Review Reports

Round 1

Reviewer 1 Report

This is a very well written and very interesting and useful clinical study with a 1-year clinical follow up that tested two innovative approaches for management and therapy of EFB and AFB: 1) application of “partial shook swarm” technique combined with OTC therapy and 2) innovative administration of OTC therapy provided in small amounts (30 ml) of syrup in flat small containers (jar caps).

Strengths:

1.      Considering recent “re-emergence” of EFB in North America this publication will certainly add one more effective “tool in a tool box” for treatment of EFB affected colonies which will still have a potential to generated honey yield during the same season (which is usually not the case in complete shook swarm treatment).

2.      New innovative administration of OTC in small volumes of syrup allows even weak colonies to consume appropriate dose of antibiotics which is usually not the case when OCT is administered within powdered sugar or within large quantities of syrup (according to label instructions).

 

This reviewer has only two small comments/corrections:

1.      Line 59-61: “Canada Foul Brood Mix, Oxytet-25-S and Oxysol 62.5 (oxytetracycline) and Tylan Soluble 60 (tylosin) are commercially available [13].”

a.      Please, add also lincomycin, which was recently registered for your also in Canada

2.      Colony strength evaluation

a.      In M&M section, please indicated how colony strengths were evaluated.

b.      Please add a title for x-axis in Figure 3.

 

Author Response

Dear Reviewer,

Thank you for your revision of the document. We improved the manuscript according to your comments and suggestions.

Best regards

Author Response File: Author Response.docx

Reviewer 2 Report

General Comments

This manuscript present data regarding the analysis of the effects of an integrated treatment of honeybee colonies infected with two bacterian species (Paenibacillus larvae and Melissococcus plutonius), using the beekeeping technique partial shook swarm combined with antibiotic (oxytetracycline) treatment. The paper discusses several interesting aspects related to importance to control these pathogens and the existent methods. The results are important because they present, for the first time, a combination of control methods and a new way of administering the antibiotic. The paper is well written for most part. However, some aspects about manuscript need to be revised. mainly related to the methodology.

 

Specific comments

 

1) Title: According to zoological nomenclature rules, add the author of the species.

2) Line 111: This part of the text is where I see the main problems with the methodology. There are parts that should be better addressed, described, for example: Why was the monitoring done for two years? How was this monitoring done? What is the purpose of this monitoring? Was the infection monitored as well? How was it identified that the animals were contaminated, what was the test used? How were the bacteria grown and identified (genetic sequencing)?

3) Line 120: Is this antibiotic concentration defined somewhere? Manufacturer's instructions or previous work?

4) Line 125: The sentence “Consumption of antibiotic solutions was measured after each week of treatment.” is out of place, put it further down where the methodology of consumption is mentioned.

5) Another experimental error was the failure to include a control group for comparison. Although the authors compared pre- and post-treatment situations, positive and negative control data should have been included.

6) Lines 150-152: In my opinion the correlation is weak. The points are too far from the line. The authors have not discussed this further, they only cite de correlation, but do not specify the type of correlation. It must be reviewed.

7) Line 157: Why only 6 colonies out of a total of 27? Did the others not produce honey? This needs to be addressed, because the way it is exposed the impression is that the production was low.

8) Captions for all figures are uninformative, should be more comprehensive and describe all the important aspects to be observed in the image.

9) Figure 3: This figure is not good. On what measure was strength based? This measurement should be on the chart. What do the + signs mean? Explain the figure better and improve the caption.

10) Line 200: “This strongly reduced internal contamination.” How was it confirmed that it reduced contamination? What tests have been performed on the bees to confirm this? Are there microscopy analyzes (light or electron) that show this? Were microbial growth cultures performed with internal fluids to support this affirmation?

11) Line 204: “development of antibiotic resistance”. Antibiotics do not develop resistance; they select for resistant colonies.

Author Response

Dear Reviewer,

Thank you for your comments and suggestions. Please find below (and in the revised manuscript) our point-to-point answers.

1) Title: According to zoological nomenclature rules, add the author of the species.

Thank you for your note. We added the author of the species in the title.

2) Line 111: This part of the text is where I see the main problems with the methodology. There are parts that should be better addressed, described, for example: Why was the monitoring done for two years? How was this monitoring done? What is the purpose of this monitoring? Was the infection monitored as well? How was it identified that the animals were contaminated, what was the test used? How were the bacteria grown and identified (genetic sequencing)?

We improved the manuscript clarifying the mentioned aspects of the methodology. Please see lines 142-150.

3) Line 120: Is this antibiotic concentration defined somewhere? Manufacturer's instructions or previous work?

The antibiotic concentration chosen is the same used for registered veterinary medicines for bees in the USA. Moreover, it is the same concentration applied in Mosca, M., Giannetti, L., Franco, A., Iurescia, M., Milito, M., Pietropaoli, M., Leto, A., Di Ruggiero, C., Mezher, Z., Palazzetti, M., Gallo, V., Croppi, S., Macrì, S. & Formato, G. (2022). Impact of Oxytetracycline on Apis mellifera Colonies: Preliminary Results on Residues and Antibiotic Resistance. Journal of Apicultural Science,66(2) 159-170. https://doi.org/10.2478/jas-2022-0013

4) Line 125: The sentence “Consumption of antibiotic solutions was measured after each week of treatment.” is out of place, put it further down where the methodology of consumption is mentioned.

Thank you for your comment. We moved the sentence in the proper place.

5) Another experimental error was the failure to include a control group for comparison. Although the authors compared pre- and post-treatment situations, positive and negative control data should have been included.

Thank you for your comment. A positive control group (untreated) was not foreseen as, according to national legislation, it is not possible to leave untreated colonies affected by AFB and EFB in the apiary.

6) Lines 150-152: In my opinion the correlation is weak. The points are too far from the line. The authors have not discussed this further, they only cite de correlation, but do not specify the type of correlation. It must be reviewed.

Thank you for the note. The correlation between the two variables is linear. In lines 286-291 we also specified that this aspect is relevant for the beekeeping practical implications: colonies approx. 5’825-8’155 bees have less chance of survival to the shook swarm technique and do not take enough OTC so in that case would be preferred the colony destruction.

7) Line 157: Why only 6 colonies out of a total of 27? Did the others not produce honey? This needs to be addressed, because the way it is exposed the impression is that the production was low.

Thank you for your comment. We improved the sentence. Please see lines 193-197.

8) Captions for all figures are uninformative, should be more comprehensive and describe all the important aspects to be observed in the image.

9) Figure 3: This figure is not good. On what measure was strength based? This measurement should be on the chart. What do the + signs mean? Explain the figure better and improve the caption.

Thank you. We improved captions and figures according to your suggestions.

10) Line 200: “This strongly reduced internal contamination.” How was it confirmed that it reduced contamination? What tests have been performed on the bees to confirm this? Are there microscopy analyzes (light or electron) that show this? Were microbial growth cultures performed with internal fluids to support this affirmation?

We opted for removing the sentence as it is related to results of a research that is not published yet.

11) Line 204: “development of antibiotic resistance”. Antibiotics do not develop resistance; they select for resistant colonies.

Thank you for your comment. We corrected the sentence.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript is interesting and scientifically sound. There are some questions/suggestions to authors to improve this document:

Please clarify, how many strong colonies and how many weak colonies were evaluated throughout the study.

How many relapses, mentioned in line 154, were from strong colonies, and how many were from weak colonies?

How was the production of honey in those 21 colonies which couldn´t recover the original production, mentioned in line 158? Could you provide the average or a range of percentages of reduction in production for these colonies?

Figures 1, 2, and 3 are barely discussed. Please include a relevant discussion for those images, otherwise please delete them.

Lines 170-205, in the discussion section, are mostly a repetition of what was previously commented on in the introduction. It would be advisable to revise and organize it.

Why did you choose to use sucrose to administer OTC? Did you evaluate using honey or pollen as an OTC administration mechanism?

You mentioned in line 275: “providing better food safety for all consumers of hive products.”. Given that OTC residuals could be potentially included as a risk for food safety, did you measure if there are traces of OTC in honey or pollen, or, are there reports which mention it?

 

Author Response

Dear Reviewer,

Thank you for your questions and suggestions. Please find below (and in the revised manuscript) our point-to-point answers.

Please clarify, how many strong colonies and how many weak colonies were evaluated throughout the study.

Thank you for your comment. Please see line 174 of the revised manuscript.

How many relapses, mentioned in line 154, were from strong colonies, and how many were from weak colonies?

How was the production of honey in those 21 colonies which couldn´t recover the original production, mentioned in line 158? Could you provide the average or a range of percentages of reduction in production for these colonies?

Thank you for your comment. We improved the sentence. Please see lines 193-197.

Figures 1, 2, and 3 are barely discussed. Please include a relevant discussion for those images, otherwise please delete them.

Thank you for your note. We cited the figures in the discussion session where the new administration technique, colonies consumption rates and colonies strength are discussed.

Lines 170-205, in the discussion section, are mostly a repetition of what was previously commented on in the introduction. It would be advisable to revise and organize it.

Thank you for your comment. We revised and moved the paragraph in the proper section.

Why did you choose to use sucrose to administer OTC? Did you evaluate using honey or pollen as an OTC administration mechanism?

Thank you for your comment. We used sucrose to administer OTC as pollen and honey can contain AFB spores, so it would need gamma ray sterilization.

You mentioned in line 275: “providing better food safety for all consumers of hive products.”. Given that OTC residuals could be potentially included as a risk for food safety, did you measure if there are traces of OTC in honey or pollen, or, are there reports which mention it?

Yes, we just published a paper on a trial where we found that the same amounts of OTC resulted in a long-term presence of residues and ARGs. Please see it here:

Mosca, M., Giannetti, L., Franco, A., Iurescia, M., Milito, M., Pietropaoli, M., Leto, A., Di Ruggiero, C., Mezher, Z., Palazzetti, M., Gallo, V., Croppi, S., Macrì, S. & Formato, G. (2022). Impact of Oxytetracycline on Apis mellifera Colonies: Preliminary Results on Residues and Antibiotic Resistance. Journal of Apicultural Science,66(2) 159-170. https://doi.org/10.2478/jas-2022-0013

 

Best regards

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors made most of the suggested corrections. The paper is considerably better. I recommend publishing in Agriculture.