Oxidative Stress and Inflammatory Responses in Horses Naturally Infected with Theileria equi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Vito Biondi and others is focused on a study of biochemical markers and markers of oxidative stress in the blood of horses naturally infected with Theileria equi. Although the authors generally demonstrate an increase in oxidative and inflammatory markers after the infection, significant revisions are necessary to ensure the manuscript is scientifically sound and the conclusions are fully based on the significant results obtained. Therefore, I believe the manuscript needs major revision.

1. Lines 90-92 "Given the exploratory nature of the study and the relatively limited sample size, no adjustment for multiple comparisons was applied. Therefore, the findings should be interpreted with appropriate caution and considered hypothesis-generating rather than confirmatory."

I would like to point out to the authors that only statistically significant results supported by a sufficient level of statistical significance are considered reliable; otherwise, they cannot be presented in the conclusions and abstract. I recommend reviewing everything and removing or rephrasing strong conclusions throughout the manuscript based on results that do not withstand multiple comparison correction where necessary.

 

2. The manuscript lacks some some important details, in particular, the infection level, duration of infection in the animals, or age of the animal. High variance within the group of infected animals can likely be explained using these variables. While it is difficult to determine the duration of infection in naturally infected animals, the degree of infection (weak or low) could be quantified, for example, by antigen or antibody levels in the blood. This could further subdivide the groups into several subgroups, which could possibly explain the high intragroup variation in marker values and improve correlations. I recommend the authors do this.
3. The abstract contains too many abbreviations. I recommend removing those that are repeated no more than once, such as on lines 19 and 20.
4. Line 320: "…clinical history, hematological and biochemical findings, serological testing by indirect fluorescent antibody test (IFAT), and molecular detection by PCR." Please provide the parameters for group division, the size of the infected and uninfected animal groups, and a table. Such descriptions would much improve the Materials and Methods chapter.

Author Response

Dear Reviewer,

Thank you very much for your careful evaluation of our revised manuscript and for your positive comments regarding the improvements made. We greatly appreciate the additional observations and suggestions, which have helped us further improve the accuracy and clarity of the manuscript. All comments have been carefully addressed as detailed below.

R. Lines 90–92: "Given the exploratory nature of the study and the relatively limited sample size, no adjustment for multiple comparisons was applied..." I would like to point out to the authors that only statistically significant results supported by a sufficient level of statistical significance are considered reliable; otherwise, they cannot be presented in the conclusions and abstract. I recommend reviewing everything and removing or rephrasing strong conclusions throughout the manuscript based on results that do not withstand multiple comparison correction where necessary.

A. We sincerely thank the Reviewer for this important comment. In response to this suggestion, we have revised our statistical analysis by applying the Benjamini–Hochberg false discovery rate (FDR) correction to account for multiple comparisons. The Statistical Analysis section has been updated accordingly, and all statistical results have been re-evaluated using the adjusted p-values. Following this reanalysis, we carefully reviewed the Abstract, Results, Discussion, and Conclusions to ensure that all interpretations are supported only by findings that remained statistically significant after FDR correction. Where appropriate, statements have been revised or moderated to avoid overinterpretation of results that did not retain statistical significance after adjustment. We believe that these revisions strengthen the statistical robustness of the study while maintaining the exploratory nature of the investigation. The revised manuscript now presents conclusions that are fully supported by the corrected statistical analyses.

R. The manuscript lacks some important details, in particular, the infection level, duration of infection in the animals, or age of the animal. High variance within the group of infected animals can likely be explained using these variables. While it is difficult to determine the duration of infection in naturally infected animals, the degree of infection (weak or low) could be quantified, for example, by antigen or antibody levels in the blood. This could further subdivide the groups into several subgroups, which could possibly explain the high intragroup variation in marker values and improve correlations. I recommend the authors do this.

A. We sincerely thank the Reviewer for this thoughtful and constructive suggestion. The age of all enrolled horses has now been reported (see line 87). As correctly noted by the Reviewer, because this study was based on naturally occurring infections, the duration of infection could not be reliably established. Regarding infection burden, all infected horses were seropositive by indirect fluorescent antibody test (IFAT), with antibody titers ranging from 1:50 to 1:1600 (see lines 87-90). However, these titers were used exclusively to confirm exposure and are not considered reliable quantitative indicators of parasite burden or disease severity in equine theileriosis. Likewise, peripheral blood smear examination was performed to estimate parasitemia but, because of its limited sensitivity in chronically infected horses, it was not considered suitable for quantitative stratification. Unfortunately, quantitative PCR, which would have allowed accurate estimation of parasite load and subgroup analysis according to infection intensity, was not available for the present study. Consequently, subdivision of infected horses based on parasite burden was not feasible. To address this important point, we have expanded the Discussion to acknowledge this limitation and to highlight that future longitudinal studies combining quantitative molecular assays with serial clinical evaluations will be essential to investigate the relationship between parasite load, disease progression, oxidative stress, and inflammatory biomarkers.

R. The abstract contains too many abbreviations. I recommend removing those that are repeated no more than once, such as on lines 19 and 20.

A. We thank the Reviewer for this useful suggestion. The Abstract has been revised to reduce the number of abbreviations. Abbreviations used only once or infrequently have been replaced with their full terms to improve readability while retaining only the most commonly used abbreviations throughout the manuscript.

R. Line 320: "...clinical history, hematological and biochemical findings, serological testing by indirect fluorescent antibody test (IFAT), and molecular detection by PCR." Please provide the parameters for group division, the size of the infected and uninfected animal groups, and a table. Such descriptions would much improve the Materials and Methods chapter.

A. We thank the Reviewer for this valuable suggestion. The Materials and Methods section has been substantially revised to provide a more detailed description of the study design and group allocation criteria. Specifically, we have clarified the inclusion and exclusion criteria, the diagnostic parameters used to classify horses as infected or healthy controls, and the clinical, hematological, biochemical, serological (IFAT), and molecular (PCR) findings considered for group assignment. We have also specified the number of animals included in each group (35 infected and 20 healthy control horses) and added a new table (Table 2) summarizing the characteristics of the study population, the group distribution, and the classification criteria. We believe that these additions substantially improve the transparency and reproducibility of the study design.

On behalf of all the authors

Dr. Fabio Bruno

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates oxidative stress and inflammatory responses in horses naturally infected with Theileria equi. The manuscript is generally well written, and the Discussion appropriately places the findings into the context of previous literature. 

However, there are some major and minor comments.

Introduction

• The causative agents of equine piroplasmosis are Theileria equi and Babesia caballi, not only equi . It is better to say “Theileriosis” instead of Equine piroplasmosis

 

 

Results

• The variables in Figures 1, 2 and 3 should be separate. The units of the variables are differential, and the numbers on the y-axis do not represent the values of the variables. On the y-axis, the units should be mentioned. In addition, the data should first be presented in tables with means/ medians and p-values.
• Figure 4: the “*” is on the p-value
• In addition, the explanation of the abbreviations of the groups, hematological and biochemical variables, inflammatory, and oxidative stress markers should be included in the footnotes.
• Table 1: “*” and “**” are better to be explained only in the footnote, not in the title.
• Table 1: What is CS?
• There are no data about the results of blood smear microscopy, IFAT, and PCR.

 

Statistical analysis

• This section should remove in the section “Materials and methods”
• How did you decide the number of horses in each group?

Materials and Methods

• In which locations were the horses living?
• Why is the number of horses in the control group different from the number in the group with the infected horses?
• Were infected horses considered horses that were positive in at least one of the methods (blood smear, IFAT, PCR) or in all of them?
• The name of the hematological analyzer, please.
• Please provide the manufacturer information for all the commercial kits.
• What were the inclusion and exclusion criteria for this research?
• Did you examine the samples for other pathogens?
• For the ELISA assays, you do not have to explain all the steps, as you state that you performed the analysis according to the manufacturers’ instructions.

General

• In the figures you write “Teileria equi”. It is “Theileria equi”.
• Abbreviations should be defined only once, at the beginning, and then used consistently.

 

References

• References are duplicated:

5 and 38, 6 and 39, 21 and 41, 22 and 42, 24 and 44, 31 and 45

Please remove duplicate entries and renumber the references accordingly.

Author Response

Dear Reviewer,

Thank you very much for your careful evaluation of our revised manuscript and for your positive comments regarding the improvements made. We greatly appreciate the additional observations and suggestions, which have helped us further improve the accuracy and clarity of the manuscript. All comments have been carefully addressed as detailed below.

Introduction

R. The causative agents of equine piroplasmosis are Theileria equi and Babesia caballi, not only T. equi. It is better to say “Theileriosis” instead of Equine piroplasmosis.

A. We thank the Reviewer for this important observation. We have replaced the term "equine piroplasmosis" with "theileriosis" where appropriate throughout the manuscript to improve accuracy.

Results

R. The variables in Figures 1, 2 and 3 should be separate. The units of the variables are different, and the numbers on the y-axis do not represent the values of the variables. On the y-axis, the units should be mentioned. In addition, the data should first be presented in tables with means/medians and p-values.

A. We appreciate this valuable suggestion. Figures 1–3 have been completely revised by separating the variables according to their respective units and biological categories. A new figure has been created, including the different variables. The y-axes now report the appropriate measurement units for each variable. Furthermore, we have added new tables presenting the descriptive statistics (mean ± SD or median and interquartile range, as appropriate) together with the corresponding p-values, while the figures are now intended to provide a visual representation of these data.

R. Figure 4: the “” is on the p-value.*

A. Thank you for pointing out this formatting error. Figure 4 has been modified and improved.

R. The explanation of the abbreviations of the groups, hematological and biochemical variables, inflammatory, and oxidative stress markers should be included in the footnotes.

A. We agree with the Reviewer. Explanations of all abbreviations used for study groups, hematological and biochemical parameters, inflammatory markers, and oxidative stress markers have been added to the corresponding figure and table footnotes.

R. Table 1: “” and “**” are better explained only in the footnote, not in the title.*

A. We thank the Reviewer for this suggestion. The title of Table 1 has been simplified, and the explanations of the statistical symbols (* and **) have been moved to the table footnote.

R. Table 1: What is CS?

A. We apologize for the lack of clarity. The abbreviation "CS" has now been defined.

R. There are no data about the results of blood smear microscopy, IFAT, and PCR.

A. We thank the Reviewer for highlighting this omission. We have now included a summary of the diagnostic results obtained by blood smear microscopy, IFAT, and PCR in the Results section, together with the corresponding descriptive data (see lines 87-89).

Statistical Analysis

R. This section should be moved to Materials and Methods.

A. We agree with the Reviewer. The Statistical Analysis section has been moved to the Materials and Methods section.

R. How did you decide the number of horses in each group?

A. We thank the Reviewer for this important question. The study was designed as an observational investigation on naturally infected horses. Therefore, the sample size was determined by the availability of animals fulfilling the predefined inclusion criteria during the study period rather than by an a priori sample size calculation. This information has now been clarified in the Materials and Methods section (see lines 360-364).

Materials and Methods

R. In which locations were the horses living?

A. We thank the Reviewer for this comment. The geographical origin and housing locations of the enrolled horses have now been specified in the Materials and Methods section (see lines 357-359).

R. Why is the number of horses in the control group different from the number in the infected group?

A. We appreciate this observation. The difference in sample size reflects the availability of healthy horses meeting the inclusion criteria during the study period. This has now been clarified in the revised manuscript (see lines 360-364).

R. Were infected horses considered horses that were positive in at least one of the methods (blood smear, IFAT, PCR) or in all of them?

A. We thank the Reviewer for requesting this clarification. The diagnostic criteria used to classify infected horses have now been clearly specified in the Materials and Methods section, indicating the combination of diagnostic tests required for inclusion in the infected group (see lines 369-375).

R. The name of the hematological analyzer, please.

A. Thank you for this suggestion. The manufacturer and model of the hematological analyzer have now been reported in the Materials and Methods section.

R. Please provide the manufacturer information for all the commercial kits.

A. We agree with the Reviewer. Manufacturer names, cities, and countries have now been provided for all commercial assay kits used in this study.

R. What were the inclusion and exclusion criteria for this research?

A. We thank the Reviewer for this important suggestion. The inclusion and exclusion criteria have now been described in detail in the Materials and Methods section (see 379-385).

R. Did you examine the samples for other pathogens?

A. We thank the Reviewer for this valuable comment. To minimize potential confounding effects, all enrolled horses were screened at the time of enrolment for the principal infectious agents included in the differential diagnosis. In particular, Babesia caballi and Anaplasma phagocytophilum infections were excluded by indirect fluorescent antibody test (IFAT). Therefore, the clinical and laboratory findings observed in the study population were attributed to Theileria equi infection. This information has been added to the Materials and Methods section of the revised manuscript (see lines 365-368).

R. For the ELISA assays, you do not have to explain all the steps, as you state that you performed the analysis according to the manufacturers’ instructions.

A. We appreciate this suggestion. The description of the ELISA procedures has been shortened, retaining only the essential methodological information while stating that all assays were performed according to the manufacturers' instructions.

R. In the figures you write “Teileria equi”. It is “Theileria equi”.

A. We thank the Reviewer for identifying this typographical error. The spelling has been corrected throughout all figures.

R. Abbreviations should be defined only once, at the beginning, and then used consistently.

A. We agree with the Reviewer. The manuscript has been carefully revised to ensure that each abbreviation is defined at its first appearance and subsequently used consistently throughout the text.

References

R. References are duplicated: 5 and 38, 6 and 39, 21 and 41, 22 and 42, 24 and 44, 31 and 45. Please remove duplicate entries and renumber the references accordingly.

A. We thank the Reviewer for carefully checking the reference list. The duplicate references have been removed, and the remaining references have been renumbered accordingly throughout the manuscript.

On behalf of all the authors

Dr. Fabio Bruno

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors carefully addressed all my comments, therefore I do not have any further suggestions

Author Response

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for taking the time to review our revised version. We greatly appreciate the positive feedback and we are pleased that the revisions have satisfactorily addressed all of the Reviewer’s comments.

Kind regards,

On behalf of all the authors

Dr. Fabio Bruno