Vitamin Compatibility with the Marek’s Disease Vaccineâ€
, Christopher J. Williams 2, Joshua Deines 2 and Edgar David Peebles 1Round 1
Reviewer 1 Report
The authors presented a manuscript that seeks to evaluate the compatibility of the Marek's disease vaccine, with vitamins with different levels of 25OHD3 or L-AA.
The manuscript is well written and easy to follow, but there are some issues that must be addressed, before acceptance for publication.
1.- Could the authors provide more information (including references) regarding the concentrations defined as high and low for both, 25OHD3 and L-AA?
2.- Could you provide more data regarding to the cell line used in the study?
3.- Statistical analysis must be described in materials and methods section
4.- I suggest to separate the results from discussion section. Also discussion must be improved
NA
Author Response
Reviewer 1:
The authors presented a manuscript that seeks to evaluate the compatibility of the Marek's disease vaccine, with vitamins with different levels of 25OHD3 or L-AA. The manuscript is well written and easy to follow, but there are some issues that must be addressed before acceptance for publication.
- Could the authors provide more information (including references) regarding the concentrations defined as high and low for both, 25OHD3 and L-AA?
Answer:
Thank you for the comments. The relevant information was inserted in the text between lines 64 and 78, and the appropriate references were added to the Reference section.
“The in ovo injection of 0.6 µg of 25OHD3 combined with commercial diluent resulted in an increase in the hatchability of live embryonated embryos [18] and the subsequent bone quality of post-hatch broilers [13]. Furthermore, immunity [15,19,20], breast meat yield [21], and live performance [14, 21] were improved in Ross 708 broilers in response to 2.4 µg of 25OHD3 as compared to non-injected- or diluent-control groups. However, in comparison to non-injected or diluent-injected control groups, there were no beneficial effects on hatch and post-hatch variables when lower than 0.3 µg or higher than 2.4 µg of 25OHD3 was in ovo-injected [19-21]. As compared to saline or non-injected control treatments, L-AA at 12 mg resulted in an increase in the hatchability [22], body weight gain [16,17], and antioxidant capacity [16] of Ross 708 broilers, while there were no positive effects on their hatching process and post-hatch live performance when administrated below 1.2 mg [16,22].”
- Could you provide more data regarding to the cell line used in the study?
Answer:
The following sentence was added on lines 97 and 98 of the Materials and Methods section: “Approximately 25,000 OU2.2 chick embryo fibroblast cells (Abujoub and Coussens, 1995) were delivered in each dose of the CEF-MDV.” The Abujoub and Coussens, 1995 reference was also added to the Reference section.
- Statistical analysis must be described in materials and methods section
Answer:
Thank you for the comment. The data for this study was generated by a Cellometer AutoT-4 device and the values indicated the percentages of live cells. The device did not provide replication at each individual time to allow for statistical analysis. Statistical analysis could only be performed if the data were pooled across time. Because the timing of the responses to treatment was important, the authors elected to plot the progression of cell viability with time so that the results could be reported more meaningfully and with more relevance and accuracy. Thus, the extracted information for a Cellometer AutoT-4 device was plotted to illustrate the percentages of live cells without conducting a statistical comparison between treatments, which allowed us to numerically compare the treatments.
- I suggest separating the results from discussion section. Also, discussion must be improved
Answer:
The Results and Discussion sections were separated in the revised draft. We found that between the two vitamin sources tested, one would be a better candidate to combine with the Marek’s Disease vaccine for in ovo injection application. We provided two reasons why L-AA failed between lines 150 and 170. Considering that the current manuscript is a research note and not a full paper, the necessary relevant information was provided in this manuscript, and the Discussion was also considered to present sufficient information for the reader.
Reviewer 2 Report
Dear Editor,
In this article, the authors deal with vitamin compatibility, respectively L-Ascorbic acid and 25-hydroxyvitamin D3, with the Marek's disease vaccine. The idea of the manuscript is very good, but there are still some points to be addressed by the authors in order to improve their manuscript.
Introduction section:
Line 35; line 43 - I recommend to remove high pathogenic avian influenza from the listed diseases in which vaccination is commonly used.
Add a few references related to in ovo vaccination:
· https://doi.org/10.1080/09712119.2023.2187396
· https://doi.org/10.1016/B978-0-12-818708-1.00012-9
Material and Methods section
A summary diagram with all the steps of this complex study is recommended.
Conclusions section
Line 161 - I recommend checking spelling.
Line 163 - The Supplementary Materials were not available for the reviewer.
Line 184 - I recommend checking spelling.
Author Response
Reviewer 2:
In this article, the authors deal with vitamin compatibility, respectively L-Ascorbic acid and 25-hydroxyvitamin D3, with the Marek's disease vaccine. The idea of the manuscript is very good, but there are still some points to be addressed by the authors in order to improve their manuscript.
Introduction:
- Line 35; line 43, I recommend to remove high pathogenic avian influenza from the listed diseases in which vaccination is commonly used.
Add a few references related to in ovo vaccination:
- https://doi.org/10.1080/09712119.2023.2187396
- https://doi.org/10.1016/B978-0-12-818708-1.00012-
Answer:
Thank you for the comment and recommended references. More relevant information was added for those references related to commercial in ovo vaccination. The provided references are linked to the in ovo injection of nutrients, and not vaccines, that we used when we discussed nutrients.
Material and Methods section
- A summary diagram with all the steps of this complex study is recommended.
Answer:
To remain in the format of a short communication and because of the actual simplicity of the procedure, in which just the live/dead CEF-MDV ratio was assessed using a cellometer, which was aptly described as a common procedure in the Materials and Methods section, the authors prefer not to include a schematic of the procedure.
Conclusions section
- Line 161 - I recommend checking spelling.
Answer:
The relevant correction was applied to the sentence.
“It was demonstrated in this study that both the low and high doses of L-AA had negative effects on CEF-MDV survival.”
- Line 163 - The Supplementary Materials were not available for the reviewer.
Answer:
Thank you for the comment. To address the comment, it was well-demonstrated and discussed that as compared to the control, that both levels of 25OHD3 used in this study were compatible with the chick embryo fibroblast cells infected by the MD virus (CEF-MDV) within the 2-hour period. However, L-AA mainly killed the CEF-MDV and most likely would not provide adequate protection for embryos. The CEF-MDV were even dramatically reduced to the low level of L-AA. This indicates that only 25OHD3 can be combined with CEF-MDV to safely deliver the acceptable doses of the virus to embryos. We addressed the aforementioned information between lines 150 and 170. Also, if the reviewer refers to “Data Availability Statement: Not applicable” on line 182, this research was conducted under collaboration between Mississippi State University, USDA, and Zoetis, in which we are not allowed to include the proprietary information of the contents of the supplementary material.
- Line 184 - I recommend checking spelling.
Answer:
The relevant correction was applied to the sentence.
The authors express their appreciation for the assistance provided by Mr. Eric Nixon and a Zoetis team of scientists. Also, our appreciation is extended to Mr. Rodney Johnson for invaluable assistance.
Reviewer 3 Report
The manuscript entitled " Vitamin compatibility with the Marek's disease vaccine" represents a considerable amount of work. The following comments need to be addressed before the manuscript is suitable for publication in Poultry Journal.
1- Why authors didn’t statistically analysis their results?
2- Why did the author use one parameter for measurement? I think is not enough for judging the efficiency of these combinations.
Author Response
Reviewer 3:
The manuscript entitled "Vitamin compatibility with the Marek's disease vaccine" represents a considerable amount of work. The following comments need to be addressed before the manuscript is suitable for publication in Poultry Journal.
- Why authors didn’t statistically analysis their results?
Answer:
Thank you for the comment. The data for this study were generated by a Cellometer AutoT-4 device and the values indicated the percentages of live cells at each of 5 time periods for each treatment. Although repeated measurements every 30 minutes were provided, single readings by the device at each time without replication of the data at each time did not allow for statistical analysis to be conducted. Statistical analysis could only be performed if the data were pooled across time. Because the timing of the responses to treatment was important, the authors elected to plot the progression of cell viability with time so that the results could be reported more meaningfully and with more relevance and accuracy. Thus, the extracted information from the Cellometer AutoT-4 device were plotted to illustrate the percentages of live cells without conducting a statistical comparison between treatments, which then allowed us to compare the treatments numerically.
- Why did the author use one parameter for measurement? I think is not enough for judging the efficiency of these combinations.
Answer:
Thank for the comment. We examined two variables (cell survivals and pH). However, cell survival was the most and reliable factor to best compare the efficacies of the treatment methods tested. A reduction in the percentage of chick embryo fibroblast cells infected by the MD virus (CEF-MDV) would indicate that an inadequate protective immunity would be elicited.
Round 2
Reviewer 1 Report
The authors addressed almost all my concerns. Nevertheless, I still have recommendations regarding data analysis.
Firstly, I suggest the authors add In M&M sections the explanation provided regarding data generated and limitations to perform statistical analysis.
Secondly, this issue (not statistical analysis) must be included in the discussion and conclusions
Na
Author Response
Reviewer 1
The authors addressed almost all my concerns. Nevertheless, I still have recommendations regarding data analysis.
Firstly, I suggest the authors add In M&M sections the explanation provided regarding data generated and limitations to perform statistical analysis.
The relevant changes were applied to the material and method section
“The data for this study was generated by a Cellometer AutoT-4 device and the values indicated the percentages of live cells. The device did not provide replication at each individual time to allow for statistical analysis. Statistical analysis could only be performed if the data were pooled across time. Because the timing of the responses to treatment was important, the authors elected to plot the progression of cell viability with time so that the results could be reported more meaningfully and with more relevance and accuracy. Thus, the extracted information for a Cellometer AutoT-4 device was plotted to illustrate the percentages of live cells without conducting a statistical comparison between treatments, which allowed us to numerically compare the treatments.”
Secondly, this issue (not statistical analysis) must be included in the discussion and conclusions
Discussion:
To-date, the viability of CEF-MDV upon its exposure to various vitamins has not been evaluated. Without statistical comparison, the plots of the progression of cell viability with time for each treatment clearly indicated that at the levels of inclusion used, L-AA had a detrimental effect on the viability of CEF-MDV.
Conclusions:
The compatibility results reported in this study using plots of cell viability, demonstrated that both low and high doses of L-AA had negative effects on CEF-MDV survival.
