Pathogenicity and enzyme screening of some selected non-dermatophytic moulds
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Ten non-dermatophytic moulds isolated from both symptomatic and asymptomatic cattle skin, including Penicillum citrinum, Aspergillus welwitschiae, Aspergillus aculeatus, Curvularia kusanol, Cladosporium teniussmum, Pestalotiopsis microspora, Fusarium oxysporum, Fusarium linchenicola, Absidia sp. and Aspergillus fumigatus, were subjected to a pathogenicity test using albino mice. These isolates were also screened for five enzymes using a standard plate method. Results from pathogenicity tests showed that Absidia sp., Cladosporium tenuissimum and Aspergillus welwitschiae were able to elicit discoloration, lesion production and alopecia on the albino mice skin, respectively, providing evidence of clinical symptoms associated with cutaneous mycoses. The enzyme screening results revealed the highest zone of activity for keratinase (65 mm), amylase (86 mm), protease (60 mm), lipase (60 mm) and cellulase (86 mm) which were observed on Pestalotiopsis microspora , Aspergillus welwitschiae , Cladosporium tenuissimum , Aspergillus welwitschiae and Aspergillus welwitschiae respectively. Pathogenicity tests showed that some of these moulds may be virulent and this can be attributed to their possession of some virulence factors, including secretion of hydrolytic enzymes.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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Please note the further clarification required from Reviewer 2. Please ensure all aspects of these requests are considered within the next revision.
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Comments to Author
Dear Dr Onyenwe, Many thanks for taking the time to respond to the comments and suggestions. I think your responses have improved the quality of the manuscript. I do however have a few more comments, questions and suggestions: For clarity and methodological rigour, I think the section on how the pathogenicity tests were carried out needs revising. More detail is required to explain how the fungal fragment was quantified- the mentioned inoculum is this the starting inoculum or what was applied directly to the skin of the mice? To clarify does standardisation occur at both points- dilution of fragment and then application of homogenised pathogen? Do you use CFU or another means to measure the Do you (can you) verify if all isolates are homogenised equally? Additionally, you have said that you use a …
Comments to Author
Dear Dr Onyenwe, Many thanks for taking the time to respond to the comments and suggestions. I think your responses have improved the quality of the manuscript. I do however have a few more comments, questions and suggestions: For clarity and methodological rigour, I think the section on how the pathogenicity tests were carried out needs revising. More detail is required to explain how the fungal fragment was quantified- the mentioned inoculum is this the starting inoculum or what was applied directly to the skin of the mice? To clarify does standardisation occur at both points- dilution of fragment and then application of homogenised pathogen? Do you use CFU or another means to measure the Do you (can you) verify if all isolates are homogenised equally? Additionally, you have said that you use a mixed population per isolate, does this mean that you house a male and female pair together? Do you then routinely infect only the males or only the females or is this different per each isolate? As only 1 mouse is used per isolate how can you be sure that what you are seeing is the result of the pathogen? There does not appear to be enough statistical power to definitively state this? I am a little confused with the timing of how the disease progresses- does monitoring only occur once lesions are observed, does this always happen around the day 15 mark? Measurement of zones of enzymatic activity: I appreciate that it may not be possible to retake this images, and formatting has definitely improved, the quality is still not great. At the point the zones of activity were measured were any pictures taken, it might be beneficial to see use these images instead. I might be confused but I am not convinced by some of the results based on these images. for example: Fig 3B does the red circle indicate the zone of activity? if so as the fungal growth is not circular, where are these measurements being made from? Additionally, fig2B does the red circle indicate what you are aiming to show as the zone of activity? if so how can this be measured as such when there are clearly other colonies within this zone? There are still quite a few errors throughout the text that need to be rectified, below are a few examples: line 36 pathogen needs to be in italics line 160 extra backslash line 181, no space in the name of the pathogen line 221 in the word opportunistic.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
All comments from the previous round of reviews have been addressed.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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I would like to draw your attention to comments regarding animal experiments and ensuring that the claims being made are supported by appropriate data.
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Comments to Author
In the current form I think that this manuscript requires some significant alterations, in order to both substantiate the claims made, as well to allow the reader to understand and reproduce what has been carried out as well as use the methods described in other work. My reasoning's can be found below. Methodological rigour, reproducibility and availability of underlying data What species of mice were used in this study? Are they males, females or was a mixed population used? Was the 'fragment' of fungal isolate that was applied to the animals skin quantified? Was what was applied standard across all pathogens used or representative of what would be seen outside of the laboratory? I- am unsure if 1 mouse per condition is appropriate to act as a representative for the claims that are being made, can you …
Comments to Author
In the current form I think that this manuscript requires some significant alterations, in order to both substantiate the claims made, as well to allow the reader to understand and reproduce what has been carried out as well as use the methods described in other work. My reasoning's can be found below. Methodological rigour, reproducibility and availability of underlying data What species of mice were used in this study? Are they males, females or was a mixed population used? Was the 'fragment' of fungal isolate that was applied to the animals skin quantified? Was what was applied standard across all pathogens used or representative of what would be seen outside of the laboratory? I- am unsure if 1 mouse per condition is appropriate to act as a representative for the claims that are being made, can you be sure that what you are seeing is the result of the pathogen alone and not due to another underlying factor, or if the differences observed between the pathogens is due to the pathogenicity of each species or due to differences in the quantity of pathogen administered? In the methods section of 4 out of the 5 enzymatic tests you state that this was carried out in duplicate, but do not state this for the protease activity screen- was this also carried out in duplicate? Is duplicate enough for this kind of work to show a definitive result? Lines 72-75 need rewording, the word primers on line 74 should come after 3' and before at on line 73. Presentation of results and how the style and organization of the paper communicates and represents key findings The quality of the some of the images, particularly in figures 2 and 3 is poor and in some cases it is quite hard to see how these images support the claims that are made. For instance in Figure 2, particularly A and C and Figure 3B shows the hydrolysis of cellulose by Absidia corymbifera which you claim gives a clearance zone of 60 mm, however with the images as the currently are these enzymatic effects are extremely hard to see. There is an error in the figure legend of Figure 1- it states that 1C is of a cow, not a mouse (pictured, and as is claimed in the text). There is also a spelling error here, this should read: 'revealing alopecia.' There is a discrepancy and confusion between the text and legend for figure 2, so clarification is needed on what exactly these show. The text seemingly states that A shows keratin hydrolysis by A fumigatus, B shows the hydrolysis of starch by A. terreus and C shows proteolysis by P microspora. Additionally, for 2C you state in the text that P. microspora does not grow, however there is clear growth on the plate in the figure. The legend states: 'the keratine hydrolysis(clear zone on agar plate) by Curvularia kusanol,, (B): shows zones of starch hydrolysis on agar plate 350 by Penicillum citrinum, ( C)Shows the keratine hydrolysis(clear zone on agar plate) by Curvularia kusanol.' I think clarification is needed on which organisms are shown in the images, both in the main text and the legend, and on which assay you are depicting in 2C. All images of plates are taken at varying distances, so with the data as it is it is hard for the reader to appreciate the results that you are trying to present. Clearer images, all taken at the same scale, perhaps showing the ruler used to measure the diameter might be more useful. There is a formatting difference between tables 1-4 and 5- table 5 has used a different font, compared to the rest of the tables. As a duplicate has been taken, please could the tables also show the range of the data between the duplicates measured? Supplementary Table 1, is not well referenced in the text so it is hard to fully understand what this is trying to show. I think it links back to lines 137-139, however line 137 states; 'twenty (25) DNA extracts…'. I think this should read twenty-five (25) as there are 25 rows in the table which I think, but may be wrong, link back to these DNA samples. Additionally, it would be good to know which of the samples A-Y correspond to the pathogens that you discuss in the manuscript. 4. Literature analysis or discussion Whilst I now understand that these non-dermatophytic molds seemingly exhibit some pathogenicity to mammals, I am unsure, based on the discussion how this impacts the cattle market? Are these pathogens commonly effecting cattle, is there a need to find a means to tackle these infections? What are the next steps? In the discussion section I am unsure why points 1 and 2 have been made to stand out? 5. Any other relevant comments. There are a number of errors throughout the text and in the references, including both spelling and grammar (including lack of spaces (line 20 should read results revealed), extra spaces or extra full stops, non-italicised names of pathogens…). Importantly, on a number of occasions the names of the pathogens used in this study are miss spelt, including: line 15, there is no space between Curvularia and kusanol and line 151 Pestalotiopsis microspora not microspore. The reference in the main text for the data (figures 1-3) is very unclear as to which pathogen is being shown, this then means that the assertions made in the main text contradict the figure they are referencing.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
1) The authors claim these moulds are highly virulent and attribute this to the secretion of the enzymes that have been tested for. I do not believe there is sufficient evidence for the claims of high virulence. Firstly only a single mouse was tested for each sample. Secondly, no standardisation or quantification of the initial inoculum is detailed. Only that a fragment was suspended in 5 ml water and rubbed onto the mouse (no details of where on the mouse nor how much of the 5 ml). Samples listed as non virulent may of received a significantly lower dosage for example. Details of how inoculations were quantified and standardised are needed before any claims of relative virulence can be made. Also it is unclear why beads added to the 5 ml broke the hyphae (termed fungal strands in the manuscript) and …
Comments to Author
1) The authors claim these moulds are highly virulent and attribute this to the secretion of the enzymes that have been tested for. I do not believe there is sufficient evidence for the claims of high virulence. Firstly only a single mouse was tested for each sample. Secondly, no standardisation or quantification of the initial inoculum is detailed. Only that a fragment was suspended in 5 ml water and rubbed onto the mouse (no details of where on the mouse nor how much of the 5 ml). Samples listed as non virulent may of received a significantly lower dosage for example. Details of how inoculations were quantified and standardised are needed before any claims of relative virulence can be made. Also it is unclear why beads added to the 5 ml broke the hyphae (termed fungal strands in the manuscript) and why this was necessary for determining virulence. 2) Concerns for the ethics of the mouse experiment. What metrics were recorded and used to determine when to cull the mouse? Were they all left for a month regardless of disease progression? 3) Results for the enzymatic assays are presented thoroughly in table form but results for the virulence assays are lacking. The only results listed are a single symptom for each of three samples and a picture of each in figure 1. Figure 1 should be more clearly labelled with the organism that caused the symptom shown (also part c is listed as a cow not a mouse). Additionally, other than stating that 7 samples showed no symptoms after a month, no other details such how long it took after inoculation for symptoms to present or health of all the mice are detailed as results from this animal experiment. 4) The authors previous manuscript (Prevalence of Non-Dermatophytic Molds Associated with Cutaneous Mycoses in Cattle in Abia and Imo) details the anatomical sites these 451 samples were recovered from and if they were from a lesion. Presenting that data in a table for the samples chosen (for the 20 selected for phylogenetic analysis and the 10 selected for virulence/enzymatic analysis) as well as an explanation for why these samples were chosen would provide context to the manuscript and findings. 5) In relation to previous reviewers comment 6, the data has been typed into a table in supplementary table 1 but the screenshot (supplementary figure 1) has not yet been deleted. Additionally, the samples are not labelled other than meaningless A-Y and there are many superfluous columns. Only sample name, ng/ul and the absorbance ratios are informative. 6) There are 3 supplementary figure 1s. The phylogenetic tree is shown twice. 7) Figure 3 is still very stretched looking at the shapes of the Petri dishes, as in previous reviewers 8th comment. 8) How were the pictures of the enzyme assays selected? Of the 50 tests performed only 5 plates are shown and two of the same keratin hydrolysis assay (suspect it is incorrectly labelled). These plates also show many colonies not just the one inoculated in the centre as detailed in the methods. How can the diameter of a clearance zone be measured if it overlaps completely with another? 9) Reference formatting is inconsistent using both [1], (author, date), and author (date). Additionally 'et al.' is inconsistently typed throughout (sometimes italicised, sometimes without space and sometimes et,al.). Reference format for final reference (36) is inconstant. 10) Substantial polishing required throughout for consistent font, typos and missed/extra spaces. Polishing of figure legends and a figure title for each would make the purpose of the figures clearer.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: No details on monitoring the health of the mice nor a point where the mice were culled to prevent any unnecessary suffering.
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Before peer review, there are some fundamental elements missing I would ask you to address. These include: 1) Ethics statement does not mention the use of animals, but the manuscript does describe these. Please ensure the ethics statement is complete and in accordance with common reporting guidelines, e.g. ARRIVE Guidelines https://arriveguidelines.org/arrive-guidelines 2) Detailed methodology on the isolation and characterisation of fungal isolates used in this study. Where did they come from, how were they sampled and how do you know with certainty that they are the different fungi you report? This is completely absent from the manuscript 3) There appears to be no information on repeats/replicates of the enzyme work. Can you detail this please. 4) Supplementary Table 1 and figure 1 appear with no context, or reference earlier on in …
Before peer review, there are some fundamental elements missing I would ask you to address. These include: 1) Ethics statement does not mention the use of animals, but the manuscript does describe these. Please ensure the ethics statement is complete and in accordance with common reporting guidelines, e.g. ARRIVE Guidelines https://arriveguidelines.org/arrive-guidelines 2) Detailed methodology on the isolation and characterisation of fungal isolates used in this study. Where did they come from, how were they sampled and how do you know with certainty that they are the different fungi you report? This is completely absent from the manuscript 3) There appears to be no information on repeats/replicates of the enzyme work. Can you detail this please. 4) Supplementary Table 1 and figure 1 appear with no context, or reference earlier on in the manuscript. Why are they here and what do they link too? Where are the methodology and related results/discussion? If they are to be included please expand on all of this. Also table 1 would need to be typed into a standard format table, not a screenshot. 5) How were the fungal isolates for all the work described cultured and prepared? 6) Generally figures are of low quality and stretched, is it possible to make sure they are standard size? Please carefully reconsider the manuscript so that it describes an ethically sound and reproducible study.
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