Prevalence and Molecular Characterization of Cryptosporidium in Diarrheic Dairy Calves: A Descriptive Study in Family Farms of Southern Santa Catarina, Brazil

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript titled “Prevalence and Molecular Characterization of Cryptosporidium in Diarrheic Dairy Calves: A Descriptive 3 Study in Family Farms of Southern Santa Catarina, Brazil” addresses an important topic in veterinary parasitology and public health. The study is relevant given the zoonotic potential of C. parvum and the economic impact of calf diarrhea. 

Major Comments    
The authors stated that the manuscricpt investigated the management practice, this approach required studying the management risk factors, which were not actually done?????
The sampling strategy limits representativeness. The authors should acknowledge this limitation more explicitly and discuss how it may affect prevalence estimates.
Results mention both C. parvum and C. bovis, but the abstract only highlights C. parvum. This inconsistency should be corrected. Moreover, sequencing need to add the gene accession number ??????
Only 62.5% of PCR-positive samples yielded usable sequences. The authors should discuss possible reasons (DNA degradation, inhibitors, low oocyst load) and implications for interpretation.
Although management data were collected, the study presents only descriptive statistics. A more robust statistical analysis (e.g., logistic regression) could identify associations between farm practices and infection rates.
The discussion sometimes repeats results rather than critically analyzing them. Greater emphasis on zoonotic implications, environmental persistence, and comparison with international studies would add value.

Minor Comments
Terms such as “semi-extensive” and “semi-intensive” are used interchangeably; standardize terminology.
Figure 1 should include a scale bar. Tables should be reformatted for clarity (e.g., consistent decimal places, clearer headings).
Some references are cited as numbers but lack full bibliographic details in the provided text. Ensure all references are complete and formatted according to journal guidelines. 

Author Response

Dear Editor and Reviewers, all adjustments are highlighted in yellow on the manuscript at this round of review.  We appreciate all comments provided.

Reviewer 1

The manuscript titled “Prevalence and Molecular Characterization of Cryptosporidium in Diarrheic Dairy Calves: A Descriptive Study in Family Farms of Southern Santa Catarina, Brazil” addresses an important topic in veterinary parasitology and public health. The study is relevant given the zoonotic potential of C. parvum and the economic impact of calf diarrhea.

 

Major Comments

    
The authors stated that the manuscricpt investigated the management practice, this approach required studying the management risk factors, which were not actually done?????

AU: We have clarified this at L-14–16: “This study describes the management practices and evaluates the molecular identification of Cryptosporidium species in dairy calves from southern Santa Catarina, Brazil, an important dairy cattle milk production region” and at L-73–75: “Considering the limited sample size, management data were used only to characterize the farms included in the study, and no risk-factor analysis was carried out”.

The sampling strategy limits representativeness. The authors should acknowledge this limitation more explicitly and discuss how it may affect prevalence estimates.

AU: Thank you for this comment. We agree and have revised the manuscript to more explicitly acknowledge this limitation. The present study was designed as a descriptive observational study, and the limited sample size, together with the sampling strategy, does not allow robust assessment of associations between management practices and Cryptosporidium infection or identification of risk factors. We have added this at L- 300–312: “This study has some limitations that should be considered when interpreting the results. The limited number of farms and animals included, together with the sampling strategy used, may have affected the precision and representativeness of the prevalence estimates. Therefore, the prevalence reported here should be interpreted with caution. In addition, fecal samples were collected only once from each animal, which may have reduced the ability to detect true-positive calves, particularly if oocyst shedding was intermittent or below the detection limit at the time of sampling. Finally, although management information was collected, this was a descriptive observational study and did not have sufficient sample size or statistical power to robustly evaluate associations between management practices and Cryptosporidium infection. For this reason, management data were used only to characterize the sampled farms, and no risk-factor analysis was performed. Future studies with larger sample sizes, repeated sampling, and designs specifically planned to evaluate risk factors are needed”.

Results mention both C. parvum and C. bovis, but the abstract only highlights C. parvum. This inconsistency should be corrected. Moreover, sequencing need to add the gene accession number ??????

AU: Thank you for this comment. You are correct. We have revised the manuscript to correct this inconsistency. Cryptosporidium bovis was removed from the text, as all successfully sequenced samples were identified as C. parvum. The gene accession number was added in L-200: “Genbank submission no. (SUB16279819)”

Only 62.5% of PCR-positive samples yielded usable sequences. The authors should discuss possible reasons (DNA degradation, inhibitors, low oocyst load) and implications for interpretation.

AU: The limited success rate of sequencing (62.5%) from PCR-positive samples can be primarily attributed to the use of a phenol-chloroform extraction method, which, although cost-effective, may not efficiently remove fecal-derived PCR inhibitors such as humic acids and complex polysaccharides that co-precipitate with DNA and interfere with downstream sequencing reactions. Additionally, the samples were stored at −20°C following initial microscopic examination and prior to molecular analysis, a condition that, while suitable for preserving oocyst morphology, may have contributed to partial DNA degradation over time, particularly in samples with inherently low oocyst loads, thereby reducing the quality and yield of amplifiable template for sequencing. The absence of commercial DNA purification kits, which typically incorporate inhibitor removal columns, likely compounded these challenges by allowing inhibitory substances to persist through the extraction process. We have added this at L- 264–268: “The sequencing outcome was likely influenced by technical challenges inherent to molecular analysis of fecal specimens, including the presence of PCR inhibitors, the use of phenolchloroform extraction instead of commercial kits with inhibitor-removal columns, and prolonged storage at −20°C prior to molecular processing [44,45]”.

Although management data were collected, the study presents only descriptive statistics. A more robust statistical analysis (e.g., logistic regression) could identify associations between farm practices and infection rates.

AU: Thank you for this comment. We agree that more robust statistical approaches, such as logistic regression or mixed models, could be useful to investigate associations between management practices and Cryptosporidium infection in studies with larger sample sizes and adequate statistical power. However, the present study was designed as a descriptive observational study, with the main objective of characterizing management practices and identifying Cryptosporidium species in diarrheic dairy calves from small- to medium-sized predominantly family-based farms in southern Santa Catarina.

Considering the limited sample size and the incomplete responses for some questionnaire items, we chose a cautious descriptive approach rather than fitting regression models that could generate unstable estimates, biased associations, or underestimate possible effects. Therefore, management data were used only to describe the farms included in the study, and no risk-factor analysis was performed.

We believe that reporting these descriptive management data together with molecular identification results is still relevant, as it provides baseline information for this region and may help guide future studies with larger sample sizes specifically designed to evaluate risk factors associated with Cryptosporidium infection.

The discussion sometimes repeats results rather than critically analyzing them. Greater emphasis on zoonotic implications, environmental persistence, and comparison with international studies would add value.

AU: Thank you for your comment. We have added this at L- 269 – 285: “The prevalence observed in the present study (48%) is comparable to figures reported in international studies. In Brazil, the intensive dairy farming systems prevalent in several regions have been associated with the circulation of zoonotic C. parvum subtypes. This finding underscores the potential for animal-to-human transmission through environmental contamination and occupational exposure [14,34].This scenario aligns with previous Brazilian studies that identified contamination of water sources on dairy farms with zoonotic C. parvum subtypes, highlighting the role of cattle as reservoirs for environmental contamination and the potential for waterborne transmission to humans [20,31]. In this context, the presence of diarrheic calves shedding high numbers of oocysts represents a significant source of environmental contamination, as these structures can be dispersed through runoff, soil, and untreated water sources commonly used in the studied region [18,38]. Nevertheless, the precise pathways by which oocysts gain access to water sources remain insufficiently characterized in this region. Whether contamination occurs through direct fecal deposition, inadequate manure management, or rainfall-induced runoff has yet to be elucidated. Consequently, further investigations are essential to identify these transmission routes and enable the implementation of effective interruption strategies [20,31]”.

 

 

 

 

 

Minor Comments

Terms such as “semi-extensive” and “semi-intensive” are used interchangeably; standardize terminology.

AU: Thank you for your comment. We have updated to “semi-extensive” throughout the manuscript.

Figure 1 should include a scale bar. Tables should be reformatted for clarity (e.g., consistent decimal places, clearer headings).

AU: We have adjusted the tables and included the scale bar in Figure 1.

Some references are cited as numbers but lack full bibliographic details in the provided text. Ensure all references are complete and formatted according to journal guidelines. 

AU: Thank you for your comment. Updated.

 

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Dear authors,

The manuscript is highly interesting, as it explores the management practices of Cryptosporidium infection in dairy calves and provides molecular identification of the microorganisms in the southern region of Santa Catarina, Brazil.

Line 106- 107: “for 30 seg each 15 min.” is “for 30 sec every 15 min”

Author Response

Reviewer 2

Dear authors,

The manuscript is highly interesting, as it explores the management practices of Cryptosporidium infection in dairy calves and provides molecular identification of the microorganisms in the southern region of Santa Catarina, Brazil.

Line 106- 107: “for 30 seg each 15 min.” is “for 30 sec every 15 min”

AU: Thank you for your comment. Updated.

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

I agree with the modifications made.

Author Response

Reviewer 3

I agree with the modifications made.

AU: Thank you for your consideration.

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors did a good job of addressing all comments successfully

The manuscript is acceptable for publication

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Mapping and Identification of Cryptosporidiosis in Dairy 2 Calves from the Southern Santa Catarina, Brazil” presented by Drescher et al. provides an information the molecular epidemiology and species identification of Cryptosporidium infection in dairy calves from the southern region of Santa Catarina, Brazil. The authors said that they have conducted molecular epidemiological studies on the parasite cryptosporidium. However, there are major concern with the study  design, statistical analysis and result presentation .

Major comments

• The type of epidemiological study is not clear and the presentation here is not correct. How could you make a random selection of the farms?
• How could you determine the sample size. The sample size is very small ???????
• Because you want to make a cross-sectional study on cryptosporidiosis in farms with no previous history of the disease, why you only selected diarrheic calves. As is known, the parasite can be found in claves without clinical symptoms.
• The age range of diseases is very wide as the parasite mainly present in the newborn calves
• In the results, authors mentioned management as a factor related to infection without previous establishment of statistical analysis.
• The study devoid mention of risk factors and related statistical analysis to say “epidemiological study”
• The authors examined calves for cryptosporidium oocysts, authors are asked to categorize the severity of infectin according to the number of oocysts/ fields.
• Authors are asked to use contingency table analysis to establish the factors associated with cryptosporidium infection
• Where is the phylogenic analysis of the cryptosporidium parvum???

 

Minor comments

The manuscript as a whole should be revised for syntax and grammatical errors.

Poor resolution of the figure 1.

Table 2 is not needed and could be replaced be a text including the frequency of C. parvum within farms.

Discussion should be rewritten again in depth.

Reviewer 2 Report

Comments and Suggestions for Authors

The study is very interesting, investigating the positivity in infected dairy calves by Cryptosporidium and the identification of the species. However, there are some major issues.

Line 13: To have a molecular epidemiology, you have to perform PCR on the total of the samples, not only the positive samples in the microscopy examination.

Lines 85-87: Please be more specific about the conditions, and mainly about the time that the microscopic examination was performed regarding the refrigeration of the samples.

Lines 100-102: Additionally, we previously said that you froze only the positive at the microscopic examination for Cryptosporidium samples. Why did you not perform PCR on all samples?

Lines 136-137: You write that you used as a positive control, a positive for Cryptosporidium in coprological examination. I would like to clarify if the sample was one of the samples collected for the present study. When we use a positive control, we do not use one of the samples of the current study.

Lines 161- 162: “68.18% (15/22) tested positive for Cryptosporidium.” In which examination, and why /22 samples?

Reviewer 3 Report

Comments and Suggestions for Authors

1. What do you mean by “mapping” in the study title?

2. What formula was used to determine the sample size and what sampling approach was applied? (This is not clear.)

3. The sample size is too small.

4. There are no geographic references.

5. There are studies from the same area on the same parasite with a larger sample size.

The comments are attached in the manuscript PDF.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

There are some paragraphs that need to be improved in terms of language.