Review Reports - Antimicrobial Susceptibility Profiles of Campylobacter jejuni and Campylobacter coli Isolated from Processed Chickens and Turkeys in Morocco

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

in your manuscript you presented the results of the investigation related to the Campylobacter spp and its prevalence in different poultry samples as well as its antimicrobial resistance.

General comments:

-title should be changes as it does not reflect the investigation in full

-writing of antimicrobials in the whole text should be checked (with/without first capital letter)

-writing of genes in the whole text should be checked (italic or not)

Abstract - please describe why each of the mentioned methods were used (L23-25); please include a conclusion at the end of the Abstract

L 27 please rephrase "significantly more"

L 28-29 please check this sentence, not clear enough

L 29-31 please change this sentence, not clear enough

Introduction - it is a bit confusing as it is not clear which statements are related to humans and which to poultry; please include more information on the Morocco poultry industry, information on the food markets and slaughterhouses procedures regarding chickens and turkeys as well as data on human campylobacteriosis.

L 41-42 please rephrase "crucial vehicle"

L 52-55 please check this sentence, not clear enough

Sample collection - please give more information on the samples (what was the size of the samples - whole cecum or a piece of organ or a swab/portion of fecal matter as stated in L97? amount of neck skin in g? what kind of muscles did you take - a piece or?) please mention the age of the birds at the slaughter, if known

L 96 how did you incubate Columbia Agar?

Selective supplement in L 88 and antimicrobial supplement in L 93- please mention the correct names of those supplements and the manufacturers

Suspected colonies in L 98, 101- please describe what did you mean by that?

L101-104 please mention reference for those methods

L 106 not clear: "strains isolated were activated"?

L 111 punctuation missing?

Antimicrobial disks and PCR reagents/primers- please mention the manufacturer

Quality control - did you also include negative control?

Results- is there any reason why you presented the results of statistical analysis in a separate chapter and not related to the results?

Strains identification - did you determined the campylobacter species by biochemical methods L 153-155?

L 161-163 please change this sentence, not clear enough

L 163-166 this comparison with the reference (7) not clear as El Baaboua investigated the prevalence of Campylobacter in meat, animal and human samples - please clarify

L 171-172 please check this sentence, not clear enough

L 181 please rephrase "highly observed"

L 203-207 the samples used in the investigation of Zhao (11) were from retail market?

Chapter 3.3. please rewrite and connect the findings in some order - by the percentage of resistance or sensitivity? also, discuss the usage of antimicrobials tested in this investigation with the usage in poultry or in humans; does poultry producers in Morocco use amoxicillin or AMC in the treatment of poultry? the same for gentamicin?

L 253-254 please check this sentence

L 257 - please rephrase "slightly higher"

L 267 clavulanic acid?

L 315 please check spelling

Conclusion - L 323-328 should be changed; please mention the effect of this research on poultry production, traditional market and/or slaughterhouse procedures?

L 209-210 please check this sentence

L 222-226 please check those sentences?

L 226 "this reduction" - did you or other researchers observed higher resistance in previous investigation?

Author Response

From: Prof. Siham FELLAHI

Head of Avian Pathology Unit,

Department of Veterinary Pathology and Public Health,

Agronomy and Veterinary Institute Hassan II,

BP 6202, Rabat -Instituts, Morocco

Email:s.fellahi@iav.ac.ma;

26Mars, 2025

Dear Ms. Marriner He,

Section Managing Editor

Poultry

We thank you and the Reviewer for the valuable comments on our paper entitled “Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco". by Zineb Soubai, Nadia Ziyate, Sami DARKAOUI, Rim Rais, FELLAHI SIHAM,Benaissa Attarassi, Nabila Auajjar. We very much appreciate the opportunity to respond to the comments and to be allowed to resubmit the paper to Poultry Journal. We have attached point-by-point replies to the reviewers’ questions and comments below and have kept all the suggestions already inserted in the manuscript file. We are now confident that the revised version of our paper will satisfy the high expectations of your journal. Please do not hesitate to contact us if you have any remaining questions or concerns.

Sincerely,

Siham FELLAHI, DVM, Msc, PhD

Professorat Agronomy and Veterinary

Institute Hassan II, Morocco

Answer to the Reviewer

We thank the Reviewers for their valuable comments and the opportunity to improve our manuscript and resubmit to the Poultry Journal. We have carefully considered the comments and revised the manuscript accordingly. The following is a point by point response to the Reviewer's questions and comments.

Reviewer 1

Comments 1: title should be changes as it does not reflect the investigation in full

Response: We thank the reviewer for this valuable comment. In response,we have carefully considered possible adjustments and ultimately adopted a proposal made by the second reviewer,“Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco” as it aligns well with the focus of the paper. We hope this revision meets expectations, and of course, we remain open to any further suggestions if needed.

Comments 2: writing of antimicrobials in the whole text should be checked (with/without first capital letter)

Response: We sincerely thank the reviewer for this valuable remark. We completely agree and have carefully reviewed the writing related to antimicrobials throughout the entire text, making the necessary adjustments, which we have highlighted.

Comments 3: writing of genes in the whole text should be checked (italic or not)

Response: We truly appreciate the reviewer’s insightful comment. We have carefully reviewed the writing of gene names throughout the entire text, ensuring the correct use of italics where appropriate. All necessary adjustments have been made and highlighted.

Comments 4: Abstract - please describe why each of the mentioned methods were used (L23-25); please include a conclusion at the end of the Abstract

Response: We thank the reviewer for this pertinent comment. We agree with the reviewer and have added in the main text a description of why each method was used:” The research of Campylobacter was performed according to the standard ISO10272-1(2017), and the confirmation of Campylobacter species was carried out using MALDI-TOF mass spectrometry, Additionally, the disk diffusion method was employed to assess antimicrobial susceptibility, while conventional PCR was utilized to detect the presence of the tet(O) gene in tetracycline-resistant strains.”, as well as a conclusion at the end of the Abstract. “The results provide an overview of the current state of antibiotic resistance in Campylobacter in Morocco. They underline the urgent need for prudent use of antimicrobials and may encourage further research into the development of new antimicrobial agents. They also highlight the importance of raising awareness of the risk of carcass contamination”.

Comments 5: L 27 please rephrase "significantly more"

Response: We thank the reviewer for this helpful comment. We have rephrased "significantly more" to better convey the intended meaning. The revised text now reads as follows: “Moreover, the results revealed a notably higher detection of Campylobacter in traditional markets (41,33%) than in slaughterhouses (19,85%)” [L32]. We hope this adjustment meets your expectations.

Comments 6: L 28-29 please check this sentence, not clear enough

Response: We thank the reviewer for valuable feedback. We have revised the sentence for clarity and improved its readability. The updated version is as follows: “Additionally, it was more prevalent in chicken samples (38,46 %) than in turkey samples (25,91%).”[L33-34]. We hope this revision addresses your concern.

Comments 7: L 29-31 please change this sentence, not clear enough

Response: We appreciate your insightful feedback. The sentence has been revised for better clarity and flow. The updated version is as follows: “Campylobacter Resistance to antimicrobials revealed a resistance rate of 99% to ciprofloxacin, 73% to ampicillin, 82% to tetracycline, 29% to erythromycin, and 0% to gentamicin, as well as to the association of amoxicillin and clavulanic acid.”[L34-37]

Comment 8: Introduction - it is a bit confusing as it is not clear which statements are related to humans and which to poultry; please include more information on the Morocco poultry industry, information on the food markets and slaughterhouses procedures regarding chickens and turkeys as well as data on human campylobacteriosis.

Response: We thank the reviewer for the valuable feedback. We have clarified the distinction between statements related to humans and poultry. Additionally, we have included more information on the Moroccan poultry industry. Furthermore, we have incorporated relevant data on human campylobacteriosis. You can find these changes highlighted in the text in the introduction section.

Comment 9: L 41-42 please rephrase "crucial vehicle"

Response: Thank you for your feedback. We have rephrased 'crucial vehicle' to improve clarity. The revised version now reads: “the main routes by which these pathogens are transmitted”. We hope this addresses your concern.

Comment 10: L 52-55 please check this sentence, not clear enough

Response: Thank you for your comment. We have reviewed the sentence and made the necessary revisions to improve clarity. “Alarming studies show that there is a link between the uncontrolled use of antimicrobial agents to prevent, control, and treat bacterial infections in animals, and the emergence and spread of resistance, and even multi-resistance, in Campylobacter strains”

Comment 11: Sample collection - please give more information on the samples (what was the size of the samples - whole cecum or a piece of organ or a swab/portion of fecal matter as stated in L97? amount of neck skin in g? what kind of muscles did you take - a piece or?) please mention the age of the birds at the slaughter, if known

Response: Thank you very much for your thoughtful comments. We would like to clarify that the whole cecum was used, and by "portion of fecal matter," we mean a loopful from the interior of the cecum, following the ISO 10272-1 (2017) standard. To ensure clarity, we have updated the manuscript to reflect this more explicitly.

Regarding the age of the birds before slaughter, we acknowledge that this information was not previously mentioned in the manuscript. Unfortunately, we do not have precise data on the exact age of the birds used in our study. However, we can confirm that slaughterhouses operate under legal regulations, which set the minimum slaughter age at 18 days for broiler chickens and 100 days for turkeys according to decree from the Minister of Agriculture, Maritime Fisheries, Rural Development, and Water and Forests (n° 2986-17, dated 7 November 2017) concerning the approval and publication of a standard specification document related to the organic production of livestock animals and beekeeping products. This is the only information we can provide in this regard. We included this information in the manuscript and highlighted changes [L109-114]

We provide information on the specific type of muscles, which in this case are the breast muscles, that we checked throughout the entire manuscript.

We mention the detail of “size of samples”, and “whole caecum in the” Culture and Growth Conditions section. “For samples such as muscle and neck skin, it was necessary to apply an enrichment step. A 10g sample was homogenously mixed with 90 ml Preston” “A loopful (10 µL) from the interior of the cecum is directly streaked into mCCDA”.

Comment 12: L 96 how did you incubate Columbia Agar?

Response: Thank you for your valuable question. We would like to clarify that Columbia Agar was incubated under microaerophilic conditions at 41.5°C, in accordance with the ISO 10272-1 (2017) standard. [L135]

Comment 13: Selective supplement in L 88 and antimicrobial supplement in L 93- please mention the correct names of those supplements and the manufacturers.

Response: Thank you for your careful review. We appreciate your attention to detail. The selective supplement used in L88 and the antimicrobial supplement in L93 are [Preston antimicrobic supplement (Biolife, Italiana, Milano-Italia) and Bolton CCDA antimicrobic supplement (Biolife, Italiana, Milano-Italia)]. We have now specified these details in the revised manuscript for clarity.

Comment 14: Suspected colonies in L 98, 101- please describe what did you mean by that?

Response: Thank you very much for your insightful question. By 'suspected colonies,' we refer to those described in the ISO 10272-1 (2017) standard. On mCCDA agar, characteristic colonies are grayish, often with a metallic sheen, flat, and moist, with a tendency to spread on drier agar surfaces. We truly appreciate your careful review and hope this clarification is helpful.

Comment 15: L101-104 please mention reference for those methods.

Response: Thank you for your feedback. I would like to clarify that the methodology was in accordance with the ISO 10272-1 (2017) standard. We have mentioned this in the manuscript and highlighted the relevant changes to ensure transparency. [L138]

Comment 16: L 106 not clear: "strains isolated were activated"?

Response: Thank you for your valuable feedback. We understand that the phrase "strains isolated were activated" may not be clear. To improve clarity, we have revised the sentence to make it more precise. The updated text now reads: “The isolated strains were subcultured using an overnight culture”. We appreciate your suggestion and hope this modification enhances readability.

Comment 17: L 111 punctuation missing?

Response: Thank you for your careful review. We have corrected the punctuation to improve clarity. We appreciate your attention to detail and your helpful suggestion.

Comment 18: Antimicrobial disks and PCR reagents/primers- please mention the manufacturer

Response: Thank you for your suggestion. We have now included the manufacturer details for the antimicrobial disks and PCR reagents/primers in the revised manuscript. We appreciate your attention to detail and your helpful feedback.

Comment 19: Quality control - did you also include negative control?

Response: Thank you for your insightful suggestion. Yes, we confirm that we used Campylobacter jejuni ATCC 33560 as a negative control,which does not contain the tet(O) gene. We appreciate your careful review and valuable feedback. [L188]

Comment 20: Results- is there any reason why you presented the results of statistical analysis in a separate chapter and not related to the results?

Response: Thank you for your valuable feedback. After reviewing your comment, we understand your concern. We initially separated the statistical analysis to provide a clearer focus on the data and its interpretation. However, after further consideration, we agreed there was no strong reason to keep it separate and have now integrated the statistical analysis directly with the results for a more cohesive presentation. We appreciate your suggestion and have made this adjustment accordingly.

Comment 21: Strains identification - did you determined the campylobacter species by biochemical methods L 153-155?

Response: Thank you very much for your thoughtful question. Yes, we initially determined the Campylobacter species using biochemical methods, as outlined. However, we further confirmed the identification using MALDI-TOF. We appreciate your careful review and your valuable feedback.

Comment 22: L 161-163 please change this sentence, not clear enough

Response: Thank you for your valuable feedback. We have carefully revised the sentence and have implemented the changes in both the Results “According to biochemical identification and MALDI-TOF confirmation, 159 (28,7%) Campylobacter isolates were recovered from 150 poultry samples, including 133 isolates of Campylobacter coli and 26 isolates of Campylobacter jejuni.” and Discussion” In the present study, the finding demonstrated that the occurrence of Campylobacter species in poultry samples was 28,7%, including 133 isolates of Campylobacter coli and 26 (isolates) of Campylobacter jejuni according to biochemical and MALDI-TOF identification. This indicates a dominance of Campylobacter coli, consistent with the results of a study conducted by El Baaboua et al [7], which reported a predominance of Campylobacter coli 108 isolates compared to Campylobacter jejuni 22 isolates.” sections. We appreciate your time and effort in reviewing our work.

Comment 23: L 163-166 this comparison with the reference (7) not clear as El Baaboua investigated the prevalence of Campylobacter in meat, animal and human samples - please clarify

Response: Thank you very much for your insightful comment. We sincerely appreciate your careful review of our work. In response to your suggestion, we would like to clarify that our intention was solely to compare the predominance of Campylobacter coli. We have now made this clearer in the text and ensured that the comparison with reference is more precise. We truly appreciate your time and effort in helping us improve the clarity of our manuscript.

Comment 24: L 171-172 please check this sentence, not clear enough

Response: Thank you for your valuable feedback. We have carefully revised the sentence to improve its clarity: [L211-212] ”Notably, (57.65%) of caecum samples were colonized by Campylobacter, with a higher occurrence in chicken caecum (64.86%) compared to turkey caecum (50,45%)”. [L 302-304]“Regarding caecum colonization, Campylobacter was detected in 57,65% of samples, with a notably higher occurrence in chicken caecum (64,86%) compared to turkey caecum (50,45%)”We appreciate your time and effort in reviewing our work and helping us enhance the quality of our manuscript.

Comment 25: L 181, please rephrase "highly observed"

Response: Thank you very much for your kind and thoughtful feedback. We truly appreciate your effort in helping us improve the clarity of our manuscript. Regarding the phrase "highly observed," we understand the need for a clearer expression. We have revised it in the text and replaced it with [L 309-311]“Comparing the incidence of Campylobacter in caecum samples from slaughterhouse and traditional markets, the Campylobacter rate was higher in traditional markets (78.43%) than in slaughterhouses (40%).”

Comment 26: L 203-207 the samples used in the investigation of Zhao (11) were from retail market?

Response: Thank you very much for your thoughtful review and insightful question. Yes, the samples used in the investigation by Zhao (11) were indeed from the retail market, specifically from 59 stores of four supermarket chains. We sincerely appreciate your time and effort in helping us improve the clarity of our manuscript.

Comment 27: Chapter 3.3. please rewrite and connect the findings in some order - by the percentage of resistance or sensitivity? also, discuss the usage of antimicrobials tested in this investigation with the usage in poultry or in humans; does poultry producers in Morocco use amoxicillin or AMC in the treatment of poultry? the same for gentamicin?

Response: Thank you for your valuable feedback. We have revised the manuscript to present the findings in a more structured manner, organizing them based on the percentage of resistance for clarity, and highlighted the changes. Additionally, we have provided a more detailed discussion on the usage of the antimicrobials tested in this investigation in the poultry sector [L 400-404]. Regarding amoxicillin-clavulanic acid (AMC), it is not authorized for use in the poultry sector. Similarly, gentamicin is also not authorized.

Comment 28: L 253-254 please check this sentence

Response: Thank you for your careful review and valuable feedback. We have carefully checked the sentence and revised it for clarity and accuracy. We appreciate your suggestion, which has helped improve the manuscript. “Previous studies [13] [14] reported resistance rates of 12.2% and 7.1%, respectively, to gentamicin”. [L 383-384]

Comment 29: L 257 - please rephrase "slightly higher

Response: Thank you for your valuable suggestion. We have rephrased "slightly higher," and it now reads as "[exhibited a higher prevalence of resistance]." We appreciate your careful review and feedback. [L386]

Comment 30 : L 267 clavulanic acid?

Response: Thank you for your insightful comment. We intended to refer to the combination of amoxicillin and clavulanic acid, and we have now clarified this in the text. We appreciate your careful review and valuable feedback.

Comment 31: L 315 please check spelling

Response: Thank you for your careful review. We have checked the spelling and made any necessary corrections, “Breast muscle” and “Ceacum”. We appreciate your valuable feedback.

Comment 32: Conclusion - L 323-328 should be changed; please mention the effect of this research on poultry production, traditional market and/or slaughterhouse procedures?

Response: Thank you for your valuable suggestion. We have revised the conclusion to include the impact of this research on poultry production, traditional markets, and slaughterhouse procedures. We appreciate your insightful feedback, which has helped improve the manuscript.

Comment 33: L 209-210 please check this sentence

Response: Thank you for your careful review. We appreciate your feedback. We have reviewed the sentence in Lines and made the necessary corrections to improve clarity. “with 73% of isolates showing resistance to ampicillin”

Comment 34: L 222-226 please check those sentences?

Response: Thank you for your valuable feedback. We have carefully reviewed the sentences in Lines and have made the necessary corrections for clarity and accuracy. “Our results indicate a slightly lower rate compared to other recent studies that reported a high resistance to ampicillin among Campylobacter species, reaching 85% and 95.2%, respectively [13][14]. Moreover, a study conducted in Tunisia revealed the presence of the blaOXA-61 resistance gene, associated with ampicillin resistance, in Campylobacter jejuni and Campylobacter coli with prevalence rates of 81% and 93%, respectively [21].”

Comment 35: L 226 "this reduction" - did you or other researchers observed higher resistance in previous investigation?

Response: Thank you for your question. Previous studies have indeed reported varying levels of ampicillin resistance among Campylobacter isolates. Some investigations have observed higher resistance rates, such as those reporting 85% and 95.2% resistance, as cited in our manuscript [13][14]. Additionally, the presence of the blaOXA-61 resistance gene, associated with ampicillin resistance, in Campylobacter jejuni and Campylobacter coliwith prevalence rates of 81% and 93%, respectively, in a study conducted in Tunisia. Our findings show a slightly lower resistance rate in comparison, which we have discussed in the context of our research."

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Both minor and major editing is recommended, as follows

-. L2-L3 The title should be more informative. An option of a new version could be "Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco".

-. L39-L40 Please include the Campylobacter species and potential origin of the pathogenic Campylobacter for humans. be mentioned. Is Campylobacter with poultry-origin only linked with gastroenteritis in humans? Please include other human clinical signs/presentations potentially related.

-. L46-L50 Are the authors referring to human cases? If yes, please be explicit mentioning this.

-. Information about potential risk points of contamination of chicken and turkey carcasses at processing plants can be added

-. L61-L65 The aim of this study should be concise and informative. The authors are recommended to rewrite this paragraph.

-. L72 In the subsection "Sample collection" is recommended to include the number of traditional markets and slaughterhouses/processing plants from which the chicken and turkey samples were obtained. Information about permission for sampling and analysis should be included by the authors.

-. L75-L76 The authors are commended to specified what meat groups were sampled to be analyzed for the bacterial isolation.

-. L100 The subsection "Biochemical identification" described the parameters to identify the suspected-Campylobacter spp. colonies. Another subheading is recommended here.

-. L106 The term "activated" is not clear. Are the authors referring to subculture colonies to confirm the Campylobacter spp. identity?

-. L109-L110 The inclusion of the city and country of the manufacturer is recommended.

-. L111-L112 The deletion of the sentence "When a ... the microorganism" is recommended.

-. L116 and L125/126 EUCAST (2023) should be included as cited reference in the text and listed as reference.

-. L116-L118 The addition of the trademark, city and country of the manufacturer of each of the antibiotic disks is recommended. The name of each antibiotic should be in lowercase.

-. L120-L121 The inclusion of the cities of the manufacturers is recommended.

-. L133 Is this 1 ul of primer composed of the mixed 0.5 of Fw and 0.5 of Rv primers? If not, please be explicit describing how many ul of each primer is included in this mix.

-. L134 The authors should include "nuclease-free" to "water".

-. L152 The section of Results and Discussion should be differentiated, to facilitate the comprehension and fluent reading the obtained results.

-. L171 "Caecum samples" are not contaminated by Campylobacter spp because this bacterium can colonize the cecal mucosa. The term "contaminated" can be applied to the neck skin and meat samples.

-. The captions of figures 1 and 2 should be slightly modified. The terms "caecum" should replace "cecum" ti unified this term with the text, and "neck" should replace "neak".

-. The authors are recommended to include and differentiate in the Results section how many isolates of C. jejuni and C. coli were isolated from chicken and turkey samples.

-. L241-L243 This paragraph is recommended to be rewrite.

-. L254-L258 This paragraph is recommended to be rewrite clarifying the results included here.

-. L259 The heading of Table 2 should be more informative. Also, the disk concentration for each antibiotic should be included.

-. L278-L281 The authors are recommended to highlight the multi-resistant antibiotic profile of some of the Campylobacter spp. isolates studied here.

-. L283 Figure 4 could be included as a pie chart. The caption can be more informative.

-. L298 Figure 5 can be deleted.

-. L311-L315 The typing of Captions of figures 6 and 7 should be reviewed. As previously suggested, caecum samples should be considered as "colonized" instead of "contaminated", as the authors mentioned.

-. L321 The term "campylobacters" could be replaced by "Campylobacter spp.". Information related to the multiresistant Campylobacter isolates can be added in the Conclusion section. Control strategies during carcass processing to mitigate the impact of Campylobacter spp contamination at traditional markets and slaughterhouses could be added. What other recommendations can be given to poultry meat consumers to reduce the impact of campylobacteriosis in humans?

Author Response

From: Prof. Siham FELLAHI

Head of Avian Pathology Unit,

Department of Veterinary Pathology and Public Health,

Agronomy and Veterinary Institute Hassan II,

BP 6202, Rabat -Instituts, Morocco

Email:s.fellahi@iav.ac.ma;

26Mars, 2025

Dear Ms. Marriner He,

Section Managing Editor

Poultry

Sincerely,

Siham FELLAHI, DVM, Msc, PhD

Professorat Agronomy and Veterinary

Institute Hassan II, Morocco

Answer to the Reviewer

Reviewer 2

Comment 1:-. L2-L3 The title should be more informative. An option of a new version could be "Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco".

Response: Thank you for your valuable suggestion. We have taken your recommendation into account and have revised the title accordingly to make it more informative. The new title is now: Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco.We hope this revision meets your expectations.

Comment 2: -. L39-L40 Please include the Campylobacter species and potential origin of the pathogenic Campylobacter for humans. be mentioned. Is Campylobacter with poultry-origin only linked with gastroenteritis in humans? Please include other human clinical signs/presentations potentially related.

Response: We thank the reviewer for their valuable feedback. We have revised the manuscript to explicitly mention the different Campylobacter species relevant to human infections, including C. jejuni and C. coli, which are the most commonly associated with gastroenteritis. [L52-53]

Regarding the clinical presentations, we have expanded our discussion to highlight that while gastroenteritis is the most common manifestation of Campylobacter infection, extra-intestinal sequelae, especially acute neurological symptoms such as Guillain-Barré syndrome and Miller Fisher syndrome,can also occur, particularly in immunocompromised individuals.[L53-55]

We appreciate the reviewer’s insightful comments, which have helped improve the clarity and comprehensiveness of our manuscript.

Comment 3: L46-L50 Are the authors referring to human cases? If yes, please be explicit mentioning this.

Response: Thank you for your comment. Yes, we are referring to human cases in the sentence. We have revised it to make this explicit: “Campylobacter infections in humans are generally self-limiting.”We appreciate your helpful suggestion. [L65]

Comment 4: -. Information about potential risk points of contamination of chicken and turkey carcasses at processing plants can be added

Response: We thank the reviewer for their valuable feedback. This point has already been discussed in the Discussion section (lines 315–322). However, if the reviewer prefers us to elaborate on it elsewhere in the manuscript, please let us know, and we would be happy to make the necessary adjustments.

Comment 5: L61-L65 The aim of this study should be concise and informative. The authors are recommended to rewrite this paragraph.

Response: Thank you for your valuable feedback. In response to your suggestion, we have revised the paragraph to make the aim of the study more concise and informative. We appreciate your input, which has helped improve the quality of the manuscript. “This study aimed to (i) assess the presence of Campylobacter coli and Campylobacter jejuni in various poultry samples collected from slaughterhouse and traditional markets in the Rabat-Salé-Kénitra region in Morocco; (ii) detect the prevalence of antimicrobial resistance of the isolated strains; (iii) investigate the presence of the tet(O) gene in strains resistant to tetracycline.” [L80-84]

Comment 6:-. L72 In the subsection "Sample collection" is recommended to include the number of traditional markets and slaughterhouses/processing plants from which the chicken and turkey samples were obtained. Information about permission for sampling and analysis should be included by the authors.

Response: We sincerely thank the reviewer for the thoughtful comment. As noted in the manuscript, the slaughterhouse was a single facility that supplies poultry meat to almost the entire Rabat-Salé-Kénitra region, but we are happy to clarify this here. Additionally, we have updated the "Sample Collection" subsection to specify that the samples from traditional markets were obtained from five different locations. Furthermore, as recommended, we have included an ethical statement to ensure transparency regarding the permissions obtained for sampling and analysis. We truly appreciate the reviewer’s suggestions, which have helped enhance the clarity and completeness of our manuscript.

Comment 7: -. L75-L76 The authors are commended to specified what meat groups were sampled to be analyzed for the bacterial isolation.

Response: Thank you for your valuable feedback. In response to your comment, we have now specified in the manuscript that the breast muscle was the meat group sampled for bacterial isolation. Your suggestion has been very helpful in enhancing the clarity of the manuscript.

Comment 8: L100 The subsection "Biochemical identification" described the parameters to identify the suspected-Campylobacter spp. colonies. Another subheading is recommended here.

Response: We thank the reviewer for their valuable suggestion. To address this, we propose adding a new subheading in the subsection "Biochemical identification":“2.4.1 Culture characteristics”, and “2.4.2 Biochemical reactions.”Please let us know if this adjustment aligns with your recommendation.

Comment 9: -. L106 The term "activated" is not clear. Are the authors referring to subculture colonies to confirm the Campylobacter spp. identity?

Response: We sincerely thank the reviewer for their valuable feedback. Yes, the term "activated" referred to the subculturing of colonies to confirm the identity of Campylobacter spp. We have revised the manuscript accordingly for better clarity.

Comment 10: -. L109-L110 The inclusion of the city and country of the manufacturer is recommended.

Response: We sincerely thank the reviewer for their valuable suggestion. We have now included the city and country of the manufacturer as recommended and highlighted this change in the manuscript.

Comment 11: -. L111-L112 The deletion of the sentence "When a ... the microorganism" is recommended.

Response: We sincerely thank the reviewer for their valuable suggestion. We have deleted the sentence as recommended

Comment 12: -. L116 and L125/126 EUCAST (2023) should be included as cited reference in the text and listed as reference.

Response: We thank the reviewer for their valuable feedback. EUCAST (2023) has been included as a cited reference in the text, highlighted, and listed in the references section.

Comment 13: L116-L118 The addition of the trademark, city and country of the manufacturer of each of the antibiotic disks is recommended. The name of each antibiotic should be in lowercase.

Response: We sincerely thank the reviewer for their valuable suggestions. We have added the trademark, city, and country of the manufacturer for each antibiotic disk as recommended. Additionally, we have adjusted the antibiotic names to be in lowercase. These changes have been highlighted in the manuscript.

Comment 14: L120-L121 The inclusion of the cities of the manufacturers is recommended.

Response: We sincerely thank the reviewer for their valuable suggestion. We have included the cities of the manufacturers as recommended and highlighted these changes in the manuscript.

Comment 15: L133 Is this 1 ul of primer composed of the mixed 0.5 of Fw and 0.5 of Rv primers? If not, please be explicit describing how many ul of each primer is included in this mix.

Response:We sincerely thank the reviewer for their thoughtful question. To clarify, the 1 µL of primer refers to 1 µL of the forward (Fw) primer and 1 µL of the reverse (Rv) primer, not a mix. We will revise the manuscript to explicitly describe this in detail for better clarity.

Comment 16: L134 The authors should include "nuclease-free" to "water".

Response:We sincerely thank the reviewer for their valuable suggestion. We have updated the manuscript to specify "nuclease-free water" as recommended and have highlighted this change.

Comment 17: L152 The section of Results and Discussion should be differentiated, to facilitate the comprehension and fluent reading the obtained results.

Response:We sincerely thank the reviewer for their valuable suggestion. To enhance clarity and readability, we have differentiated the Results and Discussion sections as recommended. These changes have been implemented and highlighted in the manuscript.

Comment 18: L171 "Caecum samples" are not contaminated by Campylobacter spp because this bacterium can colonize the cecal mucosa. The term "contaminated" can be applied to the neck skin and meat samples.

Response:We sincerely thank the reviewer for their valuable observation. We agree with this clarification and have replaced the term "contaminated" with "colonized" for caecum samples, while retaining "contaminated" for neck skin and meat samples. This change has been highlighted in the manuscript.

Comment 19: The captions of figures 1 and 2 should be slightly modified. The terms "caecum" should replace "cecum" ti unified this term with the text, and "neck" should replace "neak".

Response:We sincerely thank the reviewer for their valuable suggestion. We have modified the captions of Figures 1 and 2 by replacing "cecum" with "caecum" to ensure consistency with the text and correcting "neak" to "neck".

Comment 20: We thank the reviewer for their helpful suggestion. We have now included and differentiated the number of C. jejuni and C. coli isolates obtained from chicken and turkey samples in the Results section.Please let us know if further revisions are needed.

Response:

Comment 21: L241-L243 This paragraph is recommended to be rewrite.

Response:We sincerely thank the reviewer for their valuable suggestion. We have rewritten the paragraph to improve clarity and readability. This revision has been highlighted in the manuscript.

Comment 22: L254-L258 This paragraph is recommended to be rewrite clarifying the results included here.

Response:We sincerely thank the reviewer for their valuable suggestion. We have rewritten the paragraph to enhance clarity and better explain the results. This revision has been highlighted in the manuscript.

Comment 23: L259 The heading of Table 2 should be more informative. Also, the disk concentration for each antibiotic should be included.

Response: We thank the reviewer for their valuable feedback. We have revised the heading of Table 2 to make it more informative. Additionally, we have updated the table to include the specific disk concentrations for clarity.

Comment 24: L278-L281 The authors are recommended to highlight the multi-resistant antibiotic profile of some of the Campylobacter spp. isolates studied here.

Response:We thank the reviewer for their valuable suggestion. To address this, we have included a new table that presents the number of isolates resistant to one, two, three, and four antibiotics along with their respective resistance profiles. We hope this addition enhances the clarity of our findings.

If this approach does not fully meet your expectations, please let us know, and we will be happy to make further modifications.

Comment 25: L283 Figure 4 could be included as a pie chart. The caption can be more informative.

Response:We thank the reviewer for their suggestion. We have now modified the figure to be presented as a pie chart for better visualization. Additionally, we have revised the caption to make it more informative. We have highlighted these changes in the manuscript

Comment 26: L298 Figure 5 can be deleted.

Response:We thank the reviewer for their valuable feedback. We agree with your suggestion and have removed Figure 5 as recommended.

Comment 27:L311-L315 The typing of Captions of figures 6 and 7 should be reviewed. As previously suggested, caecum samples should be considered as "colonized" instead of "contaminated", as the authors mentioned.

Response:We sincerely thank the reviewer for the thoughtful suggestion. As recommended, we have revised the captions for Figures 6 and 7 to use the term "distribution" instead. We appreciate the reviewer’s attention to this detail, which has improved the accuracy of our manuscript.

Comment 28:L321 The term "campylobacters" could be replaced by "Campylobacter spp.". Information related to the multiresistant Campylobacter isolates can be added in the Conclusion section. Control strategies during carcass processing to mitigate the impact of Campylobacter spp contamination at traditional markets and slaughterhouses could be added. What other recommendations can be given to poultry meat consumers to reduce the impact of campylobacteriosis in humans?

Response:We thank the reviewer very respectfully for their valuable feedback. We have replaced the term "campylobacters" with Campylobacter spp. as recommended. Additionally, information related to multiresistantCampylobacter isolates has been added in the Conclusion section. We have also made efforts to ensure coherence with the recommendations of the first reviewer, who also suggested additions in the same section.Please let us know if furtheradjustments are needed.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors provide valuable insights into the prevalence and antimicrobial resistance of Campylobacter spp. in poultry from Moroccan slaughterhouses and traditional markets. The findings highlight a significantly higher contamination rate in traditional markets and alarming resistance levels, particularly to ciprofloxacin, tetracycline, and ampicillin, while no resistance was observed for gentamicin and amoxicillin/clavulanic acid.

This research has direct implications for food safety, public health policies, and antimicrobial stewardship. The findings highlight the urgent need for enhanced hygiene practices in poultry processing, stricter regulations on antibiotic use in animal farming, and continuous monitoring programs to curb the spread of resistant Campylobacter strains.

Below some concerns the authors should address.

Line 20: add “systems” after poultry production

Line 20: add a period after spp (spp.) and correct throughout the text from this point onward

Line 23: “The” with capital letter

Line 28: Use the decimal notation with a period, not a comma and correct throughout the text from this point onward

Line 30: revealed a “resistance rate of 99% to…”

Line 30: names of antibiotics in lowercase. Correct throughout the text from this point onward

Line 31: “0% to gentamicin, as well as to the association of amoxicillin and clavulanic acid.”

Line 32: profile “to” tetracycline

Line 32: tet(O) in lowercase and italics. Correct throughout the text from this point onward

Line 42: change “it has become imperative” to “it is imperative”

Line 43: add “the” between “for” and “surveillance”

Line 43: change “and the antimicrobial resistance of this bacterium” to “and its antimicrobial resistance”

Line 49: “first-line treatment”

Line 50: change “The development of antimicrobial resistance is observed in Campylobacter species, and this has serious implications for treating Campylobacter infections in humans” to “the development of antimicrobial resistance in Campylobacter species has serious implications for treating human infections”

Line 74: “between September 2022 and October 2023” or “from September 2022 to October 2023”

Line 74: remove a space before “Of”

Line 77: remove a space before “The”

Line 86: remove a space before “In”

Line 114: change “sensitivity to antimicrobials” to “antimicrobial susceptibility”

Line 120: change “on the dried plate” to “on a dried plate”

Line 121: Fisher

Line 122: change to “inoculation was performed by swabbing the surface, and the plate was allowed to dry”

Line 123: change “distributed” to placed”

Line 125: change “following” to “according to”

Line 153: Gram in capital letter

Line 153: add “and” between catalase and oxidase-positive

Line 155: indoxyl and hippurate in lowercase

Line 158: remove “an”

Line 161: findings

Line 162: remove the parentheses

Line 165: change “the same” to “similarly”

Line 171: “with chicken caecum contamination (64.86%) being notably higher than turkey caecum contamination (50.45%)

Line 175: droppings

Line 175: “reported a contamination rate of up to 71% in 105 cloacal swabs”

Line 182: put the percentages in parentheses

Line 183: “6.66% of neck skin samples tested positive in slaughterhouse, while 0% was detected in muscle samples”

Line 184: “in traditional markets, the prevalence was 17.39% and 15% in neck skin and muscle samples, respectively

Line 224: “among Campylobacter species, reaching 85% and 95.2%, respectively”

Line 236: “were associated with specific genetic mutations”

Line 275: “It is important”

Line 321: Campylobacter in capital letter and in singular form

Line 323: “the prevalence in chicken samples was higher than in turkey samples”

Line 327: change “phonetic” to “phenotypic”

Author Response

From: Prof. Siham FELLAHI

Head of Avian Pathology Unit,

Department of Veterinary Pathology and Public Health,

Agronomy and Veterinary Institute Hassan II,

BP 6202, Rabat -Instituts, Morocco

Email:s.fellahi@iav.ac.ma;

26Mars, 2025

Dear Ms. Marriner He,

Section Managing Editor

Poultry

We thank you and the Reviewer for the valuable comments on our paper entitled “Antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolated from processed chickens and turkeys in Morocco". by Zineb Soubai, Nadia Ziyate, Sami DARKAOUI, Rim Rais, FELLAHI SIHAM,Benaissa Attarassi, Nabila Auajjar. We very much appreciate the opportunity to respond to the comments and to be allowed to resubmit the paper to Poultry Journal. We have attached point-by-point replies to the reviewers’ questions and comments below and have kept all the suggestions already inserted in the manuscript file. We are now confident that the revised version of our paper will satisfy the high expectations of your journal. Please do not hesitate to contact us if you have any remaining questions or concerns.

Sincerely,

Siham FELLAHI, DVM, Msc, PhD

Professorat Agronomy and Veterinary

Institute Hassan II, Morocco

Answer to the Reviewer

Reviewer 3

Comment 1:Line 20: add “systems” after poultry production

Response:We thank the reviewer for their helpful comment. In response, we have added “systems” after poultry production

Comment 2: Line 20: add a period after spp (spp.) and correct throughout the text from this point onward

Response: We thank the reviewer for their comment. We agree with the suggestion and have made the necessary changes in the manuscript, highlighting the modification accordingly.

Comment 3: Line 23: “The” with capital letter

Response: We thank the reviewer for their careful attention to detail. In response to your suggestion, we have revised the sentence as per Reviewer 2’s recommendation. As the word "the" is no longer at the beginning of the sentence, there is no need for a capital letter.We appreciate your feedback and are happy to make any further adjustments if needed.

Comment 4: Line 28: Use the decimal notation with a period, not a comma and correct throughout the text from this point onward

Response: We thank the reviewer for their helpful comment. In response, we have corrected the notation to use a period instead of a comma, starting from line 28 and throughout the rest of the manuscript.

Comment 5: Line 30: revealed a “resistance rate of 99% to…”

Response: We thank the reviewer for their comment. We agree with the suggestion and have made the necessary changes in the manuscript, highlighting the modification accordingly.

Comment 6: Line 30: names of antibiotics in lowercase. Correct throughout the text from this point onward

Response: We thank the reviewer for their comment. We agree with the suggestion and have made the necessary changes in the manuscript, highlighting the modification accordingly.

Comment 7: Line 31: “0% to gentamicin, as well as to the association of amoxicillin and clavulanic acid.”

Response: We thank the reviewer for their valuable feedback. We agree with your suggestion and havemade the necessary changes in the manuscript, highlighting the modification accordingly.

Comment 8: Line 32: profile “to” tetracycline

Response: We thank the reviewer for their attention and correction, which have improved the manuscript. We have made the change according to their recommendation and highlighted it accordingly.

Comment 5: Line 32: tet(O) in lowercase and italics. Correct throughout the text from this point onward

Response: We sincerely thank the reviewer for their attentive review and valuable correction, which have helped improve the manuscript. We have changed tet(O) to lowercase and italics throughout the text from this point onward and have highlighted the modifications accordingly.

Comment 6: Line 42: change “it has become imperative” to “it is imperative”

Response: We sincerely thank the reviewer for their careful review and valuable suggestion. We have changed “it has become imperative” to “it is imperative” as recommended and have highlighted the modification accordingly.

Comment 7: Line 43: add “the” between “for” and “surveillance”

Response: We sincerely thank the reviewer for their careful review and valuable suggestion. We have added “the” between “for” and “surveillance” as recommended and have highlighted the modification accordingly.

Comment 8: Line 43: change “and the antimicrobial resistance of this bacterium” to “and its antimicrobial resistance”

Response: We thank the reviewer for their attention and correction, which have improved the manuscript. We have made the change according to their recommendation and highlighted it accordingly.

Comment 9: Line 49: “first-line treatment”

Response: We thank the reviewer for their attention and correction, which have improved the manuscript. We have made the change according to their recommendation and highlighted it accordingly.

Comment 10: Line 50: change “The development of antimicrobial resistance is observed in Campylobacter species, and this has serious implications for treating Campylobacter infections in humans” to “the development of antimicrobial resistance in Campylobacter species has serious implications for treating human infections”

Response: We thank the reviewer for their attention and correction. We agree with the suggestion, have made the change according to their recommendation, and highlighted it accordingly.

Comment 11: Line 74: “between September 2022 and October 2023” or “from September 2022 to October 2023”

Response: We thank the reviewer for their attention and correction, which have improved the manuscript. We have made the change according to their recommendation and highlighted it accordingly.

Comment 12: Line 74: remove a space before “Of”

Response: We thank the reviewer for their comment. We agree with the suggestion and have made the necessary changes in the manuscript, highlighting the modification accordingly.

Comment 13: Line 77: remove a space before “The”

Response: We sincerely appreciate the reviewer's thoughtful comment. We fully agree with the suggestion and have carefully implemented the necessary changes in the manuscript, ensuring that the modifications are clearly highlighted.

Comment 14: Line 86: remove a space before “In”

Response: have made the required adjustments in the manuscript and have highlighted the modifications accordingly.

Comment 15: Line 114: change “sensitivity to antimicrobials” to “antimicrobial susceptibility”

Response: We truly appreciate the reviewer’s suggestion. We have carefully considered it, incorporated the necessary changes into the manuscript, and highlighted them for easy reference.

Comment 16: Line 120: change “on the dried plate” to “on a dried plate”

Response: Thank you for your kind and helpful comment. We have taken it into account, made the suggested revisions in the manuscript, and ensured they are properly highlighted.

Comment 17: Line 121: Fisher

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 18: Line 122: change to “inoculation was performed by swabbing the surface, and the plate was allowed to dry”

Response: We truly appreciate the reviewer’s suggestion. We have carefully considered it, incorporated the necessary changes into the manuscript, and highlighted them for easy reference.

Comment 19: Line 123: change “distributed” to placed”

Response: We truly appreciate the reviewer’s suggestion. We have carefully changed it, and highlighted them for easy reference.

Comment 20: Line 125: change “following” to “according to”

Response: We truly appreciate the reviewer’s suggestion. We have carefully changed it, and highlighted them for easy reference.

Comment 21: Line 153: Gram in capital letter

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 22: Line 153: add “and” between catalase and oxidase-positive

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 23: Line 155: indoxyl and hippurate in lowercase

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 24: Line 158: remove “an”

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 25: Line 161: findings

Response: We thank the reviewer for their effort in providing valuable feedback, which has greatly improved the manuscript. After making the changes recommended by the first reviewer, we have found that the term in question no longer exists in the text.Please let us know if any further revisions are required.

Comment 26: Line 162: remove the parentheses

Response: We are deeply grateful to the reviewer for their careful attention to detail. We truly appreciate the correction and have thoughtfully incorporated the change into the manuscript.

Comment 27: Line 165: change “the same” to “similarly”

Comment 28: Line 171: “with chicken caecum contamination (64.86%) being notably higher than turkey caecum contamination (50.45%)

Response: We thank the reviewer for their effort in providing valuable feedback, which has greatly improved the manuscript. After making the changes recommended by the first reviewer, we have found that this sentence in question no longer exists in the text.Please let us know if any further revisions are required.

Comment 29: Line 175: droppings

Response: We are deeply grateful to the reviewer for their careful attention to detail. We truly appreciate the correction and have thoughtfully incorporated the change into the manuscript.

Comment 30: Line 175: “reported a contamination rate of up to 71% in 105 cloacal swabs”

Comment 31: Line 182: put the percentages in parentheses

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 32: Line 183: “6.66% of neck skin samples tested positive in slaughterhouse, while 0% was detected in muscle samples”

Response: We thank the reviewer for their thoughtful comment. We fully agree with the suggestion and have carefully implemented the necessary changes in the manuscript, ensuring that the modifications are highlighted.

Comment 33: Line 184: “in traditional markets, the prevalence was 17.39% and 15% in neck skin and muscle samples, respectively

Comment 34: Line 224: “among Campylobacter species, reaching 85% and 95.2%, respectively”

Comment 35: Line 236: “were associated with specific genetic mutations”

Response: We sincerely thank the reviewer for their thoughtful comment. We fully agree with the suggestion and have carefully made the necessary changes to enhance the manuscript, ensuring that the modifications are highlighted.

Comment 36: Line 275: “It is important”

Comment 37: Line 321: Campylobacter in capital letter and singular form

Response: We sincerely thank the reviewer for their keen attention to detail. We truly appreciate the correction of the term and have implemented the change in the manuscript accordingly.

Comment 38: Line 323: “the prevalence in chicken samples was higher than in turkey samples”

Response: We thank the reviewer for their efforts in providing valuable feedback, which has significantly improved the manuscript. After incorporating the changes suggested by the first reviewer, we found that the sentence in question no longer appears in the text. Please let us know if any further revisions are necessary.

Comment 39: Line 327: change “phonetic” to “phenotypic”

Response: We thank the reviewer for their efforts in providing valuable feedback, which has significantly improved the manuscript. After incorporating the changes suggested by the first reviewer, we found that the term in question no longer appears in the text. Please let us know if any further revisions are necessary.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for taking into consideration all the comments and suggestions given by the reviewer.

Best regards

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Thank you for all editing you performed. I have no additional comments to provide.

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the Authors for considering and adding my suggestions to the manuscript. To me it can be accepted in the present form.