The Use of Flocked Swabs with a Protective Medium Increases the Recovery of Live Brucella spp. and DNA Detection

ABSTRACT Brucellosis is a worldwide zoonosis caused by bacteria from the genus Brucella. Once established, it is very hard to eradicate this disease, since it contaminates animals, the environment, and humans, causing problems for veterinary and public health as well as wildlife protection programs. Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions, as well as from contaminated environments. The aim of this study was to compare main of the commercially used swab types for sampling and diagnostics of Brucella spp. and determine the optimal storage conditions and time frame for testing. To achieve this, we tested bacterial and molecular methods for detection of Brucella abortus, Brucella melitensis, and Brucella suis using nine swab types, all with different tip materials, treated immediately after spiking, after 72 h at +4°C, and after 72 h at −20°C. Flocked swabs showed the highest capacity to preserve bacterial viability and DNA quality, regardless the storage conditions. Flocked swabs immersed in a protective medium provided the best conditions for Brucella survival in all three storage conditions. At the same time, the efficacy of quantitative PCR (qPCR) detection for all swabs, including the positive control, was above 50%, irrespective of the storage conditions, while bacterial survival was significantly lowered when swabs were kept at +4°C or −20°C for 72 h (48.2% and 27.5%, respectively). Compared to the positive control and other types, the flocked swabs maintained higher reproducibility regarding their capacity to preserve live bacteria in all three storage conditions. IMPORTANCE In order to protect public and veterinary health from highly zoonotic bacteria such as members of the genus Brucella and prevent their dissemination into the environment, direct diagnostics are of utmost importance. However, in addition to the highly specific diagnostic tests, the sampling methods, time necessary for specimens to reach the laboratories, and transport conditions are important factors to consider in order to increase the sensitivity of performed tests, especially bacterial culturing and qPCR. This paper shows how different swab types and storage conditions influence classical bacteriological diagnostics of the most prevalent Brucella species – B. melitensis, B. abortus, and B. suis – but have little impact on molecular methods. The presented results highlight (i) the choice of swab regarding the storage and transport conditions, (ii) the importance of immediate swab treatment upon sampling, and (iii) that molecular methods do not depend on storage conditions, unlike classical bacteriological isolation.

Reviewer #2 (Comments for the Author): Overall Comments: • The paper was an interesting read and I appreciated the opportunity to review it! • Overall, it is well done, but there are a few areas/phrases that seem awkward that I would recommend changing for clarity (described in detail below). • It would be nice to have a little more discussion on why the 3 Brucella strains were selected somewhere in the paper. You give a brief glimpse into why the Thomsen strain was selected with the discussion about its lower resistance to storage, but more discussion about the other strains would be appreciated. • The positive and negative controls are discussed in the experimental design section, but only discussed so far as what they are. Since a lot of your experiment and data is comparison to the positive control, I would also recommend detailing how the positive and negative controls were tested. Was it placed on a control swab and tested as well? Was the PBS solution directly tested? Were the controls tested in quadruplicate as the swabs were? Clarification and more discussion on this would be useful. • Along those lines, the data shows that one swab type (S5) produced more live bacteria and DNA when compared to the positive control, and is pointed out several times, but there is no discussion as to how this would be possible. This should be something to clarify and discuss further. • I would like to see more data from your testing. I would recommend including a table of results showing the calculated percent recovery of live Brucella and the calculated percent recovery of Brucella DNA. Also, it would be nice to visually see the correlation of Ct values and number of CFU's for each Brucella species. • More numbers and data need to be given in the Results section. The tables and data shown are more comparisons of the values, but not the values themselves so there is nothing for the reader to assess except your statements about the data. • Was any method or corrections used in the statistical analysis for repeat testing as the swabs/testing was performed in quadruplicate? This needs to be addressed. • Daub and swab seem to be used interchangeably throughout the paper but can be confusing. I would recommend only using a single terminology consistently. • Use of duplicate/triplicate/quadruplicate should be singular, not plural as stated multiple times within the paper.
Specific Comments: • Line 24, remove "both" as you list more than 2 areas where Brucella can be persistent • Line 26, "Therefore" seems to be an awkward transition phrase here. Recommend re-writing or adding another sentence to connect the thoughts from this sentence to the one before it. • Line 27, remove "the" • Line 29-30, "...optimal storage conditions for shipment within 72 hours." I believe you mean to say optimal storage conditions and timeframe for testing. The way this is phrased is awkward and seems to mean anytime within those 72 hours, but your experiments were either immediate or 3 days with no in-between. • Line 32, "...after 72h at +4oC or at -20oC." I would recommend re-writing to something like "after 72h at +4oC and after 72h at -20oC." As written, it seems like the swabs were +4oC or -20oC. • Line 32, remove "That" • Line 34, make "swabs" singular as there was only 1 tested with protective media • Line 35, 55% is listed but there's no reference to the reader on how that compares to the other swabs to know if 55% is good or bad. I'd recommend adding additional data for the others or removing that part. • Line 42-43, What I understood this sentence to mean was diagnostics are important to protect public and veterinary health from the zoonotic potential of Brucella, but the phrase "prevent the spill over into the environment" is awkward and confusing. If wanting to include a comment about the environment, maybe rephrasing as protecting the environment or explaining where the spill over would be coming from would be helpful for clarification. • Line 43, switch "is" to "are" • Line 45, add a comma after laboratories and remove "one the" • Line 48, remove "worldwide" • Line 59, numbers are used and spelled out, use one form consistently (I.E. "12 Brucella species" and "three are considered...") • Line 59-60 states 3 Brucella species are pathogenic to humans but does not list what these are. The species are listed in line 60 but discussed in the context of animal reservoirs. There needs to be a link or better clarification to these species as human pathogens. • Line 61, add "respectively" after "pigs" and before "causing" • Line 61-63, I believe you are trying to say that animals rarely show symptoms, but when clinical signs are displayed, they include what you have listed, but how it's currently phrased is a bit confusing and contradictory. I would also recommend including a descriptor infectious/non-infectious for the long-lasting vaginal discharge. • Line 63, clarify "Excretion" more -I assume you are discussing excretion of the Brucella bacteria, but excretion could be construed as bodily excretions as well since discharges were discussed in the previous sentence. • Line 66, change "get" to "become" • Line 67, add "contaminated" between "with" and "animal" and replace "and" with "or a" • Line 67, add a comma after "humans" and add "a" after usually • Line 69, add "a" between "has" and "low" • Line 70, change "abscess" to "abscesses" • Line 71, clarify the phrase "tendency of the disease to present in a chronic...". Is this in people? In animals? In both? As it is, I'm assuming it is describing humans. • Line 73, remove the comma after "miscarriage" but I would rearrange the sentence to start with the phrase "In animals, isolation can be done..." and move the "after miscarriage or other clinical manifestations" phrase to the end of the sentence. • Line 74, EU and OIE are used and though they are common abbreviations, I would recommend spelling them out the first time they are used here. • Line 75, add a comma after "years" • Line 76, remove "more and more" • Line 77, clarify "bacterial detection" method. Are you meaning culture or PCR or both? • Line 78, replace "have" with "has" and end the sentence after "survival". Start a new sentence at "Freezing" • Line 79, replace "as" with "is" • Line 81, add a qualifier to "personnel" -is the personnel sampling? Or personnel testing? Or other? • Line 82, replace "is" with "the" and "even more" with "especially" • Line 85, "Referent" should this be "Reference"? also clarification on the "not more than 48 hours after sampling" phrase is needed. Is this for sample submission (I.E. when the sample is originally mailed) or for sample arrival at the lab? • Line 88, "Which means that on average" is awkwardly phrased here. Using something more like "With this timeframe" or "With this submission timeline" • Line 89, replace "the" with "a" • Line 92, end the sentence after "detection", remove "whereas" and start the next sentence with "PCR..." • Line 99, add a comma after "objectives" • Line 100 discusses 2 storage conditions but does not mention timeframes which many comparisons are used later to the immediate testing. Maybe rephrase to include 2 timeframes and 2 temperature conditions? • Line 110, "used compared" only need "used" or "compared" here • Line 118, replace "dilutions" with "dilution" and "were" with "was" • Line 122, it would be nice to add more information as to why this size of swab was selected, even if it is something as simple as this size is what is used for official testing, or commonly used in private practice, etc. Also, I'm sure more than 8 swabs were identified in review of commercially available swabs. Maybe some discussion as to how many were reviewed and how these 8 were identified for use (any criteria the others did not meet for inclusion, limiting to number by material type, etc.) should be included? • Line 130-131, "until full capacity of the tip was reached" needs more clarification and discussion. How was this determined? Evidenced by dripping from the swab? What signaled the endpoint? • Line 142, clarify the "two independent analyses" phrase. Did multiple individuals perform the spiking and testing? Was it the same individual doing multiple runs? • Line 147, make "Swabs" singular and "tip" plural • Line 147, clarify which swab tips were removed? Was this just for the eSwabTM or was it for all swabs. Since the previous sentence does not mention swab removal from the PBS clarification is needed. • Line 151, replace "dilutions" with "dilution" • Line 153, "at least in duplicate" needs revised. Does this mean the protocol was different for different swabs? How did data collection change if there were swabs with 2 plates and other swabs with more than 2 plates? If this was how it was done, there needs to be further explanation. If only 2 plates were used, this needs to be stated that way. • Line 164, remove "Total" and replace "were" with "was" • Line 172, add "the" between "to" and "appropriate" • Line 198, remove the first "The" • Line 199, replace the second "the" with "a" • Line 203, percentage listed is mean recovery rate, but before is listed as mean survival rate, please keep consistent terminology • Line 203-204, again recovery rate is discussed, same comment as above about consistent terminology • Line 205, discusses recovery ratios -same comments as above • Line 207, add "the" between "between" and "three" • Line 208, would rephrase as "The bacteriology recovery of all three Brucella strains..." and provide the p-value to show the significance • Line 217, give the percentages for the recovery rates from the different storage temperatures as done in the line before for immediately treated swabs • Line 219, add "when" between "than" and "frozen" • Line 219-221 is phrased awkwardly, maybe rephrase as "Live bacteria were isolated with at least 20% recovery from all directly treated swabs". Also, if you want to include the one swab type exception, explain which one it was any what differences there were. Also, I would give the percentages for the live bacteria recovery from the other storage conditions here for an easy comparison for the reader. • Line 222-224 can be condensed by removing as the comparisons listed are by storage conditions and are redundant. • Line 232, add a comma after "one" • Line 233, replace the last "the" with "a" • Line 237, replace the ";" with "and was" and add "the" between "from" and "control" • Line 238, replace "none" with "no" and add "the" between "to" and "control" • Line 239, remove "it" • Line 232-239, add percentages for the recovery rates. This is done some in the next section discussing the qPCR and should be done for the live bacteria recovery as well. The results section should have numbers or percentages shown to back the statements. • Line 242, add numbers or percentages to the comparison of dry, non-flocked swabs to controls. • Line 243, give values for the comparison of S4 to S7 • Line 245, remove the comma after "isolation" and add "a" after "(S5)" • Line 247-249, give numbers and/or percentages. Provide the data • Line 254-262, same comment as above • Line 264-265, move the "except S5" to the end of the sentence and give the numbers to show the exception • Line 267, "swabs" should be singular • Line 263-270, give numbers and/or percentages. Provide the data. • Line 273, replace "sampled" with "are used for sampling" • Line 274, "on to" should be one word • Line 276, add a comma after "manual" • Line 277, replace "and" with "or" • Line 278-281 Sentence/thought should be expounded upon more. The fact that Brucella is excreted in the billions or trillions after miscarriages is important because...there's always excess bacteria? Why is the bacterial excretion fact important to transport information or lack thereof? • Line 282, remove "a" • Line 286, "...after 72h at +4oC or at -20oC." I would recommend re-writing to something like "after 72h at +4oC and after 72h at -20oC." As written, it seems like the swabs were +4oC or -20oC. • Line 286, remove "most" • Line 290, remove the comma after "swabs" • Line 294, "rescued" is used instead of "recovered" as previously used. Please use consistent terminology • Line 294, "sample" should be plural • Line 295, add "over" between "However" and "the" and add a comma after "decade" • Line 296, the phrase "and assured the constant conditions" is awkward. Recommend clarifying or rephrasing that part. • Line 298, remove "significantly" • Line 308-309, The use of refrigeration in a shorter timeframe should be addressed here. Have studies shown shorter times than 72 hours to be advantageous? Is this an area for further study? • Line 315, remove the comma after "methods" • Line 326, add "an" between "is" and "even" • Line 329, remove "In the same time" and start the sentence at "The higher..." • Line 332, replace "a" with "the" and add "the" between "than" and "others" and make "others" singular • Line 336, "diagnostics" should be singular • Line 337, remove "bacteria" • Line 343, "fibbers" should be "fibers" • Line 344, switch the order of "impacts" and "significantly" • Line 347, "protect" should be "protects" • Line 351, "within" should be "with" • Line 352, "diagnostics" should be singular • Line 350-353 should be expanded to discuss the fact that only 1 swab with protective media was tested and address the fact that additional testing of swabs with protective media should be performed to determine the best protective media and to confirm that repeatability to the results here • Line 353, add "to" between "seems" and "play" • Line 354, remove the comma after "DNA" • Line 356, add "an" between "in" and "environment" • Line 360, remove "a" • Line 364, "swab" should be plural • Line 367, add "compared to the" between "as" and "positive" • Line 371, remove "as" • Line 377, List "(S6)" after "Ca-alginate" as done previously throughout the paper • Line 378, add "the" between "Although" and "Ca-alginate" • Line 379, clarify "it" -referring to the swab or testing and remove "the" • Line 380, remove "These" and start the sentence with "Swabs" • Line 372-383, discussion of additional testing of the swabs at their lowest absorbance level and how that could change results should be included. Also, discussion of additional testing of these swabs using a matrix more like the discharges sampled when used in the field should be done. • Line 392, remove "in neither" • Line 393, add a comma after "conclusion" • Line 394, remove "in the same time" and the "the" between "prevent" and "spill" • Line 397, remove "present" • Line 399, "swab" should be plural • Line 423-424, check that this is cited correctly. 2016 and 2019 are listed, but the most recent version I have been able to find is 2018.
• Figure 1 and Figure 2 confidence intervals are listed in the legends with the asterisks, but how they are used in the figures is very confusing. The CI is reported between groups, but this is not clear in the figures. Possibly using different symbols for each comparison could help. • Line 557, remove "various" • Line 562-563, remove "regardless the strain" as this figure only shows the relationship between live bacteria/DNA and temperature condition • Line 563, "Full and dashed lines..." please add this to the legend for Figure 1. • Line 569, "PC" is used as an abbreviation for positive control, but it is "CP" in the figures. Please use abbreviations consistently • Line 571, "depend" should be "depended" • Line 572, "show" should be "showed the" • Line 575, remove the comma after "results" • Line 576, add a comma after "Still" and "release" should be "released" and "is" should be "was" • Line 578, "negatively" and "affected" should have their order reversed in the sentence • Line 591, the ";" after "S2" should be a comma, remove "even", end the sentence after "isolated", and remove "while only from" and add "were the only cases where" • Line 592, remove the second "from" • Line 593, add "had" between "flocked)" and "significantly", also "significantly" is used quite a lot here, maybe find another word/phrase to use so it's not as repetitive. • Line 592-593, sentence needs a little revision and more bacteria were isolated from S5 compared to what? Clarify in the sentence • Line 593 remove "From" and start the sentence with the swab types then add "had" before "significantly" • Line 594, remove the first "were" • Line 594-596, it makes it seem like S5 was the only swab to have growth when others did not. Is this what you were trying to say? Please revise for clarification if it is not. • Line 596, move "Throughout the storage conditions" to the end of the sentence. • Line 601, "this variation was less significant when they were frozen at..." is the -20oC compared to the +4oC or to the positive control or to the DT? Please clarify. • Line 604, CI were listed out in previous figures where this only shows one CI, can you show the range as the previous figures with different symbols? • Line 613, remove "the amplification of IPC" • Line 619, "difference" should be "different" • Line 620, replace "with" with "compared to the" Staff Comments:

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In this study, Freddi et al examine the impact of different commercially available swabs on Brucella culture and PCR detection. It highlights the importance of collection media and storage conditions in diagnostics. My comments are as follows: Major comments: 1. The entire manuscript needs to proofread for grammar and sentence structures, especially the Abstract, Importance, and Introduction sections 2. The swabs were assessed by inoculating them with ~10 6 Cfu/50 uL of bacteria. Why was this the appropriate Cfu to assess these swabs? 3. The authors conclude that the flocked swabs with protective media enhance the recovery of Brucella in culture and for molecular detection. This study would be of a greater impact if the authors could collect some prospective data from the field to support their observations.

None
Dear Dr Lainhart, The authors are grateful to the reviewers for their critical evaluation and their helpful suggestions and corrections. We have made changes according to their constructive comments and introduced modifications to the manuscript to clarify our work. All issues raised by the reviewers have been addressed here below. The corrections are visible in the revised manuscript (deleted are marked with yellow and added text in blue).
If you consider that additional changes would be needed we rest at your disposal.
All the best!

Luca Freddi
Reviewer #1 (Comments for the Author): In this study, Freddi et al examine the impact of different commercially available swabs on Brucella culture and PCR detection. It highlights the importance of collection media and storage conditions in diagnostics. My comments are as follows: Major comments: 1. The entire manuscript needs to proofread for grammar and sentence structures, especially the Abstract, Importance, and Introduction sections All grammar and wording mistakes have been checked and revised in the new version.
2. The swabs were assessed by inoculating them with ~106 Cfu/50 uL of bacteria. Why was this the appropriate Cfu to assess these swabs?
Because it is 10^6 CFU/50µl is enough quantity of bacteria to ensure positive detection (by qPCR and bacterial culture) for all types of swabs immediately processed after spiking. Nonpublished preliminary tests identify ~10^6 CFU/50µl of bacteria as the minimal concentration for spiking swabs. In the same time, it is a small enough quantity to have negative values if the swabs do not retain Brucellae or their DNA. In authors experience with field samples, when contaminated with 10^6 CFU/50µl the isolation rate is above 95%. As show in Fig. 2A, only from a very small number of swabs positive Brucella culture cannot be ensured, after directly treatment.
3. The authors conclude that the flocked swabs with protective media enhance the recovery of Brucella in culture and for molecular detection. This study would be of a greater impact if the authors could collect some prospective data from the field to support their observations.
The aim of this study was to compare the different swab types in the as equal as possible conditions. Therefore, laboratory conditions, where each swab could be charged with the same quantity of Brucellae is the ideal for selecting the ones that can be used in the field conditions.
According to the reviewer's comments, the importance to compare selected swabs in the field conditions is highly interesting and will be focus on soon.

Minor comments None
Reviewer #2 (Comments for the Author): Overall Comments: • The paper was an interesting read and I appreciated the opportunity to review it!
We would like to thank the reviewer for this positive evaluation.
• Overall, it is well done, but there are a few areas/phrases that seem awkward that I would recommend changing for clarity (described in detail below).
All grammar and wording mistakes have been checked and revised in the new version.
• It would be nice to have a little more discussion on why the 3 Brucella strains were selected somewhere in the paper. You give a brief glimpse into why the Thomsen strain was selected with the discussion about its lower resistance to storage, but more discussion about the other strains would be appreciated.
According to reviewer remark, the text has been modified: Line 62 -Sentence added: These three species of Brucella, are mandatory reportable diseases throughout the world, and still have high prevalence among domestic and wild animals, causing threat for public health (11).
Line 366 -Sentence added: From our experience, besides lower detectability in non-and selective media, Thomsen strain grows slower and takes several days to reach visible CFUs.
• The positive and negative controls are discussed in the experimental design section, but only discussed so far as what they are. Since a lot of your experiment and data is comparison to the positive control, I would also recommend detailing how the positive and negative controls were tested. Was it placed on a control swab and tested as well? Was the PBS solution directly tested? Were the controls tested in quadruplicate as the swabs were? Clarification and more discussion on this would be useful.
We apologise, authors considered that positive and negative controls should be tested in the same way as analysed materials. To correct that from line 137 several additions were made: The same amount of bacteria was also used to spike 2 ml of PBS as a positive control (PC) (50°µl of bacteria + 1950°µl of PBS), and a 2 ml non-spiked PBS was used as a negative control. Three storage conditions were selected to compare swabs, PCs and negative controls: immediately processed after spiking (DT); stored for three days at +4°C (72h_+4°C) and -20°C (72h_-20°C). For all storage conditions, of each Brucella strain used, four swabs per type as well as four PCs were spiked. In order to avoid the biological effect of swab material and variations of bacteria pipetting, two independent analyses were performed for each individual swab, accounting in total of eight analyses per swab type, bacterial strain, and storage condition.
To compare culture and molecular analysis, spiked swab tips were immersed in 2 ml of PBS, vortexed for 30 seconds and incubated at 37°C overnight, to discharge bacteria. Only to the eSwab TM 1 ml of PBS was added because it already contains 1 ml of transport medium. PCs were only incubated at 37°C overnight, without additional PBS.
• Along those lines, the data shows that one swab type (S5) produced more live bacteria and DNA when compared to the positive control, and is pointed out several times, but there is no discussion as to how this would be possible. This should be something to clarify and discuss further.
We agree with the reviewer regarding the importance to understand how the eSwab TM (S5) can provide better bacterial and DNA protection than other treated swabs. This topic was discussed extensively in the discussion session (lines 349-368). The swab S5 has the modified Amies medium that plays important role in protecting bacteria and DNA. In particular, the presence of the emulsifier in the medium can explain a better preservation of live bacteria and DNA over time. Moreover, the flocked swab (as swab S4) contain a nylon flocked fiber which can increase from two-to three-fold the absorption of yield targeted cell and improve diagnostic sensitivity.
• I would like to see more data from your testing. I would recommend including a According to reviewer remark, authors provide the mean, minimal and maximal values of bacterial and DNA recovery rates regarding the Brucella strains, storage conditions, and swab type used in additional supplementary Table S2. Moreover authors are ready and willing (as we stated) to share the raw data upon individual request for future studies or analyses.
• More numbers and data need to be given in the Results section. The tables and data shown are more comparisons of the values, but not the values themselves so there is nothing for the reader to assess except your statements about the data.
The results section has been revised to add data (where possible) on mean values for bacteriology and qPCR recovery rate. For data not shown, authors provide the mean, minimal and maximal values of bacterial and DNA recovery rates regarding the Brucella strains, storage conditions, and swab type used in additional supplementary table S2.
• Was any method or corrections used in the statistical analysis for repeat testing as the swabs/testing was performed in quadruplicate? This needs to be addressed.
There was no need for corrections. As S1 table indicates, the IPC was always in the acceptable range of 3Ct differences (the maximal difference observed was 2Ct). Therefore, the authors considered that there is no need for correction in live bacteria recovery, because of the discrepancies in results.
• Daub and swab seem to be used interchangeably throughout the paper but can be confusing. I would recommend only using a single terminology consistently.
Daub and swab are the nouns for the same objects, and are used interchangeably in order to avoid repetitions, make the sentences wordy and confusing. As manuscript has been proofread, we removed word daub wherever was possible.
• Use of duplicate/triplicate/quadruplicate should be singular, not plural as stated multiple times within the paper. Corrected.
Specific Comments: • Line 24, remove "both" as you list more than 2 areas where Brucella can be persistent Done • Line 26, "Therefore" seems to be an awkward transition phrase here. Recommend re-writing or adding another sentence to connect the thoughts from this sentence to the one before it.
Corrected to: Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions as well as from contaminated environment.
• Line 27, remove "the" Done • Line 29-30, "...optimal storage conditions for shipment within 72 hours." I believe you mean to say optimal storage conditions and timeframe for testing. The way this is phrased is awkward and seems to mean anytime within those 72 hours, but your experiments were either immediate or 3 days with no in-between.
According to reviewer remark, the text has been corrected to: …... and determine the optimal storage conditions and time frame for testing.
The text has been corrected following reviewer suggestion.
• Line 34, make "swabs" singular as there was only 1 tested with protective media Corrected.
• Line 35, 55% is listed but there's no reference to the reader on how that compares to the other swabs to know if 55% is good or bad. I'd recommend adding additional data for the others or removing that part.
According to reviewer remark, the text has been corrected to: The flocked swab immersed in protective media, provides the best conditions for Brucella survival in all three storage conditions.
• Line 42-43, What I understood this sentence to mean was diagnostics are important to protect public and veterinary health from the zoonotic potential of Brucella, but the phrase "prevent the spill over into the environment" is awkward and confusing. If wanting to include a comment about the environment, maybe rephrasing as protecting the environment or explaining where the spill over would be coming from would be helpful for clarification.
According to reviewer remark, the text has been corrected to: In order to protect public and veterinary health from highly zoonotic bacteria such as Brucella genus, and prevent the dissemination into the environment, the direct diagnostics are of utmost importance.
• Line 45, add a comma after laboratories and remove "one the" Corrected.
• Line 59, numbers are used and spelled out, use one form consistently (I.E. "12 Brucella species" and "three are considered...") According to standard style guides, the authors use words for cardinal numbers less than 10 and numerals for 10 and above.
• Line 59-60 states 3 Brucella species are pathogenic to humans but does not list what these are.
The species are listed in line 60 but discussed in the context of animal reservoirs. There needs to be a link or better clarification to these species as human pathogens.
According to reviewer remark, the text has been corrected to: Out of the 12 currently described Brucella species, B. abortus, B. melitensis and B. suis are considered to be highly pathogenic for humans (4, 5). These three species of Brucella, are mandatory reportable diseases throughout the world, and still have high prevalence among domestic and wild animals, causing threat for public health. They affect mainly cattle, small ruminants and pigs respectively, causing abortions and infertility (6, 7).
• Line 61-63, I believe you are trying to say that animals rarely show symptoms, but when clinical signs are displayed, they include what you have listed, but how it's currently phrased is a bit confusing and contradictory. I would also recommend including a descriptor infectious/non-infectious for the long-lasting vaginal discharge.
The text has been modified to: Infected animals rarely show clinical symptoms. Those usually occur during pregnancy in females, as miscarriages which can also be followed by long-lasting vaginal discharge and in worst case end-up in sterility (6).
• Line 63, clarify "Excretion" more -I assume you are discussing excretion of the Brucella bacteria, but excretion could be construed as bodily excretions as well since discharges were discussed in the previous sentence.
The text has been modified to: Excretion of Brucella is reported in sick as well as asymptomatic animals.
• Line 67, add "contaminated" between "with" and "animal" and replace "and" with "or a" Corrected.
• Line 67, add a comma after "humans" and add "a" after usually Corrected.
• Line 71, clarify the phrase "tendency of the disease to present in a chronic...". Is this in people?
In animals? In both? As it is, I'm assuming it is describing humans.
According to reviewer remark, the text has been modified: The tendency of the disease to present a chronic and persistent form in humans, results in granulomatous disease capable of affecting any organ system (1).
• Line 73, remove the comma after "miscarriage" but I would rearrange the sentence to start with the phrase "In animals, isolation can be Corrected..." and move the "after miscarriage or other clinical manifestations" phrase to the end of the sentence.
According to reviewer remark, the text has been modified: In animals, isolation of Brucellae can be done via collection of genital swabs after miscarriage or other clinical manifestations.
• Line 74, EU and OIE are used and though they are common abbreviations, I would recommend spelling them out the first time they are used here. Corrected.
• Line 77, clarify "bacterial detection" method. Are you meaning culture or PCR or both?
According to reviewer remark, the text has been modified to: Swab type and storage temperature could influence successful bacterial isolation and molecular detection • Line 78, replace "have" with "has" and end the sentence after "survival". Start a new sentence at "Freezing" The sentence has been modified following reviewer's suggestion.
• Line 81, add a qualifier to "personnel" -is the personnel sampling? Or personnel testing? Or other?
The sentence has been completed following reviewer's suggestion.
• Line 85, "Referent" should this be "Reference"? also clarification on the "not more than 48 hours after sampling" phrase is needed. Is this for sample submission (I.E. when the sample is originally mailed) or for sample arrival at the lab?
According to reviewer remark, the text has been revised to: OIE guidelines instruct that field samples should be delivered to the diagnostic laboratories as soon as possible (11), while the EU Reference Laboratory for Brucellosis recommends a maximum delay of 48 hours between sampling and its arrival to the laboratory… • Line 88, "Which means that on average" is awkwardly phrased here. Using something more like "With this timeframe" or "With this submission timeline" According to reviewer remark, the text has been modified to: With this submission timeline, diagnostics are performed, on average, three days after sample collection.
• Line 100 discusses 2 storage conditions but does not mention timeframes which many comparisons are used later to the immediate testing. Maybe rephrase to include 2 timeframes and 2 temperature conditions?
According to reviewer remark, the text has been revised to: In order to achieve the objectives, we tested bacterial culture and qPCR diagnostics on nine different spiked swabs, treated immediately or after 72h and stored in two temperature conditions.
• Line 122, it would be nice to add more information as to why this size of swab was selected, even if it is something as simple as this size is what is used for official testing, or commonly used in private practice, etc. Also, I'm sure more than 8 swabs were identified in review of commercially available swabs. Maybe some discussion as to how many were reviewed and how these 8 were identified for use (any criteria the others did not meet for inclusion, limiting to number by material type, etc.) should be included?
According to reviewer remark, the text has been modified to: Among commercially-available swabs, only swabs with regular sized tips (~5 mm wide and ~16 mm long) commonly used for sampling, were selected. Only swabs with a transport tube have been selected, without considering the different types of handles. Furthermore, for the same type of tip material, only one representative was selected, belonging to the main brands on the market.
• Line 130-131, "until full capacity of the tip was reached" needs more clarification and discussion. How was this determined? Evidenced by dripping from the swab? What signaled the endpoint?
According to reviewer remark, the text has been revised to: The absorbance of each swab type was calculated by adding 5 µl of PBS until full capacity of the tip was reached, evidenced by dripping from the swab.
• Line 142, clarify the "two independent analyses" phrase. Did multiple individuals perform the spiking and testing? Was it the same individual doing multiple runs?
According to reviewer remark, the text has been modified to: In order to avoid the biological effect of swab material and variations of bacteria pipetting, two independent analyses were performed by the same person for each individual swab, accounting in total of eight analyses per swab type, bacterial strain, and storage condition.
• Line 147, clarify which swab tips were removed? Was this just for the eSwabTM or was it for all swabs. Since the previous sentence does not mention swab removal from the PBS clarification is needed.
According to reviewer remark, the text has been modified to: All swab tips were then removed after vortexing step and the Working Suspension (WS) was used for bacterial plate culturing and DNA extraction.
• Line 153, "at least in duplicate" needs revised. Does this mean the protocol was different for different swabs? How did data collection change if there were swabs with 2 plates and other swabs with more than 2 plates? If this was how it was done, there needs to be further explanation. If only 2 plates were used, this needs to be stated that way.
Corrected to: All dilutions were inoculated in duplicates.
• Line 198, remove the first "The" Corrected.
• Line 199, replace the second "the" with "a" Corrected.
• Line 203, percentage listed is mean recovery rate, but before is listed as mean survival rate, please keep consistent terminology Corrected.
• Line 203-204, again recovery rate is discussed, same comment as above about consistent terminology Corrected.
• Line 205, discusses recovery ratios -same comments as above Corrected.
• Line 208, would rephrase as "The bacteriology recovery of all three Brucella strains..." and provide the p-value to show the significance According to reviewer remark, the text has been revised to: The bacteriology recovery of all three Brucella strains (mean values 55.8%, 50.5% and 37.7%, for 16M, 544 and Thomsen respectively) was significantly lower than molecular detection (mean values 66.9%, 67.2% and 69.1%, respectively; p-values< 0.00001, compared to both conditions), especially for Thomsen (data not shown).
• Line 217, give the percentages for the recovery rates from the different storage temperatures as done in the line before for immediately treated swabs According to reviewer remark, the text has been modified to: The recovery rate of immediately treated swabs (mean value 68.2%) is significantly higher than recovery from daubs stored at +4°C or -20°C (mean values 48.2% and 27.5% respectively; p-values< 0.00001, compared to both conditions).
• Line 219-221 is phrased awkwardly, maybe rephrase as "Live bacteria were isolated with at least 20% recovery from all directly treated swabs". Also, if you want to include the one swab type exception, explain which one it was any what differences there were. Also, I would give the percentages for the live bacteria recovery from the other storage conditions here for an easy comparison for the reader.
The paragraph has been revised following reviewer's suggestion.
• Line 222-224 can be condensed by removing as the comparisons listed are by storage conditions and are redundant.
Because of the amount of comparisons presented, the authors feel the additional clarification, and somewhat repetition is useful in this paragraph.
• Line 233, replace the last "the" with "a" Corrected.
• Line 237, replace the ";" with "and was" and add "the" between "from" and "control" Corrected.
• Line 232-239, add percentages for the recovery rates. This is done some in the next section discussing the qPCR and should be done for the live bacteria recovery as well. The results section should have numbers or percentages shown to back the statements.
The paragraph has been revised following reviewer's suggestion to include result section.
• Line 242, add numbers or percentages to the comparison of dry, non-flocked swabs to controls.
The data were shown at the top of the paragraph.
• Line 243, give values for the comparison of S4 to S7 The data were shown at the top of the paragraph.
• Line 247-249, give numbers and/or percentages. Provide the data The paragraph has been revised following reviewer's suggestion to include result section.
• Line 254-262, same comment as above The paragraph has been revised following reviewer's suggestion to include result section.
• Line 264-265, move the "except S5" to the end of the sentence and give the numbers to show the exception Corrected.