Retained Placenta as a Potential Source of Mastitis Pathogens in Dairy Cows

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Lines 74-79 the sampling method seems a bit strange. In my opinion the number of animals are quite low. There are much more cows diagnosed with RP on commercial farms. This may be the reason of the non significant Results and the tendencies.

Many results were mentioned in the discussion chapter. Please look a them carefully, and re-write the Results chapter.

Line 77: how did the authors detect RP if there was no rectal examination except for the temperature measurement. Was it visible?

Line 138: fever occured 54% of the Animals (6/11) (15 animals were mentioned altogether previously)

Line 164: 9/15 it is still confusing

Line 166: it would be useful to know the ratio of environmental pathogens.

Line 182-184: The Results chapter does not mention this finding. 

Line 195: please explain the abbreviation

Line 200: this is a result.

Lines: 268-269: previously, they were mentioned mostly as environmental pathogens, therefore, I cannot see the connection.

Author Response

We thank the thorough review from all three reviewers. We have implemented all corrections and answered to each suggestion. In fact, all amendments improve notably our manuscript. A revised version of the manuscript is attached with the different changes made in different colors by referee, to be easily found.

 

Reviewer 1:

Lines 74-79 the sampling method seems a bit strange. In my opinion the number of animals are quite low. There are much more cows diagnosed with RP on commercial farms. This may be the reason of the non significant Results and the tendencies.

Thanks for the very wise comment. A clarifying sentence has been added in the revised version of the manuscript, in the discussion section (Lines 307-308):” A limitation of this study was the reduced number of animals studied and that we only worked with cows with RP, without previous or posterior udder health evaluation. It was therefore not possible to associate RP and mastitis.”

The collection samples were obtained under specific conditions (no previous antimicrobial treatment, no calving assistance, no concomitant diseases) in seven relatively small farms (about 80-100 cows in productions) during the veterinary consultation of others animals. In fact, this study was originally delineated with more samplings (including cows presenting mastitis), but at final we only used samples rigorously obtained and processed to ensure the quality of the outcomes. This low sample can influence the inferential analysis (logistic regression) for some non-significant values mainly that those < 0.20 (low statistical power); our results can be considered as a preliminary assessment of association both factors (parity and fever) with each microorganism. However, the main aim of this study was to evaluate “… the presence of mastitis pathogens in dairy cows with RP, using RT-PCR. “ (Lines 78-79). This aim was reached.

Many results were mentioned in the discussion chapter. Please look a them carefully, and re-write the Results chapter.

Thanks for your care. We have carefully reviewed the whole Results and Discussion section for this issue.

Line 77: how did the authors detect RP if there was no rectal examination except for the temperature measurement. Was it visible?

Thanks for this question. Now, in lines 86-90 this issue has been clarified: “RP was diagnosed by visualization of fetal membranes hanging from the vulva > 24h after calving and their unsuccessfully removal after slight tracking procedure to confirm cotyledon-caruncle attachment. The transrectal palpation was avoided to minimize the risk of further contamination for sampling.”

Line 138: fever occured 54% of the Animals (6/11) (15 animals were mentioned altogether previously)

We have improved the sentence of Line 151 to clarify the number of animals: “Fever was detected in 54.5% of the 11 cows with Strep. uberis…”.

Please, note that the multivariate logistic regression was made for each bacterium. For the presence of Strep. uberis, one cow was inconclusive, 3 cows were negative and 11 cows positive (n=14). In the text, we only reported the proportion of cows with Strep. uberis presenting fever (n=11). The fig. 1 full elucidates this issue. We hope that the sentence and figure will be clear for readers.

Line 164: 9/15 it is still confusing

We improved the readability of the sentence in Lines 177-178: “In this study, 60% (9/15) of the mastitis pathogens that could be identified by the MastiType Multi RT-PCR kit were detected in the uterine fluid of cows with RP.”

Please note that the MastiType Multi RT-PCR kit could identify 15 mastitis pathogens and the sample size was 15 uterine fluids (cows).

Line 166: it would be useful to know the ratio of environmental pathogens.

Thanks for the suggestion. We have now changed the sentence adding the full number of animals affected in L180: “… (66.7%; 6/9) …”.

Line 182-184: The Results chapter does not mention this finding. 

This result is from a study of Bicalho et al. [34]. Now, we added in Line 199 “…in their study [34].” to avoid confusion.

Line 195: please explain the abbreviation

The abbreviation was removed. Now, Lines 205-206: “… (odds ratio = 44.8; p < 0.01).”.

Line 200: this is a result.

Thanks for the comment. We added in Lines 239-241: “This large number of species are also found in the environment and can partially justify the high proportion of Staphylococcus spp. (93.3%) observed in our study by contamination through vagina at early postpartum.”

The results of Line 200, now Line 243, is from [51]. In our study we were not able to identify CNS as a single group, only Staphylococcus spp.

Lines: 268-269: previously, they were mentioned mostly as environmental pathogens, therefore, I cannot see the connection.

Thanks for this criticism. In Lines 311-316, we have added new information to clarify the importance of environmental pathogens: “Further and larger comparative studies are required to evaluate the real impact of the uterine secretions on the mastitis prevalence during postpartum. Factors such as environmental bacterial load on the animal surface and facilities (e.g., milking room and ma-chines, calving parks), bacterial virulence and animal susceptibility should be taken in consideration for a holistic approach.”   

Additionally, we improved the discussion in Lines 225-236 to give more support for this connection: 

“Nonetheless, the common possible source for RP and mastitis does not rule out the possibility that RP play a key role aggravating mastitis through an increased level of contamination of the environment of the udder of the affected cows regarding E. coli and other bacteria.

Suriyasathaporn et al. [48] observed a tendency for increased risk of cows with RP to present clinical mastitis (risk ratio = 2.88; 95% CI = 0.96 - 8.64; p = 0.06), at same period, then cows without RP. The risk to develop severe mastitis was higher (RR = 5.4) in cows with RP than in healthy cows. Also, cows with RP up to five days after calving were more likely to present clinical mastitis up to 120 days postpartum (odds ratio = 9.45; 95% IC = 8.62 - 10.27; p < 0.001) than cows without RP [49]. A large epidemiological study addressing environmental, bacterial and host factors is required to evaluate more in deep this relationship between 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The work emply classical investigation methods comprises PCR. Today this approach could be increased by NGS technology application to have a complete vison of the microbioma present in the placenta. 

I think the authors could be added something about the application of the new sequence technology

Author Response

We thank the thorough review from all three reviewers. We have implemented all corrections and answered to each suggestion. In fact, all amendments improve notably our manuscript. A revised version of the manuscript is attached with the different changes made in different colors by referee, to be easily found.

 

Reviewer 2:

The work emply classical investigation methods comprises PCR. Today this approach could be increased by NGS technology application to have a complete vison of the microbioma present in the placenta.

I think the authors could be added something about the application of the new sequence technology

Thanks for the suggestion: now in Lines 49-57: “Uterine microbiota is indeed more diverse than once was thought and due to the significant impact of related reproductive disorders, its characterization has long been a focus of research. The techniques have been traditional culture-based techniques and more advanced molecular ones, such as real time polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) or ribosomal RNA clone libraries [12–14]. More recently, next-generation sequencing (NGS) technologies (sequencing of the rrs gene encoding 16S rRNA), conducting in-depth sequencing and facilitating the analysis of thousands of sequences per sample, permitted the description of previous unculturable bacteria of the complex uterine bacterial community [15–18].”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The paper under review deal with the retained placenta as a potential source of mastitis pathogens in dairy cows. The topic of the investigation is in the scope of the journal. The study showed high prevalence of Escherichia coli, Staphylococcus pp., yeasts, Trueperella pyogenes/Peptoniphilus indolicus, Streptococcus uberis and Streptococcus dysgalactiae. Streptococcus uberis was related to the presence of fever. The evaluated paper contributes new information to the field.

 

Remarks

The study has serious limitations.

The first limitation pointed out in the Discussion is the small number of animals. The second limitation is  the lack of examination of  udder health status (scc, clinical and bacteriological examinations). Was there a relationship between the bacteria in the uterine fluid and the health of the udder or not? The limitation should be discussed!

Retained placenta is a high risk factor for clinical mastitis in dairy cows. The underlying cause for both diseases is a severe negative energy balance and periparturient immune suppression. This should be more widely presented in the Discussion.

E. coli is one of the main etiologic agents of acute clinical mastitis during early lactation in dairy cattle. This should be indicated in the Discussion.

Author Response

We thank the thorough review from all three reviewers. We have implemented all corrections and answered to each suggestion. In fact, all amendments improve notably our manuscript. A revised version of the manuscript is attached with the different changes made in different colors by referee, to be easily found.

 

Reviewer 3:

Remarks

The study has serious limitations.

The first limitation pointed out in the Discussion is the small number of animals. The second limitation is the lack of examination of udder health status (scc, clinical and bacteriological examinations). Was there a relationship between the bacteria in the uterine fluid and the health of the udder or not? The limitation should be discussed!

We thank the comment of the reviewer. We have added a sentence regarding the limitations of the study pointed out by the reviewer in the discussion. However, we think the described results are worthy to be shared with the scientific community as a novel link between different pathologies in our herds.

The changes added in the manuscript are in Lines 307-316:

“A limitation of this study was the reduced number of animals studied and that we only worked with cows with RP, without previous or posterior udder health evaluation. It was therefore not possible to associate RP to mastitis. However, in overall, a large proportion of mastitis pathogens was detected in uterine fluids supporting our hypothesis that cows with RP may serve as source of intramammary infection, indirectly. Further and larger comparative studies are required to evaluated the real impact of uterine secretions on the mastitis prevalence during postpartum. Factors such as environmental bacterial load in animal surface and facilities (e.g., milking room and machines, calving parks), bacterial virulence and animal susceptibility should be taken in consideration for a holistic approach.”

Retained placenta is a high risk factor for clinical mastitis in dairy cows. The underlying cause for both diseases is a severe negative energy balance and periparturient immune suppression. This should be more widely presented in the Discussion.

Thanks to approach these subjects. In Lines 213-225, we have added the discussion on the importance of NEB, metabolic diseases, immune system and oxidative stress: “During the transition period and mainly around parturition, the increased energy demand favors the incidence of metabolic diseases, compromising immunity and resistance to infections by pathogenic and opportunistic agents. The condition of RP increases the occurrence of postpartum infectious diseases by uterine colonization and development of E. coli and other microorganisms [9,39]. In the same way, the negative energy balance during this period impairs the immune response in the mammary gland of lactating dairy cows, increasing the susceptibility to mastitis [40–42] and RP [39]. As well, the oxidative stress has been associated to negative energy balance [43,44] and metabolic disruptions [45] in dairy cows. It was suggested that the link RP-mastitis may be related to the weakened immune system [46], trough peripheral leukocytes impairment [47]. Moreover, the chronological conjugation of these factors with bacterial load, including bacterial virulent factors [35] can have a relevant impact on the incidence of these diseases.“.

1. coli is one of the main etiologic agents of acute clinical mastitis during early lactation in dairy cattle. This should be indicated in the Discussion.

Thanks for this remark. Now, in Lines 206-210 we have added: “E. coli is the most frequently found gram-negative pathogen causing acute clinical mastitis during early lactation in high-producing dairy cows. It invades the udder through the teats, proliferate and induce a local and systemic acute phase response that may cause irreversible tissue damage in the mammary gland, and severe clinical symptoms, sometimes even leading to the animal death 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript was corrected as adviced. This, it van be accepted in it's present form.