-->PONE-D-26-07402-->-->The etiological role of the yeast-like fungus Candida metapsilosis in the development of chronic pododermatitis in captive steppe eagles-->-->PLOS One

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: N/A

Reviewer #4: N/A

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #2: Yes

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Reviewer #1: Dear Author

Thank you for submitting this work interesting both from medical mycology and veterinary point of view

I have a few minors points that need correction

I have however a few major points that need to be adressed before article potential acceptation

Minor points

Be careful to write all Genera and species in italic (see L70 Staphyloccocus aureus and check all the manuscript)

Be more straightforward in your title : yeast-like fungis C. metapsilosis = yeast C. metapsilosis

L96 : would have been informative if you were more precise on what antimicrobials were used. Here we can only guess about antibiotics but which ones ?

L139 : Levomekol is a commercial name. Be more precise (the main component is chloramphenicol which also adresses my point above)

L162 : seeding at 28°C. OK this is acceptable. But pathogenic yeasts can also grow at higher temperature. Why haven't you tried 30 or 35°C . This would have strenghtened your discussion about thermotolerance -line 303-

some pictures especially from macro and micro cultures are blurry.

Now the major issues

The MM for the biochemical analyses is lacking. We dont' know how your performed them. Household approaches ? , API20 Candida ? ID32 C ?

The main issue is your conclusion based on BLAST results

1- some of your ITS sequences are very short, too short for a successful sequencing. A correct ITS sequening is supposed to give your between 700/750 to 850/900 bp. Not around 250. Futhermore you do not explain if you have only a forward, reverse or both sequencing. Usually, in front of unknown colonies, a forward AND reverse ITS is wanted.

2- this has a direct impact on your species identification

While your PV945930.1 has a lenght of 750 pb, a 100% coverage E0.0 and 99.28% of identity (meaning that it is a C.metapsilosis) the same cannot be said with your other sequences

PV981955. has a lenght of 239 pb, a E which is not 0.0 and an identity of 95.44%. Below 98 (or 99% if we want to be conservative) it is unlikely to be the correct specie blasted. Correct genera, ok, but the specie identification is highly questionable. Which impacts your whole discussion

The same can be said for the PV945931.1 sequence. The lenght and results are a bit better but you are in a grey area (96.13% of identity)

3-Figure 7 : this is not a CLSI result. Either you use antifungal discs (not a golden standard method) or microdilution method (CLSI-golden laboratory standard). I'd advise to present CLSI results.

There are quite a few conceptual sentences that are misleading. Probably lost in translation

L41 Our results indicate the emergence of new pathogenic yeast strains : well, as it is written, C.metapsilosis is new. Which is not the case. refer to sentence L31 which is way more clearer

same for sentence here : Candida metapsilosis was first isolated from wild steppe eagles of the

Kazakh population living in captivity, diagnosed with bacterial pododermatitis,

and from bedding, establishing their etiological role. The sentence is misleading

rework it in a similar way to ; This is the first isolation of C.metapsilosis from wild steppe eagles of the

Kazakh population living in captivity. Their etilogical role in these birds pododermatitis condition was established.

Regards

Reviewer #2: General comments

This manuscript presents a case series of three steppe eagles with chronic pododermatitis from which Candida metapsilosis was isolated. While the novelty of the pathogen is noteworthy, the study's major weakness is its inability to establish causation. It is an observational study with mycological identification, not an experimental infection study that can prove an "etiological role." The title and conclusions are overreaching. The manuscript requires major revisions to reframe the narrative as a descriptive case report with molecular characterization.

Specific Comments

Title

1) Consider "Isolation and characterization of Candida metapsilosis from chronic pododermatitis lesions in captive steppe eagles in Kazakhstan."

Abstract

1) The phrase "Our results indicate the emergence of new pathogenic yeast strains... responsible for pathology" is an overstatement. The data show an association, not definitive responsibility. The presence of the fungus does not prove it is the primary pathogen; it could be a secondary opportunistic colonizer of a wound caused by poor husbandry.

Keywords

1) Replace "Candida albicans" with "Candida metapsilosis" as the primary keyword.

Introduction

1) The introduction fails to state a clear, testable hypothesis or a specific aim for the study. It simply describes what the authors did.

2) The opening sentence about hunting with golden eagles being a "national idea" is too culturally specific for an international journal like PLOS One and detracts from the scientific focus.

Materials and Methods

1) The description of sample collection ("scraping the skin and cutting away necrotic tissue... after properly sterilizing the affected area with 70% alcohol") is insufficient. How was the site sterilized to differentiate infection from contamination? Was a biopsy taken, or just a surface swab/scraping? The method of sterilizing the surface before sampling is crucial to argue that the isolated yeast was truly from the deep tissue and not a surface contaminant.

2) The manuscript relies heavily on morphological and biochemical traits. However, for cryptic species like C. metapsilosis, which is part of the C. parapsilosis complex, molecular identification is the gold standard. The authors did this, which is good, but the methodology for the biochemical tests is vague. For the keratinophilic activity (hair perforation test), which specific protocol was used? Was it in vitro? How was it quantified to be deemed "high"? For the antifungal sensitivity testing (CLSI method), the specific standards (e.g., M27 for yeasts, not M38, which is for filamentous fungi) must be correctly cited. The authors cite M38-A2 and M44-A2. M44 is for disk diffusion, but the results are presented in a way that suggests a dilution method. Which method was actually used, and how were the breakpoints for "sensitive," "weakly sensitive," etc., determined? This is not standard CLSI terminology for yeasts.

Results

1) The results are presented in a narrative style that mixes observations with interpretation. Figures 3, 4, and 5 show primary cultures and microscopy, but the quality of the images (especially Fig. 5, which appears pixelated in the PDF) is poor and needs improvement.

2) The BLAST results are presented confusingly. The authors mention homology with a C. parapsilosis sequence (KC777375.1) used in a mouse model. Why compare to a C. parapsilosis sequence if you have identified it as C. metapsilosis? A direct comparison to confirmed C. metapsilosis reference strains (e.g., ATCC 96144) or other GenBank accessions would be more appropriate. The varying query coverage (750bp vs 669bp vs 442bp) is not explained.

3) The data in Figure 6 is presented without error bars or replicates. Was this experiment performed in triplicate? The authors state that strains have "high" keratinophilic activity, but the figure shows a bar graph. What does the y-axis represent? Quantification of this assay is missing.

Discussion

1) The discussion continues to overstate the findings. Phrases like "the uniqueness of C. metapsilosis isolation... explains the difficulties in diagnosis" are speculative.

2) The authors fail to adequately address the elephant in the room: were these eagles sick because of C. metapsilosis, or was C. metapsilosis present because the eagles were sick and immunosuppressed due to terrible husbandry? The wet bedding, poor nutrition, and stress are classic predisposing factors for opportunistic infections. The discussion must acknowledge that the fungus is likely an opportunistic pathogen exploiting a compromised host and environment, rather than the primary cause.

3) The discussion regarding the pseudohyphae formation in one strain is interesting but over-analyzed. The authors attribute it to "genetic differences," but without whole-genome sequencing, this is pure speculation. It could easily be due to the specific in vitro conditions at the time of subculture.

4) The discussion provides a good list of other Candida species found in birds but does not critically compare the pathological findings (e.g., histopathology) of those cases with the current one. No histopathology was performed in this study, which is a major limitation that should be mentioned.

Limitations

1) The authors did not include a dedicated "Limitations" section. This is a serious omission. The following must be acknowledged: (i) Small Sample Size: A case series of three birds from the same aviary. (ii) Lack of Histopathology: Without histologic examination of the tissue, it is impossible to confirm tissue invasion by the fungus and differentiate it from surface colonization or contamination. (iii) No Control Group: There were no healthy birds sampled from the same or different environments to determine if C. metapsilosis is a normal part of the eagle's skin microbiome or environment. (iv) Confounding Factors: The birds had severe husbandry issues and had received prior antibiotic treatment, which are major risk factors for fungal overgrowth. It is impossible to disentangle the role of the fungus from these factors.

Conclusions

1) Rewrite the conclusion to be more measured. State that C. metapsilosis was isolated from the lesions and characterized, and that its pathogenic potential (keratinolytic activity) suggests it may play a role in the chronicity of the disease, especially in immunocompromised birds kept in poor conditions.

References

1) The references are extensive and relevant to the field. However, some are quite old (e.g., 1975, 1978). While citing classic papers is fine, the discussion should be balanced with more recent literature (post-2020) on the C. parapsilosis complex.

Language and Writing Style

1) "unpretentiousness to the substrate" -> "undemanding nutritional requirements"

2) "an unpleasant sticky body crumpled down" -> This phrase in the medical history is nonsensical and needs clarification.

3) The sentence structure is often overly complex and repetitive.

Reviewer #3: Dear authors

thank you very much for this article.

I have several suggestion, questions, and corrections in the pdf file.

How can you be sure that C. metapsilosis was the only causative agent? You mention that you also isolated other fungi.

You should review the CLSI document; Figure 7 does not comply with the guidelines set forth in the documents.

Is it possible to construct a phylogenetic tree? The percentages for the two strains are too low to be considered the same species (metapsilosis).

Several other comments in the file.

The figures are of very poor quality. They appear blurry.

kind regards

Reviewer #4: The manuscript adds to knowledge of what we know about the role of the yeast-like fungus Candida metapsilosis in the development of pododermatitis in steppe eagles in Kazakhstan. However, the authors can improve the quality of their write up in the conclusion section. By summarizing their key findings. And removing some sections such as (line 428 to 433, page 21. This section could be part of the discussion in the manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Submitted filename: PONE-D-26-07402_reviewer.pdf

Isolation and characterization of Candida metapsilosis from foci of chronic pododermatitis in captive steppe eagles in Kazakhstan

PONE-D-26-07402R1

Dear Dr. Nessipbayeva,

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