Biochemical and Computational Assessment of Acute Phase Proteins in Dairy Cows Affected with Subclinical Mastitis

Round 1

Reviewer 1 Report

The title of the manuscript suggests bioinformatic analysis only - but the Authors were used traditional tests as well - the reviewer suggests changing of the title.

line 39: what was the basis of division of cows into groups?

lines 50-55 are not clear and not following smoothly the previous part of the abstract, please think about joining sentence addition.

line 110 not many literature data

line 113 cross-bred HF

line 116 - potential, as they are not exclusive for mammary gland

Please add concluding sentence to the Introduction part.

In M&M part we need more details dedicated to the tested animals - no data in Figs as well

line 151-154; please give more details of sampling 

line 172 what is the ph of full fresh milk according to the India standards? As in EU we take 6.6-6.8 as a correct ph

line 194 please move up this part dedicated to samples collection - was it one time point of milk sampling? Any exclusions from the study? Any bacterial screening/culturing tests?

line 279-part dedicated to SCC - numbers in idividual cows/quarters?

Any tests dedicated to basic milk components - which are also changed during masistis?

lines 504& 518 please avoid comparisons with such historical analyses

The discussion part is too brief and do not covers all aspects of the research. Please underline novelties of the study, as well as limitations and future perspectives.

Conclusions part is too long - needs re-writting

too low number of samples to talk about substitution of SCC parameter

Remember about the world trend to antibiotics usage reduction - important point to discussion

Main part of references list is composed from actual publications. The reviewer advice addition of : DOI:https://doi.org/10.3168/jds.2021-21267

The Reviewer do not see serious language problems in this manuscript

Author Response

We thank the esteemed reviewer for providing valuable suggestions in improving the quality of revised manuscript. Point by point response has been provided.

Author Response File: Author Response.pdf

Reviewer 2 Report

General comments

The introduction need be re-write: the first part are generalities (70-71) and the second part is not clear and consistente (the justification for this study is required and accuratly suported by literature, repoting the aims of the study at the end). In M&M, the SCM and groups need be accurately defined. Only after know rigorously both issues an adequate review of the manuscript can be done. This manuscript is not appropriately structured and as consequence is not consistent. Also, in our opinion, this manuscript can be divised into two studies (manuscripts). I suggest to fully rewrite the manuscript avoiding these major issues. Please see some specific comments to pursue this task.  Please avoid duplication of information between the main text and tables/figures. The potential new version needs to be fully re-reviewed

Specific comments

L36-55: Please rewrite the abstract in a correct sequence: aim(s), (M&M), main findings and conclusions.

L77-78: Reorder the tests. Eg “…by on-farm test such as California mastitis test (CMT), pH and electrical conductivity (EC), and laboratoty test such as somatic cell count (SCC), microbiological cultures and PCR.”

L81: “…mainly due…”

L83: You mean enter in mammary ducts?

L97-101: reference are required.

L106: Such as inflamation or infection?

L110: There are several studies reporting  APP’s in milk, and someones reporting albumin.

L140-143: This information is not relevant for the purpose of the study.

L140-146: Please merge into to a subsection, eg, “animals” where you can caracterize the parity, etc.

L151-153: We dont known how many farms were involved in the study.

L155-193: these test are very well described in literature. I suggest only to report them and cite a reference for each one. Also for some parts of sample collection.

L214-217:  At least a reference is required.

L231-277: This is part of another study? See L136-134 (Introduction). This manuscript is not appropriately structured.

L280: You need to report how you defined the SCM according to the results from these 4 tests.

L288-290: you also need to define the healty and affected groups: your examination is a quarter level but the groups are at animal level.

L298-321: you state terms such as “numerically higher variation” (L299), higher distribution (L301) without to test these diferences between groups.Also in some parts (L306-317), only descriptive statistic was reported without any statistical inference. Similar procedures are observed in the Serum APP’s section.

Author Response

We thank the worthy reviewer for providing valuable suggestions in improving the quality of revised manuscript. Point by point response has been provided

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for following the suggestions of the reviewer and improving of the manuscript.

please check carefully the language used in the manuscript

Author Response

We are highly thankful to the worthy reviewer for providing valuable suggestions to improve the quality of the manuscript.

Reviewer 2 Report

Dear authors,

thanks for providing the revised version. A significant improvement was done. Nevertheless, some issues persist. Please read  the specific comments careffuly. Most of the concerns are about methodology, but I think they can be probably solved in a next version.

Specific comments:

L37-38: Please remove this sentence. The classification methods of the groups are not need in the abstract. Define the abbreviations belows.

L47-48: Please uniformize the P values at p<0.05, p<0.01 and p<0.001 levels, and p>0.05 (eg., p<0.001 instaed of p<0.0001). Check the whole manuscript for this issue.

L48-50: Please, only use centesimal to present correlations (eg, r = 0.22 instaed of r = 0.218). Check the whole manuscript for this issue.

L53-55: These 3 sentences are not necessary in the abstract. Please remove. If you to present this information here please move them up (abstract= background, objectives, M&M, results and conclusions).

L56: You used SCC to identify SCM. So, you cannot conclude  that estimation of SCC is an efective tool. You only need to report that all studied APP´s are suitable to detect SCM.

L57: IMI is not defined before. Please remove (the bacteria were not identified).

L77: What you mean with “cultural tests”? bacteriological tests? SCM are screened by on-farm tests (California mastitis test (CMT), pH, electrical conductivity (EC). Positive samples were send to lab for SCC, and for bacteriological culture (or PCR).

L79: “…to detect SCM…”. Please remove “/IMI (intramammary infection)”. The IMI, by definition is detected by the identification of bacteria.

L85 “… IMI (intramammary infection) …”.

L108: “On the other hand, at our best knowledge, …”.

L121: It is preferable to use SCM (detected by SCC) instead of IMI (bacterial culture were not performed). Check the manuscript.

L121-135: Please state the objectives of this work sequential and clearly (e.g., this study aimed to… and to…). This part (L121-135) can be moved to discussion (or above in introduction) section with the respective modifications.

L141-143. This is a repetition. For example, you can start (L138) by “This cross-sectional study was…” and removing these sentences.

L150: At this step is not “composite milk” (only milk). Only when you obtain a mix from all 4 quarters can be considered “composite milk”. Few (2-3) squirts were firstly removed to avoid contaminations, right?

L153: Not for SCC (it was performed in Lab)

L155: We presume that non additive was added. So, the time lapse between obtention and laboratory prosecution should be reported.

L164-167: This information was used for what?

L183: Please define score 1, 2 and 3.

L209-212: The issue about classification remains unclear. Apparently, the cows are grouped only according SCC? I.e., >200,000 cells/mL = SCM. The previous on-farm tests were performed only as an on-farm preliminary screening; and the classification was done by SCC. If this is the case, L276 and 291 are not necessary. Also, the screening tests reported between L171-192 in M&M are not necessary (they are very well described on-farm tests).

L278: Cows were affected by mastitis not infected.

L353: Infected?

L361: Relatively?

L368 “…, and Malb…”

L492: You mean excellent.

L495: right, but this is different to measure SCC in composite or quarter milk.

L531: I suggest “multifactorial” instead of “multietiological”. There are different factors, but only one species of bacteria can be associated in several cases.

L566: Please, see above my comments for on-farm tests (CMT, Electrical conductivity, pH). Moreover, you used these tests at quarter level and SCC at composite milk level. Moreover, in practice is not usual to observe that all tests always agree between them.

L853 (Table 2). See my previous comment. I think that is preferable to consider on-farm test as a preliminary screening and consider the SCC as the main test to form the groups. The agreement between tests was not preformed (and possibly is not adequate to compare test using composite vs. quarter milk levels).

L924 (Table 12). Define MS. Also, in L455.

Author Response

We are highly thankful to the worthy reviewer for providing valuable suggestions to improve the quality of the manuscript.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Dear authors,

thanks for submitting this revised version (v3). I believe that , now, it is suitable for publication in this journal.

As minor corrections (these corrections are necessary)

L46-48 (abstract): “Further, SCC was correlated with ferritin (r = 0.26, p<0.002), CRP (r = 0.19, p <0.05) and Malb (r = 0.21, p<0.01) of milk. Also, milk SCC was correlated with serum ferritin (r = 0.28, p<0.001).

“…, CRP (r= 0.16, p>0.05), and Malb48 (r = 0.16, p>0.05)” need be removed (correlations were not significant)

L359: "...(r = 0.26**, p<0.01), CRP (r = 0.19*, p<0.05)

L363: Please correct to “p>0.05”

Please be consistent to report p-values in the whole document.

Author Response

Comments and Suggestions for Authors

As minor corrections (these corrections are necessary)

L46-48 (abstract): “Further, SCC was correlated with ferritin (r = 0.26, p<0.002), CRP (r = 0.19, p <0.05) and Malb (r = 0.21, p<0.01) of milk. Also, milk SCC was correlated with serum ferritin (r = 0.28, p<0.001).

 “…, CRP (r= 0.16, p>0.05), and Malb48 (r = 0.16, p>0.05)” need be removed (correlations were not significant)

L359: "...(r = 0.26**, p<0.01), CRP (r = 0.19*, p<0.05)

Response: We are highly thankful to the worthy reviewer for providing valuable suggestions during the course of revision and as seen we have felt a great improvement in the quality of manuscript. Many things in the manuscript that we missed were added after revision, which only an expert can pinpoint.

It is more important to know the correlation between the milk SCC and APP’s in milk and sera. As there is no significant p value in the correlation data, means there is no difference between the data sets but this does not imply that there is no correlation also. This correlation is not to be ignored if the underlying implications of such a weak correlation make sense to be reported to the research community. The correlation that we have reported tells us that we are looking at two variables that appear to have little to no collinearity. For such a low correlation statistical significance is bound to be low.

This correlation analysis part is also an important part of study as many studies are unware of such analysis and it will be helpful for many researchers who want to work on SCM and want to replicate such studies. We firmly believe it should be kept in the manuscript.

L363: Please correct to “p>0.05”

Response: As per suggestions of the worthy reviewer, changes have been incorporated in the revised manuscript.

Please be consistent to report p-values in the whole document.

Response: In this study, different statistical analysis like descriptive statistics, ROC, and correlation tests has been analyzed and for all of them p-values will vary in the manuscript.