other methods to detect ER. It seems that a conclusion about the used methods is missing. Line 479: Supp Fig: The figure is missing the probabilities for each branch separation. They are essential as it allows the reader to identify with separation are real.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Dear Author, You involved ELISA in your experiment but you didn't mention to the results clearly in your data, I highly recommend to add the results related to ELISA in clear chart or table Brief lines for the sonicated bacteria (is it already prepared or there is a procedure for that
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
Comments to Author
Dear Author, You involved ELISA in your experiment but you didn't mention to the results clearly in your data, I highly recommend to add the results related to ELISA in clear chart or table Brief lines for the sonicated bacteria (is it already prepared or there is a procedure for that
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
1. The authors note in 2 different places in the document that the sequence information has been submitted to EBI and provided the project number. Providing the project number is helpful for follow-up work and metanalyses. Instrument manufacturers (e.g., Illumina) as well as database versions were referenced in the Methods sections. Providing specific manufacturers and version control can be crucial, especially when next-generation sequencing platforms are used; kit chemistry and sequence databases can change rapidly and affect the outcome of the same analyses (e.g., an updated/more curated version of a sequence database can fix mis-annotated genomes which could have given spurious results). Of note that should be addressed, I could not find mention of control samples for DNA extraction and/or …
Comments to Author
1. The authors note in 2 different places in the document that the sequence information has been submitted to EBI and provided the project number. Providing the project number is helpful for follow-up work and metanalyses. Instrument manufacturers (e.g., Illumina) as well as database versions were referenced in the Methods sections. Providing specific manufacturers and version control can be crucial, especially when next-generation sequencing platforms are used; kit chemistry and sequence databases can change rapidly and affect the outcome of the same analyses (e.g., an updated/more curated version of a sequence database can fix mis-annotated genomes which could have given spurious results). Of note that should be addressed, I could not find mention of control samples for DNA extraction and/or sequencing. Given the various sample types and locations there likely should have been several separate controls (e.g., PBS-only, swab opened to air and then processed without touching anything). I did appreciate the note about the one sample that returned no sequence reads, as some authors may have chosen to simply not mention this. However, dovetailing with the above note about not seeing mention of controls for DNA extraction and sequencing, I'd be curious what level of reads blank barcodes or controls had relative to real samples. 2. The use of 3 different methods of detection, real-time PCR, 16S rRNA amplicon sequencing, and ELISA helps to give context when a specific method may have a bias or limit of detection issue (e.g., a specific representative strain isn't available for cross reference or the amplicon region isn't sufficiently differentiating). It could potentially be helpful to display at least some representative sequence alignments in addition to the text and trees presented. 3. A good balance throughout the article between the 3 different identification methods (PCR, sequencing, and ELISA). 4. Citations ranged across many groups and authors. It does seem there's a slight error in citation #25 ("Chidtens" likely to be 'Chickens'?) 5. Isolation, sequencing, identification, and characterization of strains seems like the necessary next step. Without metagenomics or isolates many further in silico or in vitro characterization and hypothesis generation are hindered. Given that the authors demonstrate an ability to enrich in broth for detection it is likely that they could at least enrich enough for metagenomic sequencing and MAG generation, if not isolation and banking/sequencing.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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