Exposure of Dairy Cows to Coxiella burnetii in Greece: Surveillance Results and Association of Bacterial Presence with Environmental Variables
, Ioannis Gouvias 1, Marios Lysitsas 1, Ilias Bouzalas 2, Stefania Tampach 3, Eleni Malissiova 4, Alexis Giannakopoulos 1, Constantina N. Tsokana 1, Dimitrios Vourvidis 5, Anna Kyrma 5 and Charalambos Billinis 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript “Preliminary results of dairy cow exposure to Coxiella burnetii in Greece” by Valiakos et al., presents findings related to C. burnetii in bulk milk tank samples from dairy cattle farms and anti-C. burnetii antibodies in bulk milk tank samples and serum of dairy cattle experiencing reproductive issues from those farms. The authors describe an interesting analysis of environmental parameters associated with C. burnetii in dairy farms that will be very useful to C. burnetii fields of study. The manuscript is clear, relevant and well-structured. The references are appropriate. The manuscript is scientifically sound with an appropriate experimental design and conclusions are consistent.
The major areas of concerns for improvement include the need for statistical analysis to be presented throughout the manuscript and inclusion of additional relevant information in the materials and methods to allow for reproducibility.
Specific comments
The title is not very descriptive of the manuscript content. It is not clear why this is a preliminary study and makes no mention of the main findings of study (environmental variables).
Abstract
- Line 20: Please indicate which gene(s) are the qPCR target.
- Lines 24-25: Please include the data for the PCR positive farms (ie how many were positive out of how many tested and included statistics).
- Lines 26 and 27: Please include statistics.
- Line 27: Please clarify why quotations are used for C. burnetii positive. (and later in line 198-199)
- Line 28: Please clarify what the 97m is a measurement of (ie. what is the reference datum? Sea level, ground level?) and include statistics.
- Lines 28 and 30: Please included data and statistics.
- Line 32. Please include data and statistics.
Introduction
- Pg 2, Lines 62-64: Acute Q fever can manifest as serious illness as well. Although it is rare, the authors should include this information.
- Pg 2, Line 84: C. burnetii should be italicized.
Figure 1
- Please expand the figure legend to describe what the colors mean and what each dot represents.
Materials and Methods
- Line 100: Please include statistics for the median number of animals per farm.
- Lines 112 and 113: degress C is superscript in one line but not in the next. Please make consistent.
- Line 107: What part of the animal was blood collected from? What volume of blood was collected? What tube type was used to collect the blood in? How long was the blood in the tube prior to centrifugation? Were there issues with hemolysis due to the transportation time? What was the average transportation time?
- Was the 1.2 mL of lactoserum used for the ELISA assay(s) or qPCR. The methods state that 50mL of milk was processed into 1.2mL lactoserum and the remainder 150 was stored at -20C. How was the 1.2 mL lactoserum split across the three assays (x2 ELISA assays and x1 PCR assay)?
- 2.3 serological assays
o What antigens are used in each kit (ie phase 1, phase 2, both)?
o Why were two different kits used?
o Line 133: What make/model of photometer was used?
o Line 135: Was the positive/negative criteria just for the serum samples? What about the milk samples?
- 2.4 Molecular assays
o What was the Ct threshold?
- 2.7 Ecological niche model
o How are C. burnetii positive farms defined? Is it based on any positive sample among the serology, milk PCR, or milk antibodies? If so, there are only 2 negative farms, this should be discussed as a limitation and how this can be overcome in the modeling.
o How granular is the environmental variable data? Are these within regions or at each individual farm?
Results
- Please include statistics throughout (confidence intervals, etc.)
- Sometimes C. burnetii is abbreviated, sometimes it is written out. Recommend being consistent.
Figure 2
- What do the dark blue vs light blue bars represent?
- What does the red bar represent?
- There looks to be some lettering cut of from the upper right corner. Was that a key that was cut off?
Figures 3 and 4
- What are the units of measure on the x-axis?
Discussion
- The authors should include a section about the limitations of study.
- Line 275 – What is meant by fingerprinting of the pathogen?
- Please discuss how/whether the environmental variables identified in this study may be confounded by the ability to farm cattle and how this was controlled for (ie are there more cattle in areas with the wettest quarter because that allows for better growth of grass and is therefore more suitable to farming cattle? Does this then lead to an increase in C. burnetii due to increased numbers of cattle?)
Author Response
Reviewer 1
The manuscript “Preliminary results of dairy cow exposure to Coxiella burnetii in Greece” by Valiakos et al., presents findings related to C. burnetii in bulk milk tank samples from dairy cattle farms and anti-C. burnetii antibodies in bulk milk tank samples and serum of dairy cattle experiencing reproductive issues from those farms. The authors describe an interesting analysis of environmental parameters associated with C. burnetii in dairy farms that will be very useful to C. burnetii fields of study. The manuscript is clear, relevant and well-structured. The references are appropriate. The manuscript is scientifically sound with an appropriate experimental design and conclusions are consistent.
The major areas of concerns for improvement include the need for statistical analysis to be presented throughout the manuscript and inclusion of additional relevant information in the materials and methods to allow for reproducibility.
Answer: We thank the reviewer for his comment, we have tried to modify the manuscript according to all his suggestions. Please see the specific per comment answers.
Specific comments
The title is not very descriptive of the manuscript content. It is not clear why this is a preliminary study and makes no mention of the main findings of study (environmental variables).
Answer: We thank the reviewer for his comment, we added the preliminary results as our research on the subject is ongoing. However, after your comment we decided to modify as follows “Exposure of dairy cows to Coxiella burnetii in Greece: Surveillance results and association of prevalence with environmental variables”.
Abstract
- Line 20: Please indicate which gene(s) are the qPCR target.
Answer: The info has been added, please see Line 21.
- Lines 24-25: Please include the data for the PCR positive farms (ie how many were positive out of how many tested and included statistics).
- Lines 26 and 27: Please include statistics.
Answer: The info has been added, please see Lines 26-28.
- Line 27: Please clarify why quotations are used for C. burnetii positive. (and later in line 198-199)
Answer: We thought that the use of quotations would be more appropriate to demonstrate the farms where the bacterium was detected by PCR in milk. However, as it might be confusing for the reader, we decided to remove them, and change the term to the more appropriate C. burnetii PCR positive.
- Line 28: Please clarify what the 97m is a measurement of (ie. what is the reference datum? Sea level, ground level?) and include statistics.
Answer: The relevant info has been added, please see lines 29-30.
- Lines 28 and 30: Please included data and statistics.
- Line 32. Please include data and statistics.
Answer: We agree with the reviewer, relevant data has been added in parentheses.
Introduction
- Pg 2, Lines 62-64: Acute Q fever can manifest as serious illness as well. Although it is rare, the authors should include this information.
Answer: We agree with the reviewer, relevant data has been added in lines 67-69.
- Pg 2, Line 84: C. burnetii should be italicized.
Answer: We agree with the reviewer, this has changed accordingly.
Figure 1
- Please expand the figure legend to describe what the colors mean and what each dot represents.
Answer: After discussion with the team, we decided that this image may be confusing for the reader. We changed the figure with one that all sampled farms are demonstrated as well as the PCR positive ones. Moreover we added a Figure 2 where the farms used for the modelling are demonstrated with the color re.
Materials and Methods
- Line 100: Please include statistics for the median number of animals per farm.
Answer: We agree with the reviewer, relevant data was added.
- Lines 112 and 113: degress C is superscript in one line but not in the next. Please make consistent.
Answer: We agree with the reviewer, relevant change was made.
- Line 107: What part of the animal was blood collected from? What volume of blood was collected? What tube type was used to collect the blood in? How long was the blood in the tube prior to centrifugation? Were there issues with hemolysis due to the transportation time? What was the average transportation time?
Answer: 10 ml of blood were collected aseptically from the coccygeal vein of the cows and placed in red top serum tubes. The blood was in the tube before the centrifugation 16 hours in average and the transportation time was 12 hours in average. No issues of hemolysis were detected in the samples. This info was added in the manuscript.
- Was the 1.2 mL of lactoserum used for the ELISA assay(s) or qPCR. The methods state that 50mL of milk was processed into 1.2mL lactoserum and the remainder 150 was stored at -20C. How was the 1.2 mL lactoserum split across the three assays (x2 ELISA assays and x1 PCR assay)?
Answer: We apologize for the confusion, the lactosera were used for the IDEXX ELISA kit, per manufacturer’s instructions. The whole milk was used for the ID-VET ELISA Kit and the PCR.
- 2.3 serological assays
o What antigens are used in each kit (ie phase 1, phase 2, both)?
Answer: Both kits contain a mix of Phase 1 and 1 antigens, relevant info has been added in the text, please see 2.3
o Why were two different kits used?
Answer: We apologize for the confusion, ELISA testing was performed by two laboratories, each one having a different commercial kit at its disposal. Please see relevant comment in line 132
o Line 133: What make/model of photometer was used?
Answer: Two ELISA instruments were used: MR-96A (Mindray, Shenzhen, China) and Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, USA). Relevant info has been added in the manuscript please see
o Line 135: Was the positive/negative criteria just for the serum samples? What about the milk samples?
Answer: Thank you for your comment, The cutoffs were the same for both serum and lactoserum samples, please see the correction in line 147.
- 2.4 Molecular assays
o What was the Ct threshold?
Answer: According to the manufacturer’s recommendation the sample was considered as positive for C. burnetii if a characteristic amplification curve was observed in FAM, with an approximate threshold of <40. The text was changed in line 178.
- 2.7 Ecological niche model
o How are C. burnetii positive farms defined? Is it based on any positive sample among the serology, milk PCR, or milk antibodies? If so, there are only 2 negative farms, this should be discussed as a limitation and how this can be overcome in the modeling.
Answer: We apologize for the confusion. We changed the term Coxiella burnetii positive to Coxiella burnetii PCR positive. Modelling was based on the absolute presence of the bacterium in time and place. As the presence of antibodies cannot determine with certainty the exact time (and even in some cases place) of exposure, GIS analysis was based only on the PCR positive farms and in the area where sampling was considered adequate for such an analysis (northern Greece).
How granular is the environmental variable data? Are these within regions or at each individual farm?
Answer: Environmental parameters are within regions not in each farm. Climate layers (climate grids) is a set of global with a spatial resolution of 1 km2 . This data was added in 2.6 (line 193).
Results
- Please include statistics throughout (confidence intervals, etc.)
Answer: We agree with the reviewer, CI95% have been added, please see Table 1.
- Sometimes C. burnetii is abbreviated, sometimes it is written out. Recommend being consistent.
Answer: We agree with the reviewer, relevant changes were made, in most cases the name was abbreviated.
Figure 2
- What do the dark blue vs light blue bars represent?
- What does the red bar represent?
- There looks to be some lettering cut of from the upper right corner. Was that a key that was cut off?
Answer: The Jackknife Figure (Figure 3 in the revised version) shows the impact of each variable on the entire model and gives the function and signification of each variable. Blue color shows the independent contribution of this variable to the model and light blue give the effect to the model if this variable is not included. The red bar represents the gain using all variables. This info has been added to Figure legend, please see line 263
Figures 3 and 4
- What are the units of measure on the x-axis?
Answer: The units on the x-axis are in mm of precipitation in Figure 3 (Figure 4 in the revised version) and degrees of Celsius in Figure 4 (Figure 5 in the revised version). The relevant info has been added in the Figure legends.
Discussion
- The authors should include a section about the limitations of study.
Answer: We aggre with the reviewer, a relevant paragraph was added, please see line 374.
- Line 275 – What is meant by fingerprinting of the pathogen?
Answer: We thank the reviewer for the comment, and apologize for the confusion caused, using the word “fingerprinting”. The sentence was rephrased in order to demonstrate more clearly what the authors wanted to depict: The demonstration of current circulation of the bacterium by using molecular investigation technique in BTM samples.
- Please discuss how/whether the environmental variables identified in this study may be confounded by the ability to farm cattle and how this was controlled for (ie are there more cattle in areas with the wettest quarter because that allows for better growth of grass and is therefore more suitable to farming cattle? Does this then lead to an increase in C. burnetii due to increased numbers of cattle?)
Answer: Thank you for your comment. The model showed that areas with relatively low precipitation and high minimal temperature are the ones that show a high prevalence of the bacterium presence. To make this clearer for the reader, we added a relevant comment in lines 334 and 335. These values (low precipitation and high temp) are favoring the spread if the bacterium if we take into account the ecological and biological features of C. burnetii (endospore forms).
Reviewer 2 Report
Comments and Suggestions for AuthorsI appreciate the opportunity to review the manuscript titled “Preliminary results of dairy cow exposure to Coxiella burnetii in Greece.” In this manuscript, the authors did a serological and molecular analysis of bulk milk and blood samples from 60 farms in Greece. In this study, the authors analyzed the association between positivity and environmental parameters, concluding that low altitudes and precipitations during the wettest months are risk factors.
Some aspects require attention:
1. Since “Preliminary results” suggests the results are unconfirmed, it would be prudent for the authors to revise the title.
2. In line 18, prevalence is defined as the proportion of sick animals to the total number of animals at risk. However, since this study solely focuses on animals with reproductive issues, the term “prevalence” is not accurately applied.
3. There is no mention in the text about the incorporation of internal control by the authors. Due to the lack of an internal control like B-actin or GAPDH, the verification of DNA presence in the negative samples cannot be guaranteed. An internal control is necessary because milk contains several PCR inhibitors.
4. As widely recognized, the risk of tick exposure rises as altitude decreases. In the discussion, the authors must address the crucial role of ticks in the transmission of C. burnetii.
5. Reference 20 is incomplete and lacks the necessary details.
6. In the supplementary table change serum by sera.
7. Ensure that all scientific names are italicized.
Author Response
I appreciate the opportunity to review the manuscript titled “Preliminary results of dairy cow exposure to Coxiella burnetii in Greece.” In this manuscript, the authors did a serological and molecular analysis of bulk milk and blood samples from 60 farms in Greece. In this study, the authors analyzed the association between positivity and environmental parameters, concluding that low altitudes and precipitations during the wettest months are risk factors.
Some aspects require attention:
- Since “Preliminary results” suggests the results are unconfirmed, it would be prudent for the authors to revise the title.
Answer: We thank the reviewer for the comment, title has been revised.
- In line 18, prevalence is defined as the proportion of sick animals to the total number of animals at risk. However, since this study solely focuses on animals with reproductive issues, the term “prevalence” is not accurately applied.
Answer: We thank the reviewer for the comment, sentence has been revised accordingly.
- There is no mention in the text about the incorporation of internal control by the authors. Due to the lack of an internal control like B-actin or GAPDH, the verification of DNA presence in the negative samples cannot be guaranteed. An internal control is necessary because milk contains several PCR inhibitors.
Answer: We are sorry for not reporting. ADIAVET kit detects in VIC and HEX the internal endogenous control GAPDH, normally found in the samples. DNA extraction and amplification for each sample are considered to be valid if at least a characteristic amplification curve is observed for C. burnetii (FAM) or for the internal control (VIC or HEX). Relevant text was added in lines 178.
- As widely recognized, the risk of tick exposure rises as altitude decreases. In the discussion, the authors must address the crucial role of ticks in the transmission of burnetii.
Answer: We totally agree with the reviewer; In Lines 331 we mention the role of ticks that may important in areas like the one under study. We further enhanced this reference, please see lines 332-336.
- Reference 20 is incomplete and lacks the necessary details.
Answer: We thank the reviewer for the comment, reference was corrected.
- In the supplementary table change serum by sera.
Answer: We thank the reviewer for the comment, it was corrected.
- Ensure that all scientific names are italicized.
Answer: We thank the reviewer for the comment, names were corrected.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI want to thank the authors for considering my suggestions. I could notice the changes in the manuscript. While the title has been modified, it's important to reconsider the term 'prevalence.' As previously mentioned, prevalence traditionally denotes the proportion of sick animals to the total number of animals at risk, however, since this study solely focuses on animals with reproductive issues, the term “prevalence” is not accurately applied.
Author Response
Dear Reviewer,
Thank you for your comments. We agree with the needed change of the word prevalence, we have performed the appropriate changes, please see title and lines 50, 327 and 353.
On behalf of the authors,
George Valiakos
