PONE-D-22-29069AA-amyloidosis in cats (Felis catus) housed in sheltersPLOS ONE

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PLOS ONE

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Maria Mazza: 10/19 RC Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta. 

Stefano Ricagno: this study was partially supported by Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors,

please find below my comments.

Lines 130-131: why is Congo Red used? It is necessary to specify that "staining with Congo Red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections" and to correctly quote the bibliography.

Lines 165-166: "Multiple techniques (Congo red staining, western blotting)". Being only two methods, I would eliminate "multiple techiques".

Lines 209-2011: it is not clear how the "disease duration index" was calculated. It is reported that "disease duration index was calculated arbitrarily" and that "The index represented the proportion of time a cat was sick during its stay in the shelter". Is the index therefore the mathematical ratio? Why is it written "arbitrarily"?

Line 263: "A co-localization staining" between what (Thioflavine S, DAPI or IgG)? Please specify better.

Lines 126-147: negative controls were not used in the immunofluorescence experiments and this is not a correct methodological approach for this method. The experiment requires negative controls.

Fig 2: the images in the file that I have downloaded are of low quality, in some points pixelated and this does not allow to evaluate the results obtained.

Fig 2: to understand the significance of the results, negative controls showing the histology of non-affected individuals are required.

Lines 314-323: I personally believe that a scientific work must show all the results obtained also to verify the truthfulness of the results and the correctness of the interpretations and conclusions. The number of samples examined by western blot is high (bile sample from 24) but not so high to make it impossible to present all the results, both positive and negative. I believe that the results of all western blots should be presented and not only those of some representative results, as shown in Fig. 3. Please report all results obtained (if necessary use supplementary materials).

Figure 3: "pre-stained molecular weight markers" are used in the experiment but are not visible in the result. Molecular weight numbers lack a dash "-" that indicates exactly the height that the molecular weight refers to so that the molecular weight of the bands can be accurately assessed.

Figure 3: What exactly is the molecular weight of the positive bands? I do not think it is ever made explicit in the text.

Figure 3: the experiment lacks the negative control consisting of the use of only the secondary antibody.

Lines 405-407 the molecular weight of the bands is not explained.

Lines 419-420: "This observation is in contrast with observations in client-owned cats". Where does this observation come from? Are you referring to data reported in this study or to results of other not cited works?

Reviewer #2: The manuscript “AA-amyloidosis in cats (Felis catus) housed in shelters” describes the detection in a high percentage of amyloid fibrils in tissues and bile of cats that died in shelters for different reasons. The authors conclude that the long staying in the shelter and co-presence of diseases increase the chance of amyloid deposition and add some speculations.

The topic of cat amyloidosis is interesting also for several human diseases, and the manuscript data might be of general interest. However, It has been difficult and frustrating to follow the sampling and result description as it was confounded which animal had which phenotype or was analyzed. Moreover without a plain clear and transparent display of all the raw data, the point of all the work is only what I just synthetized and what is even more critical the individual results are of little use as they are presented. Material and Methods together with the Results section must be improved to obtain a clearer exposition and results convincing. So, I suggest to the authors to add all the raw data in a Supplementary material Table, to allow a clear understanding of their analyses and conclusions.

In my opinion, to be re-considered for acceptance the paper must address this request of raw data disclosure, together with the comments that follow.

INTRODUCTION

Lines 69 and 72. As your reference (3) is about cats, please anticipate “In cats” to line 69 and remove it from line 74 as follows: “In cats the exact pathogenesis…[3]. Many conditions…”.

Line 74. “but only a limited number of cats eventually develop the disease [9]”.

Assuming “the disease” refers to amyloidosis, the reference [9] is misplaced and generates misleading information. It should be moved after “high production of SAA”, in case.

Thus, the sentence in line 74 needs a reference in support.

Tamamoto et al 2013 do not have amyloidosis among their considered diagnostic categories, nor report analyses made to detect the presence/absence of amyloidosis in their samples. They mention amyloidosis only in their discussion as follows: “SAA protein is known as a precursor of amyloid A fibrils, and sustained high SAA levels may give rise to secondary amyloidosis, so-called reactive amyloid A amyloidosis. (8) The development of amyloid A amyloidosis is considered an important prognostic factor. (5)…..However, all of these facts were reported in human medicine, and it remains unclear whether it can be applied to cats.”

Line 76. The author might want to complete the sentence: “the differences in SAA genetic sequence proved to not contribute to the pathogenesis of feline AA-amyloidosis both in Japanese domestic cats [10] and in Abyssinian breed [doi: 10.1038/s41598-021-87168-0.]

Line 81-83. Please detail more specifically (see underlined words): “the development of AA-amyloidosis in mice induced by injection of an inflammatory stimulant appears to be accelerated by the oral administration of semipurified amyloid fibrils extracted from murine, humans and cattle affected tissues and lead to the hypothesis of both intra-species and cross-species transmissibility [13].

Please add “intra species” because the focus is on the cat, the cross-species is interesting, but here less important.

Line 96. “3 consecutive”. Please change “three”, and what do you mean by “consecutive”? In a short period?

Line 97. Please correct and add: “within six months from the admission”

MATERIAL AND METHODS

Line 104 and line 229 (Results). Please add to the main text in MM the number of cats analyzed (79).

Moreover, in Results, the authors report 80 collected cats, but then you exclude one because of late sampling, which is the only parameter of exclusion stated in MM. So, no reason to talk of 80 cats, here again please report 79.

Please specify in MM that necropsy report/clinical anamnesis were available for n …(?)…/79 cats

In addition, it might be of interest which approximate percentage of each shelter total population were those cats and some general description of shelter structure and management if they might have or not have an impact on the cat promiscuity and epidemiology.

Lines 104, 231, and 354. “Northern Italy”

Line 202. Please, do you mean “both for each shelter and the entire population of sampled cats”?

RESULTS

Overall it is difficult to follow the data throughout the results section. The authors are warmly invited to produce a synoptic table in the supplementary material with all the raw data of each cat, even with missing data (see below)

Lines 238-244. This paragraph is confounding.

In Table S1 how many cats showed the 47 causes of death reported? It should be assumed 47 cats. In Table S1 are reported, “other diseases” (36 diseases) on 22 cats from shelter C. Why these data are not merged in a single table? Are the cats with phenotype 47+22=69 or what?

Please remove S1T and S2T and list in one Supplementary single table all the 79 cats, their individual phenotypic data collected when available (disease and cause of death), positiveness to Congo (negativeness and additional score or at least the presence/absence of amyloid) and the shelter.

The authors are also strongly encouraged to integrate this new Table with cat age when accepted in the shelter (if available), time of permanence in the shelter, age at death

Table 1. please specify in table 1 legend the number of cats such as “Table 1. Gender distribution, age and length of stay of the 79 cats in the three shelters”.

Line 267- 273. “Of the 22 cats with available information on other diseases”, what about the cats listed in S1T (n.47 )? Were the remaining without phenotype, which ones (see above)?

Line 272-274 “Of the 22 cats with available information on other (36?) diseases, 12 showed AA-amyloidosis”. Does this Mean that 10/22 = 45,6% of those affected by other diseases cats did not develop amyloidosis? Please clarify and in case comment.

“…with 11 cats (91.7%) being affected by one or more concurrent diseases.” Meaning that in a subset of 12, out of a total of 22 cats all with phenotype, only 91% had phenotype? Please clarify.

Table 2. Please specify also in Table 2 legend “in 79 cats”

Line 307. Please specify which diseases were the 65 cats affected by, in case they were (in addition to FILV, FeLV, and FCoV). It should be perfect in the New Supplementary table reporting the phenotypes to add a column where the cats analyzed are ticked

Line 314. Please specify which diseases or viral infections were the 24 cats affected by, apart from amyloidosis. It should be perfect in the New S. table reporting the phenotypes to add another column where the cats analyzed are ticked

Lines 335 – 338. Please specify which subset of cats (numerosity, shelter, etc). Please specify it also in Fig. 4

DISCUSSION

Line 417. “It is probable that some of the cats with AA amyloidosis were asymptomatic for the disease”

In domestic cat amyloidosis is essentially “asymptomatic” until almost the end, and the late symptoms are not pathognomonic. The diagnosis is basically by biopsy or necropsy and Congo red stain. If deposits in tissues were found the cat was affected by amyloidosis, if not the cat was not affected. Then please clarify the rationale of this sentence.

Line 420. “Whether bile excretion of AA-fibrils represents a vehicle of transmission explaining the high frequency of the disease in shelter cats remains to be elucidated.” Did the authors collect gastro-intestinal mucosa and feces, having planned to analyze bile in a prospective study? In case this may be specified as further investigation.

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Reviewer #1: No

Reviewer #2: No

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AA-amyloidosis in cats (Felis catus) housed in shelters

PONE-D-22-29069R1

Dear Dr. Ferri,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Maria Stefania Latrofa

Academic Editor

PLOS ONE

Additional Editor Comments:

The manuscript is considered accepted. It requires a minor change as noted by reviewer 2.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have adequately addressed my comments raised in the previous round of review.

I have only one remark that should be addressed and was not completely, in my opinion: in my previous review I requested to specify in MM how many cases were necropsied.

The authors kindly replied that "...The technical staff was instructed on how to adequately collect samples but were unable to perform full necropsies; hence, reports of the full necropsy are not available". This is very understandable, but I feel it is important to specify this in the Materials and Methods with a short sentence such as

New Line 112 "...tissue samples had been collected within six hours of death, not allowing for a full necropsy to be recorded"

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Reviewer #1: No

Reviewer #2: No

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