Animal, Herd and Feed Characteristics Associated with Blastocystis Prevalence and Molecular Diversity in Dairy Cattle from the North of France

Round 1

Reviewer 1 Report

Comments to Authors

In this manuscript, Audebert et al. investigate the occurrence and molecular diversity of the microeukaryotic Stramenopile Blastocystis sp. in a large population of dairy cattle in Northern France. No molecular-based studies have explored previously the presence of Blastocystis sp. in this host species in France. Blastocystis detection was accomplished by means of a qPCR assay targeting the ssu rRNA gene of the parasite. A subset of obtained amplicons were analysed by Sanger sequencing to determine the diversity and frequency of Blastocystis subtypes circulating in the surveyed cattle population. In addition to this, the Authors assessed whether husbandry management practices were associated to a higher risk of Blastocystis infection/carriage. Overall, the manuscript is interesting and timely, as very little information is currently available on the epidemiology of Blastocystis sp. in livestock in Europe. The text is nicely organized and reads well. However, some issues still require attention or addressing.

Major issues

1. Line 21: please clearly mention in the abstract section the diversity and frequency of the Blastocystis STs found in the present study. State also the percentage of positive samples that could not be assigned to a given ST because of mixed samples involving two of more Blastocystis STs.
2. Line 49: please note that detection method also plays a significant role (same comment for lines 77 and 78). Because PCR is far more sensitive than microscopy examination, I would recommend splitting this figure in two, one for PCR-based studies, and the other for microscopy-based studies. Provide mean values and ranges.
3. Line 52: please note that at present 27 genetically distinct Blastocystis STs are regarded as valid. See Stensvold et al. Trends Parasitol. 2020;36:229-232; Maloney et al. Microorganisms 2021;9:997; Maloney et al. Microorganisms 2021;9:1343. Please update the information provided in this paragraph.
4. Line 79: please comment whether this random selection took into consideration farm representability.
5. Line 155: Figure 2 represents prevalence data according to herd size. However, her size is not mentioned in the paragraph comprising lines 154-161. Please comment.

Minor issues

1. Lines 19 and 154: please consider the terms inter- and intra-herd. Please homogenize through the whole manuscript.
2. Lines 226-227: in addition to average herd size, please provide also the range of animals in the sampled farms.
3. Line 257: please provide the kit used to purify the obtained amplicons and the manufacturer, including town and city.
4. Line 258: please clearly state that obtained chromatograms were visually inspected for the presence of double peaks and overlapping traces.
5. Reference section: please note that scientific names should be italicised. Amend through the whole section.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have generated a significant amount of data pertaining to the prevalence of Blastocystis sp. in Northern France. This data provides an excellent assessment of not only the prevalence of this parasite, but potential risk factors and subtype distribution are also explored. The authors have provided a good body of work that will be very useful in the monitoring and understanding of Blastocystis sp. and the data gathered has given rise to a number of areas for further study. Overall, this manuscript will provide a platform for further research, and I recommend its acceptance pending very minor corrections.

1. Details of the statistical analyses used to determine P values should be added to all figure legends as has been done in Figure 1.
2. In section 3.3, it would be better to note the quantity of DNA used in qPCR in μg and not μl.
3. Figure legend in Figure S1 states “The size of the dots is based on the number of samples.”. On the assumption that it has been accidentally added to the legend this should be removed. Figure S2 should also have the details of statistical tests added to the legend.

Author Response

We greatly appreciate the reviewer’s positive comments and have carefully revised the manuscript according to the suggestions. 

1. Details of the statistical analyses used to determine P values should be added to all figure legends as has been done in Figure 1.

As suggested, we added the corresponding tests in Figures 2, 3 and S2.

1. In section 3.3, it would be better to note the quantity of DNA used in qPCR in μg and not μl.

We actually performed DNA extractions in 96-well plates using mostly the same amount of fecal samples for each animal. In our experience, using this DNA extraction method, the amount of DNA extracted from fecal samples and used for qPCR is between 2 and 20 ng/µl (thus between 4 and 40 ng of DNA per qPCR).

1. Figure legend in Figure S1 states “The size of the dots is based on the number of samples.”. On the assumption that it has been accidentally added to the legend this should be removed. Figure S2 should also have the details of statistical tests added to the legend.

The reviewer is absolutely right. We have removed this sentence in Figure S1.

Details of the statistical tests have also been added to Figure S2.

Reviewer 3 Report

I recommend that the authors explain why they decided to identify using real time PCR. It should be added to the section "Materials and Methods" that the diagnosis of Blastocystis is morphologically demanding and it is necessary to have sufficient experience morphologically identify small cysts, which we can also confuse with other small cysts forming parasites. I suggest this because of the completeness of detecting this parasite.

Author Response

We thank the reviewer for this comment.

Indeed, Blastocystis detection can also be performed microscopically, by experienced staff. However, since we were interested in studying not only the prevalence but also the molecular diversity of Blastocystis found in cattle, we used a molecular approach (with the sequencing of PCR amplicons). It should also be noted that molecular methods are known to be more sensitive than microscopic detection ones.

Reviewer 4 Report

This article is a good study about the prevalenve of Blastocystis sp in cattle in France.  

Author Response

We thank the reviewer for this positive comment.