<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">A comparative in silico analysis of the vlhA gene regions of Mycoplasma gallisepticum  and Myc. synoviae isolates from commercial hen farms in Mexico. </xhtml:span>
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Avian mycoplasmosis, caused by Mycoplasma synoviae and Mycoplasma gallisepticum, poses significant economic challenges due to respiratory issues, reduced egg production, and soft eggshells. The variable lipoprotein haemagglutinin (VlhA) protein, crucial for pathogenicity, comprises conserved (MSPB) and variable (MSPA) regions. The aim of this study was to identify the conserved region of vlhA gene sequences in field strain. We examined vlhA sequences from field strains collected in central Mexico (Jalisco and Mexico City). Specifically, we analysed 124 deformed eggs and 10 laying hens from 9 farms with Hy-line and Bovans breeds. Using polymerase chain reaction (PCR) targeting the mgc2 and 16S ribosomal RNA (rRNA) genes, we characterised 24 field strains, 4 of which were Myc. synoviae and 20 of which were Myc. gallisepticum (20). We analysed the vlhA regions, based on the AF035624.1 reference sequence, with ATTC strains as positive controls. Additionally, we validated the PCR with 20 negative samples from Mycoplasma isolation without the need of cultivation. We identified two amplification regions: MSPB and MSPA. Bioanalysis revealed relationships between our field samples and avian Mycoplasma sequences in GenBank, alongside similarities with lipoproteins present in Acholeplasma laidlawii PG8 and Escherichia coli. Given the significance of the VlhA protein in pathogenicity and immune evasion, the identified conserved sequences hold potential as therapeutic targets and for phylogenetic studies.
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All reviewer and editor comments have been sufficiently addressed.
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Comments to Author
I have no further comments or suggestions.
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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There is a consensus that the presented data would be valuable to the scientific community, though reviewers have raised some concerns, primarily about the methods used to generate the presented data.
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Comments to Author
General: An informative and fair in-silico analysis on Mycoplasma bringing a relevant contribution to the literature by characterising 24 in-field strains. The overall methodologies, investigative techniques and analyses are appropriate in the setting, with meaningful discussion points. There are minor referencing concerns, which have been addressed further below. There are numerous grammatical errors, and the publication requires a careful line-by-line appraisal before final publication. Whilst many of these have been highlighted below, there are repeats which are not mentioned. Fewer than the grammatical errors are specific writing recommendations, as some ideas are conveyed in a slightly complex fashion. Overall, a good publication, needing predominantly grammatical editing. 1. Methodological …
Comments to Author
General: An informative and fair in-silico analysis on Mycoplasma bringing a relevant contribution to the literature by characterising 24 in-field strains. The overall methodologies, investigative techniques and analyses are appropriate in the setting, with meaningful discussion points. There are minor referencing concerns, which have been addressed further below. There are numerous grammatical errors, and the publication requires a careful line-by-line appraisal before final publication. Whilst many of these have been highlighted below, there are repeats which are not mentioned. Fewer than the grammatical errors are specific writing recommendations, as some ideas are conveyed in a slightly complex fashion. Overall, a good publication, needing predominantly grammatical editing. 1. Methodological rigour, reproducibility and availability of underlying data Good reference has been made as to how the in-silico analyses have been performed. The data allows for adequate reproducibility. Protocol standardisations for PCR are appropriate, and there is good referencing of all reagents. The complementary study for the PCR test is well structured. There is good explanation of the hemadsorption, and the hemagglutination protocols. allowing for good reproducibility. 2. Presentation of results The results are presented in an appropriate fashion. There is good usage of diagrams. It would be useful to add sub-headings for all the main points in the discussion. E.g. Line 311 - Subheading: Surface Protein Variation, and Line 322 - Subheading: Verification of Cluster Analysis by Complementary Study. 3. How the style and organization of the paper communicates and represents key findings The impact statement serves as a useful adjunct to the reader regarding the purpose of the paper. The abstract however lacks this key piece of information and should be able to stand alone without the impact statement. A short line regarding the overall purpose of the paper would be a useful final addition to the abstract. 4. Literature analysis or discussion The literature referenced is appropriate. Useful for the introduction may be other recent studies referencing Mycoplasma and its pathogenic role, particularly Al-Baqir et al. (2023), "Mycoplasmosis in Poultry: An Evaluation of Diagnostic Schemes and Molecular Analysis of Egyptian Mycoplasma gallisepticum Strains". 5. Grammatical corrections Edits: Line 24 - Reduced production of what? Line 192 - Rephrasing of sentence, difficult to read at present Line 237 - "The result of negative controls was carried on in 3 times being positives the 2-strain mentioned." - Please rephrase statement for easier reading. Line 228 and 229 - Good usage of semi-colon. This may be deployed in other areas of the paper when numerous commas are used to make reading easier. Line 288 - "Although the design of primers that amplify the vlhA gene with the sequence AF035624.1 from Myc. synoviae typing is critical due to mutations that can be found in the sequences of the field strains, especially those designed in the MSPA region for their recombination with paralog genes [14, 32]." - The usage of although is supposed to lead to a sub-ordinate clause that contrasts with the main clause. That is not seen here. Lines 586 - 589 - The description for Figure 3 may be shortened as you have already explained the 24 strains analysed. Line 626 - "Sequence of Gen Bank in Phylogenetic tree" may be converted into your third table and referenced thereafter in Figure 5 and Figure 6. Grammatical errors: Line 25 - Italicisation of gene names (vlhA), and repetitions to be altered throughout submission. Line 62 - First comma can be removed Line 69 - Gene italicisation Line 71, 72 - Please include et al. when referencing publications with multiple authors, e.g. Noormohammadi et al. Line 117 - Correction of in-text referencing style Line 127 - Correction of double space 152 - Insertion of "et al." Line 157 - 158 - Please reference the webpage appropriately both in-text and in the main reference section. This links to the company website only and is not a helpful reference. Line 561 - "Egg withe" - spelling
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
Summary Maya-Rodríguez et al set out to analyze field isolates of Mycoplasma synoviae and Mycoplasma gallisepticum, which can cause avian mycoplasmosis and impact egg yields of chickens, from central Mexico. Using molecular analysis, they identify mycoplasma infections in ~20% of their samples tested. Further bioinformatic analysis of part of the vlhA gene of these isolates showed similarities with known isolates from other areas of the world. Major Considerations 1. The clarity of the manuscript can be confusing at times. I highly suggest proofreading for errors in consistency as well as having a native English speaker edit the manuscript for grammar. 2. I have significant concerns about figure 1, particularly about potential errors that make it difficult to review the methodology, analysis, and …
Comments to Author
Summary Maya-Rodríguez et al set out to analyze field isolates of Mycoplasma synoviae and Mycoplasma gallisepticum, which can cause avian mycoplasmosis and impact egg yields of chickens, from central Mexico. Using molecular analysis, they identify mycoplasma infections in ~20% of their samples tested. Further bioinformatic analysis of part of the vlhA gene of these isolates showed similarities with known isolates from other areas of the world. Major Considerations 1. The clarity of the manuscript can be confusing at times. I highly suggest proofreading for errors in consistency as well as having a native English speaker edit the manuscript for grammar. 2. I have significant concerns about figure 1, particularly about potential errors that make it difficult to review the methodology, analysis, and conclusions: 2a. Did the authors run a water only control? I am concerned that amplification from unrelated strains could be due to contamination or non-specific amplification. When blasting the vlhA gene against E coli, I get no hits. 2b. The authors mention in line 235 that Ecoli was positive and reference Figure 1B but I do not see that in Figure 1B. 2c. In table 1, the authors mention a 397bp product, but in figure 1 mention a 307bp product. Is this a typo, or was band size calculated? If it was calculated, what were the methods for doing so? 2d. I do not see any data for MSPA amplification. Is this meant to be figure 1B (labeled as MSPB in the legend)? If so, the authors need to show all controls and at least a subset of samples for both PCRs. 3. The polymerase used (MyTaq) is a low fidelity polymerase that could impact sequencing results by introducing mutations. Can the authors comment on any effort to ensure resulting PCRs did not accrue mutations (IE if PCR was performed 3-5x on the same isolate, and those sequences were compared directly, how robust is the fidelity of amplification?). 4. Can the authors summarize identified mutations in a table or alignment schematic with the reference sequence? I am particularly confused as to how the authors identify a stop codon to isoleucine mutation when they do not amplify the 3' end of the gene. They way I read lines 248-249 indicates to me that the reference sequence had a stop codon, and the authors identify a mutation to isoleucine? 5. In lines 271-273 the authors state "Additionally, we validated the sensitivity of the PCR test through a complementary study, demonstrating that it is not necessary to isolate mycoplasmas for PCR testing." This seems like an overstatement. The author's PCR is not specific to mycoplasma (E coli and A laidlawii were also presumably amplified), Minor Considerations 1. Be consistent with gene and protein format. Genes should be lower case, italicized (vlhA) while protein products should be upper case, non-italicized (VlhA). 2. Check references for accuracy. IE: lines 90 and 91 reference citation 19 to support that Mgall has 43 vlhA pseudogenes. However, reference 19 does not mention this at all, reference 18 does. 3. If E coli, S aureus and A laidlawii were used as negative controls in the molecular tests, these results should be reported on in table 2. 4. Unsure what has been "complemented" in the vlha complementary study. I believe the authors might mean comparative study of partial vlhA sequences? 5. Figure two could be labeled more clearly. What is the large arrow denoting? What is the star with f2 denoting? 6. It would be beneficial to expand discussion on the amplification of Acheloplasma and how it compares to previous studies. Did previous studies include Acheloplasma or other Mollicutes? Or is inclusion of this species a strength of the paper? 7. Why was E coli not included in the bioinformatic study? 8. I think the manuscript would be greatly aided by display and alignment of the actual sequences that were used in the bioinformatic analysis.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
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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Thank you very much for submitting your manuscript to Access Microbiology. After reading your manuscript, I agree with you that this is a valuable contribution to the field and that it is appropriate for the scope of this platform. However, I am afraid that it contains a number of flaws in the use of English language that could undermine the readability of the text and the overall quality of the work. I would suggest to the authors to ask a native English speaker colleague to proof it and do the corrections they consider appropriate. Alternatively, there are different language editing services that will do this for you at a cost. In case the authors are interested in this option, the Microbiology Society is partnered with Editage (please see https://www.editage.com/microbiologyresearch/) to offer a discounted pricing. We will be happy …
Thank you very much for submitting your manuscript to Access Microbiology. After reading your manuscript, I agree with you that this is a valuable contribution to the field and that it is appropriate for the scope of this platform. However, I am afraid that it contains a number of flaws in the use of English language that could undermine the readability of the text and the overall quality of the work. I would suggest to the authors to ask a native English speaker colleague to proof it and do the corrections they consider appropriate. Alternatively, there are different language editing services that will do this for you at a cost. In case the authors are interested in this option, the Microbiology Society is partnered with Editage (please see https://www.editage.com/microbiologyresearch/) to offer a discounted pricing. We will be happy to consider a revised version of this work with the amendments explained above.
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