Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Comments

The present work aimed to investigate the molecular occurrence of hemoplasmas in equids from Croatia. Although the manuscript is well-written, additional PCR assays followed by sequencing and phylogenetic inferences are needed to make this report more scientifically-sound.

Specific Comments

• Lines 20 and 28: live on the surface of red blood cells

• Line 134 and throughout the MS: Mycoplasma should be italicized (except when following the word Candidatus

Line 165: Did the authors perform red blood cells counting on the positive animal? What about the results of blood smears?

- What was the size of obtained sequence? Please, present the BLASTn results, including best match %, E-value, and query cover.

- Authors should run additional PCR assays targeting the near-complete or a larger fragment of the 16S rRNA. Moreover, authors should run a PCR targeting the 23S rRNA and sequencing the obtained amplicon.

- Phlylogenetic analyses based on both near-complete 16S rRNA and partial 23S rRNA should be performed in order to make this report more robust and scientifically sound.

Please, deposite  the obtained sequences in Genbank database and provide the accession number.

- Line 196: hematophagous arthropod 

• Was the positive horse parasitized by ticks at the time of blood sampling?

Lines 199-210: That’s the reason why authors should perform additional PCR assays. Phylogenetic analyses should be performed with larger fragments of the 16S rRNA, aiming at obtaining more accurate phylogenetic positioning. In addition, another molecular marker (e.g. 23S rRNA) will help in untangling the phylogenetic positioning of hemoplasmas in non-usual hosts, such as horses and donkeys.

- What was the size of the sequnces used by [8] in the phylogenetic analyses? Please, discuss that.

Line 226: A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in Bradypus tridactylus ( https://doi.org/10.1111/tbed.14523)

- Scientific names should be italicized throghout the MS, including those presented at the reference list.

- When Candidatus, only the word Candidatus should be italicized and the Candidatus species should appear between ‘’

Author Response

The present work aimed to investigate the molecular occurrence of hemoplasmas in equids from Croatia. Although the manuscript is well-written, additional PCR assays followed by sequencing and phylogenetic inferences are needed to make this report more scientifically-sound.

Response: We thank the reviewer for this constructive comment. We acknowledge that phylogenetic analyses would further strengthen the scientific robustness of the study. However, the primary aim of the present work was to investigate the molecular occurrence of hemoplasmas in equids from Croatia rather than to perform detailed phylogenetic inference. To increase the reliability of species identification, we performed additional PCR assays targeting the 23S rRNA gene for all animals, in addition to the initial 16S rRNA screening. While this approach allowed confident molecular identification, more extensive sequencing and phylogenetic analyses were beyond the scope of the current study and are addressed as perspectives for future research in the manuscript.

Specific Comments

• Lines 20 and 28: live on the surface of red blood cells

Response: Thank you for noticing this. The text has been corrected to specify that hemoplasmas live on the surface of red blood cells.

• Line 134 and throughout the MS: Mycoplasma should be italicized (except when following the word Candidatus

Response: All occurrences of Mycoplasma and species names have been checked and corrected to italic format throughout the manuscript and reference list.
Candidatus is now formatted correctly (only Candidatus italicized; species name in quotation marks).

Line 165: Did the authors perform red blood cells counting on the positive animal? What about the results of blood smears?

Response: We thank the reviewer for this comment. Blood smear examination was performed, whereas red blood cell counts were not assessed, as all animals were clinically examined at the time of sampling and did not exhibit any clinical signs consistent with anemia. The primary objective of the study was the molecular detection and identification of hemotropic mycoplasmas rather than the evaluation of hematological parameters.

- What was the size of obtained sequence? Please, present the BLASTn results, including best match %, E-value, and query cover.

Response: The obtained 16S rRNA fragment was approximately 572 bp in length, while the 23S rRNA fragment was 831 bp. A summary of the BLASTn results, including percentage identity, query coverage and E-value for the best matches, has been added to the Results section.

 - Authors should run additional PCR assays targeting the near-complete or a larger fragment of the 16S rRNA. Moreover, authors should run a PCR targeting the 23S rRNA and sequencing the obtained amplicon.

Response:We thank the reviewer for this recommendation. As suggested, we performed an additional PCR assay targeting a ~800 bp fragment of the 23S rRNA gene using primers 23S_HAEMO_F and 23S_HAEMO_R according to Mongruel et al. (2022).
The amplicon was successfully sequenced and confirmed Mycoplasma wenyonii, supporting the initial 16S rRNA result. These new data have been incorporated in the Methods and Results.

- Phlylogenetic analyses based on both near-complete 16S rRNA and partial 23S rRNA should be performed in order to make this report more robust and scientifically sound.

Response: To ensure the robustness and reproducibility of our findings, the positive sample was re-amplified in duplicate and independently re-sequenced, yielding identical results. Furthermore, all samples were additionally screened using a PCR protocol targeting an approximately 800 bp fragment of the 23S rRNA gene, employing the primers 23S_HAEMO_F (5′-TGAGGGAAAGAGCCCAGAC-3′) and 23S_HAEMO_R (5′-GGACAGAATTTACCTGACAAGG-3′), as described by Mongruel et al. (2022). Although this protocol resulted in amplification in 24 samples, none of the obtained amplicons could be confirmed by sequencing. These findings indicate a lack of sufficient specificity of the primers used in the alternative protocol for the detection of hemotropic mycoplasmas in the investigated host species. After repeating all experiments, the results originally reported in the manuscript were consistently reproduced and therefore considered reliable.

This section has been expanded with an explanation that the use of additional markers improves confidence in species identification, especially in non-typical hosts such as equids.

We consider that phylogenetic analysis would not provide additional insight into the infection of horses with Mycoplasma wenyonii, as the phylogenetic position of this species is already well established. Therefore, the inclusion of further phylogenetic analyses is unlikely to contribute substantially to the interpretation of the present findings

Please, deposite  the obtained sequences in Genbank database and provide the accession number.

Response: The obtained sequences have been deposited in GenBank. Accession numbers have been added to the manuscript.

Added sentence: The sequences were submitted to GenBank under the accession numbers 16S rRNA: PX832228 and 23S rRNA: PX797409

- Line 196: hematophagous arthropod 

Was the positive horse parasitized by ticks at the time of blood sampling?

Response: The positive horse was not parasitized by ticks at the time of sampling. This information has been added to the material& methods and Results section.

Added sentences: All animals were examined for the presence of ectoparasites, including ticks, at the time of blood sampling.

No ticks were detected in any of the infected mare.

Lines 199-210: That’s the reason why authors should perform additional PCR assays. Phylogenetic analyses should be performed with larger fragments of the 16S rRNA, aiming at obtaining more accurate phylogenetic positioning. In addition, another molecular marker (e.g. 23S rRNA) will help in untangling the phylogenetic positioning of hemoplasmas in non-usual hosts, such as horses and donkeys.

Response: To ensure the robustness and reproducibility of our findings, the positive sample was re-amplified in duplicate and independently re-sequenced, yielding identical results. Furthermore, all samples were additionally screened using a PCR protocol targeting an approximately 800 bp fragment of the 23S rRNA gene, employing the primers 23S_HAEMO_F (5′-TGAGGGAAAGAGCCCAGAC-3′) and 23S_HAEMO_R (5′-GGACAGAATTTACCTGACAAGG-3′), as described by Mongruel et al. (2022). Although this protocol resulted in amplification in 24 samples, none of the obtained amplicons could be confirmed by sequencing. These findings indicate a lack of sufficient specificity of the primers used in the alternative protocol for the detection of hemotropic mycoplasmas in the investigated host species. After repeating all experiments, the results originally reported in the manuscript were consistently reproduced and therefore considered reliable.

This section has been expanded with an explanation that the use of additional markers improves confidence in species identification, especially in non-typical hosts such as equids.

We thank the reviewer for the comment; however, we consider that phylogenetic analysis would not provide additional insight into the infection of horses with Mycoplasma wenyonii, as the phylogenetic position of this species is already well established. Therefore, the inclusion of further phylogenetic analyses is unlikely to contribute substantially to the interpretation of the present findings

- What was the size of the sequnces used by [8] in the phylogenetic analyses? Please, discuss that.

Line 226: A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in Bradypus tridactylus ( https://doi.org/10.1111/tbed.14523)

Response: The suggested reference has been cited and the information has been incorporated into the manuscript accordingly.

 Scientific names should be italicized throghout the MS, including those presented at the reference list.

Response: Corrected throughout the manuscript, including the reference list.

 When Candidatus, only the word Candidatus should be italicized and the Candidatus species should appear between ‘’

Response: Corrected throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Thank you very much for providing this interesting manuscript on mycoplasma in horses.

 

Simple Summary: Please include the numbers of horses and donkeys

 

Introduction:

Line 50: which arthropodes? Please be more specific

 

Material and Methods:

Maybe include some subtitels to make this part clearer

 

Only 26 donkeys were included in the study. Is this number of animals high enough to assume that donkeys do not host hemoplasmas? Did you do some power calculations beforehand to decide on the group size?

 

The results section appears somewhat short. Maybe more specific information on the results could be included.

Were there any difficulties encountered (also with the PCRs?)?

 

There is no title for the figure. Please add this.

Does each dot correspond to one horse?

 

Discussion:

Line 180f: „…robust evidence for the apparent absence of equid-specific hemoplasmas“ – how can there be absence if the prevalence is in some studies up to 26.5%. Since you only examined one country of 194, you cannot assume that there is absence in all countries and regions. Maybe there is a higher prevalence in other regions? There are several diseases that exist in some parts of the world (e.g. Ehrlichia equi – Potomac horse fever)

Author Response

Thank you very much for providing this interesting manuscript on mycoplasma in horses.

 Response: We thank the reviewer for their positive and encouraging comment on our manuscript.

Simple Summary: Please include the numbers of horses and donkeys

 Response: Numbers have been included in the revised Simple Summary (817 horses and 26 donkeys).

Introduction:

Line 50: which arthropodes? Please be more specific

Response: We clarified the description by specifying hematophagous arthropods 

Material and Methods:

Maybe include some subtitels to make this part clearer

Response: Subsections have been inserted to improve clarity (2.1. Study Area, Animals and Study Design, 2.2. Sample Collection and DNA Extraction).

Only 26 donkeys were included in the study. Is this number of animals high enough to assume that donkeys do not host hemoplasmas? Did you do some power calculations beforehand to decide on the group size?

Response: We agree that the number of donkeys is limited. This has been acknowledged as a limitation in the

The results section appears somewhat short. Maybe more specific information on the results could be included.

Response: Results have been expanded to include amplicon sizes, BLAST results, and findings from the repeated and additional PCR assays. The sequences were submitted to GenBank under the accession numbers 16S rRNA: PX832228 and 23S rRNA: PX797409

 

Were there any difficulties encountered (also with the PCRs?)?

Response: A statement was added indicating that no technical difficulties were encountered apart from expected low DNA yield in hemoplasma-negative samples. All samples were additionally screened using a PCR protocol targeting an approximately 800 bp fragment of the 23S rRNA gene, employing the primers 23S_HAEMO_F (5′-TGAGGGAAAGAGCCCAGAC-3′) and 23S_HAEMO_R (5′-GGACAGAATTTACCTGACAAGG-3′), as described by Mongruel et al. (2022). Although this protocol resulted in amplification in 24 samples, none of the obtained amplicons could be confirmed by sequencing. These findings indicate a lack of sufficient specificity of the primers used in the alternative protocol for the detection of hemotropic mycoplasmas in the investigated host species. In contrast to the initial PCR protocol targeting the 16S rRNA gene fragment, which showed no amplification-related issues

 

There is no title for the figure. Please add this.

Response: We thank the reviewer for this important comment. We added title and description

Figure 1. The map illustrates the sampling locations of horses and donkeys, providing an overview of the spatial distribution of samples and demonstrating coverage of all regions of Croatia. Dots represent sampling sites rather than individual animals.

Does each dot correspond to one horse?

 Response: A title and legend have been added. Each dot represents location, not a single  horse.

Discussion:

Line 180f: „…robust evidence for the apparent absence of equid-specific hemoplasmas“ – how can there be absence if the prevalence is in some studies up to 26.5%. Since you only examined one country of 194, you cannot assume that there is absence in all countries and regions. Maybe there is a higher prevalence in other regions? There are several diseases that exist in some parts of the world (e.g. Ehrlichia equi – Potomac horse fever)

Response: We thank the reviewer for this important comment. We agree that the term ‘absence’ must be interpreted with caution. Our intention was not to suggest a complete global absence of hemotropic Mycoplasma infections in horses. Rather, we aimed to emphasize the lack of robust evidence for the existence of equid-specific hemoplasma species. In several studies, hemotropic Mycoplasma spp. were not detected in horses at all (Valente et al. [13], Altay et al. [14], Ferreira et al.19), while in other investigations, Mycoplasma species detected in horses were phylogenetically related to non-equid hosts and not considered equid-adapted species. This issue is discussed in more detail in the Discussion section.

We fully acknowledge that our study is geographically limited to a single country and that regional differences in prevalence cannot be excluded. However, the available molecular evidence to date suggests that horses most likely act as incidental rather than natural hosts for hemotropic mycoplasmas.

Furthermore, comparison with Neorickettsia risticii (formerly Ehrlichia risticii), the causative agent of Potomac horse fever, should be made with caution, as this organism is an obligate intracellular Gram-negative bacterium with a complex life cycle involving trematodes as endosymbionts, which is fundamentally different from the biology and transmission of hemotropic Mycoplasma spp.

Reviewer 3 Report

Comments and Suggestions for Authors

The Authors describe the results of a molecular screening for hemoplasmas (HM) among an extensive population of croatian equids (817 horses and 26 donkeys) characterized by different geographical origin, age, and husbandry practice. The study highlights a very low HM prevalence with only one horse infected by Mycoplasma wenyonii. The manuscript is well written with a clear description of materials, methods and results. I have these two major concerns: 1) Considering that the observed prevalence of HM in Croatia (0.12%) is far less respect countries, especially  Germany (26.5%), have the Authors ever supposed that this may depend on the method or the target region of 16SrRNA chosen, different from those adopted in other surveys? Anyway, I suggest strongly to add in lines 213-218 or 232-233 this hypothesis among those listed in order to explain the extremely low prevalence of HM in Croatia (it would also be interesting to confirm the results using different PCR protocols); 2) Among the Methods it is stated that direct microscopic examination was used: methodology and results of such analysis are lacking (there were discrepancies between PCR and microscopic method, especially for the horse positive for Mycoplasma wenyonii? Remember to address briefly the results of microscopic analysis, if added, in Discussion chapter).

Minor concerns:

From line 46: if the acronym HM is adopted for refererring to hemotropic mycoplasmas, as made in the following line 53, it’s advisable to use it thorought all the text (line 61, 67, 75, 149 etc…; check everywhere it’s necessary);

Line 134: Mycoplasma genus, here and in all references (pages 7-8), in italic;

Page 4: Add title and legend for the Figure;

Line 159: in Methods (line 124) it seems that only one blood msample was taken from each animal while here it’s stated that the horse positive was sampled twice, in April (for PCR or microscopic examination or both?) and July (for PCR). I suggest to clear in Methods chapter this point, in other words if this double sampling was made only for this animal or also for others in the study;

Lines 165 and 168: M.wenyonii in italic;

Lines 245-247 “For research…should be used”; I believe that this sentence should be deleted as it’s only a rule (check with the Journal).

Author Response

The Authors describe the results of a molecular screening for hemoplasmas (HM) among an extensive population of croatian equids (817 horses and 26 donkeys) characterized by different geographical origin, age, and husbandry practice. The study highlights a very low HM prevalence with only one horse infected by Mycoplasma wenyonii. The manuscript is well written with a clear description of materials, methods and results. I have these two major concerns: 1) Considering that the observed prevalence of HM in Croatia (0.12%) is far less respect countries, especially  Germany (26.5%), have the Authors ever supposed that this may depend on the method or the target region of 16SrRNA chosen, different from those adopted in other surveys? Anyway, I suggest strongly to add in lines 213-218 or 232-233 this hypothesis among those listed in order to explain the extremely low prevalence of HM in Croatia (it would also be interesting to confirm the results using different PCR protocols); 2) Among the Methods it is stated that direct microscopic examination was used: methodology and results of such analysis are lacking (there were discrepancies between PCR and microscopic method, especially for the horse positive for Mycoplasma wenyonii? Remember to address briefly the results of microscopic analysis, if added, in Discussion chapter).

Response: We thank the reviewer for this insightful comment. We have added a paragraph in the Discussion acknowledging that differences in prevalence between countries may be influenced by PCR target choice, amplicon length, and diagnostic sensitivity. We now explicitly include this as one of the hypotheses explaining the low prevalence observed in Croatia.

All samples were additionally screened using a PCR protocol targeting an approximately 800 bp fragment of the 23S rRNA gene, employing the primers 23S_HAEMO_F (5′-TGAGGGAAAGAGCCCAGAC-3′) and 23S_HAEMO_R (5′-GGACAGAATTTACCTGACAAGG-3′), as described by Mongruel et al. (2022).

Results: No mycoplasmas on the surface of erythrocytes were detected in the examined blood smears.

Discussion: In contrast to successful PCR detection, microscopic examination of blood smears did not reveal the presence of mycoplasmas. This discrepancy is most likely attributable to a low level of parasitemia, which is common in subclinical or chronic infections. These findings underscore the higher sensitivity of molecular methods for the detection of low-intensity infections and support the use of PCR as the method of choice for epidemiological studies and surveillance of hemotropic mycoplasmas.

Minor concerns:

From line 46: if the acronym HM is adopted for refererring to hemotropic mycoplasmas, as made in the following line 53, it’s advisable to use it thorought all the text (line 61, 67, 75, 149 etc…; check everywhere it’s necessary);

Response: The acronym HM has now been used consistently throughout the manuscript to refer to hemotropic mycoplasmas, as suggested. All relevant sections have been carefully checked and corrected accordingly.

Line 134: Mycoplasma genus, here and in all references (pages 7-8), in italic;

Response: Mycoplasma genusi has been italicized throughout the manuscript, as suggested.

 

Page 4: Add title and legend for the Figure;

Line 159: in Methods (line 124) it seems that only one blood msample was taken from each animal while here it’s stated that the horse positive was sampled twice, in April (for PCR or microscopic examination or both?) and July (for PCR). I suggest to clear in Methods chapter this point, in other words if this double sampling was made only for this animal or also for others in the study;

Reponse Microscopic examination is now described in Methods.
No hemotropic organisms were observed; this is stated in the Results and addressed briefly in the Discussion.

Lines 165 and 168: M.wenyonii in italic;

Response: Mycoplasma wenyonii has been italicized throughout the manuscript, as suggested.

Lines 245-247 “For research…should be used”; I believe that this sentence should be deleted as it’s only a rule (check with the Journal).

Response: The sentence in lines 245–247 has been removed in accordance with the reviewer’s recommendation

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors followed the majority of this reviewer suggestions.