First Evidence of Vibrio tapetis as Causal Agent of Disease Outbreak in Meagre (Argyrosomus regius)
Round 1
Reviewer 1 Report
First of all, this manuscript would greatly benefit from a professional/scientific English language editing. I am not a native speaker but even for me some phrases and sentences sound vaguely and grammatically incorrect in several instances.
In this manuscript Jose R. López et al., diagnosed the disease outbreak in winter in meagre in Huelva, Spain. Authors identified the etiological agent as Vibrio tapetis which caused high mortality in meagre which is recorded for the first time. This paper lacks different levels of diagnosis such as Level-I , II and III which is followed by OIE. Level I and Level II diagnosis includes gross clinical observation and histopathology which is an important diagnostic test for finding out the disease severity. Along with that this study lacks the study of viral, fungal and parasitic diseases associated with meagre. Please refer the paper Soares F., Roque A., Gavaia P.J. Review of the principal diseases affecting cultured meagre (Argyrosomus regius) Aquac. Res. 2018.
Authors claim that this is first evidence but previously it was reported by Cardenas, S. (2011). Cultivo de corvina (Argyrosomus regius) (p.96). Publicacion Madrid: Fundaci on Observatorio Espa nol de Acuicultura, Con- ~ sejo Superior de Investigaciones Cientıficas. 96 p. ISBN: 978-84-00- 09291-7. Here, the Cardenas have tried but failed. Better to cite this paper in your paper. As they have detected the presence of Vibrio tapetis by Cardenas (2011) in meagre at IFAPA (Spain). However, an experimental infection made with this agent to confirm the potential of V. tapetis to affect meagre proved unsuccessful.
This paper lacks water quality parameters such as DO, Ammonia, pH, salinity and others which can cause severe acute mortality of the fishes.
Authors have conducted LD50 study by using Sea bass (Dicentrarchus labrax) which doesn’t justify as both belongs to different order. This paper also lacks histopathological study of different organs. This is the major drawback of this paper.
Here, I have point out the some of the key points, they need to be corrected with clearer and concisely in the revised MS. Overall, manuscript can be considered for the publication after incorporation of all the major corrections and clarifications. (Major revisions)
1. Line number: 10-11
Comment: The authors should rewrite the sentence with the number of fish mortality.
2. Line number: 19-23.
Comment: LD50 study by using Sea bass (Dicentrarchus labrax) which doesn’t justify as both belongs to different order
3. Line number: 19-23
Comment: Why only two dosages (1x105 and 3x108 cfu/fish) were used in this study?
4. Line number: 23-24
Comment: Whether it’s first evidence or not? Please clarify?
5. Line number: 28-30
Comment: Please specify the significant economic loss caused by Vibrio tapetis?
6. Line number: 56-59
Comment: It would be better to add the water quality parameters data which is an important criterion. Also, specify the numbers of fish dead and alive in the tank as you have mentioned about 45% mortality was recorded in Abstract section.
7. Line number: 123-135
Comment: Why authors have used European seabass (Dicentrarchus labrax) instead of meagre? And why only two doses were used?
8. Line number: 138-147
Comment: It would be better to add the meagre figures for the manuscript as the etiological agents were having similar characteristics?
9. Line number: 138-147
Comment: This study lacks the viral, fungal and parasite presence in meagre?
10. Line number: 209-214
Comment: I would be better to put the figures of infected seabass and the control at least to compare both externally and internally. It is better to perform histopathology to observe the tissues architectural changes in infected and control to strengthen and support the research findings
11. Line number: 233-235
Comment: Is this a serious loss? What about remaining 55 % of meagres?
12. Line number: 280 -295
Comment: There are many experiments to check virulence, it would have given clear idea about significant mortality and the changes in cellular levels. This is one of the important factors to be consider for first evidence against Vibrio tapetis.
Author Response
Reviewer 1:
First of all, this manuscript would greatly benefit from a professional/scientific English language editing. I am not a native speaker but even for me some phrases and sentences sound vaguely and grammatically incorrect in several instances.
The manuscript has been revised by a native English speaker professor.
In this manuscript Jose R. López et al., diagnosed the disease outbreak in winter in meagre in Huelva, Spain. Authors identified the etiological agent as Vibrio tapetiswhich caused high mortality in meagre which is recorded for the first time. This paper lacks different levels of diagnosis such as Level-I , II and III which is followed by OIE. Level I and Level II diagnosis includes gross clinical observation and histopathology which is an important diagnostic test for finding out the disease severity. Along with that this study lacks the study of viral, fungal and parasitic diseases associated with meagre. Please refer the paper Soares F., Roque A., Gavaia P.J. Review of the principal diseases affecting cultured meagre (Argyrosomus regius) Aquac. Res. 2018.
Authors claim that this is first evidence but previously it was reported by Cardenas, S. (2011). Cultivo de corvina (Argyrosomusregius) (p.96). Publicacion Madrid: Fundacion Observatorio Espanol de Acuicultura, Con- ~ sejo Superior de Investigaciones Cientıficas. 96 p. ISBN: 978-84-00- 09291-7. Here, the Cardenas have tried but failed. Better to cite this paper in your paper. As they have detected the presence of Vibrio tapetis by Cardenas (2011) in meagre at IFAPA (Spain). However, an experimental infection made with this agent to confirm the potential of V. tapetis to affect meagre proved unsuccessful.
Regarding V. tapetis, the article by Soares et al. (2018) reproduces the information from Cardenas (2011); therefore, we have referred to the original publication (in the Introduction section). However, it must be taken into account that this publication, in which the obtaining of an isolate from a single specimen is mentioned, is not a peer-reviewed article, and there is no description of Materials and Methods or Results. Therefore, among other issues, it is not possible to know which methodology was used in the identification and therefore to know if it was conclusive or not. The publication itself indicates that these are unpublished data.
This paper lacks water quality parameters such as DO, Ammonia, pH, salinity and others which can cause severe acute mortality of the fishes.
Authors have conducted LD50 study by using Sea bass (Dicentrarchuslabrax) which doesn’t justify as both belongs to different order. This paper also lacks histopathological study of different organs. This is the major drawback of this paper.
The explanations of the issues raised are given in the specific sections that follow. Regarding the absence of a histopathological study, we agree with the reviewer that it would have been convenient to obtain samples to carry it out. This is an aspect that has been included in subsequent protocols.
Here, I have point out the some of the key points, they need to be corrected with clearer and concisely in the revised MS. Overall, manuscript can be considered for the publication after incorporation of all the major corrections and clarifications. (Major revisions)
- Line number: 10-11
Comment: The authors should rewrite the sentence with the number of fish mortality.
Of the 24 fish remaining in the tank, 11 died. This data has been included in section 3.1.
- Line number: 19-23.
Comment: LD50 study by using Sea bass (Dicentrarchus labrax) which doesn’t justify as both belongs to different order.
Both species belong to the Order Perciformes; however, we agree that the ideal would have been to carry out the tests on meagre, instead of sea bass. Unfortunately, it was not possible to carry out the trials on meagre.
- Line number: 19-23
Comment: Why only two dosages (1x105 and 3x108cfu/fish) were used in this study?
Due to strong economic restrictions, we were very limited in terms of the number of fish available.
- Line number: 23-24
Comment: Whether it’s first evidence or not? Please clarify?
As previously indicated, the publication by Cardenas (2011) mentions the recovery of an isolate of V. tapetis, however it does not include Material and Methods or Results to assess how the identification was done, if it was conclusive or preliminary. The publication itself, that is not peer-reviewed, indicates that these are unpublished data. In any case, to avoid any possible misunderstanding, the text (including the title) has been modified indicating that it is the first time that V. tapetis has been found associated with a disease outbreak in meagre.
- Line number: 28-30
Comment:Please specify the significant economic loss caused by Vibrio tapetis?
Although the mentions of the economic losses derived from the massive mortalities caused by V. tapetis are numerous, we have not found any source that values them in euros or dollars. Therefore, we have removed this reference from the text.
- Line number: 56-59
Comment: It would be better to add the water quality parameters data which is an important criterion. Also, specify the numbers of fish dead and alive in the tank as you have mentioned about 45% mortality was recorded in Abstract section.
Water quality parameters at the moment of the disease outbreak have been included in section 2.1. The number of fish dead (11 of a total of 24) has been included in section 3.1. The percentage of mortality was calculated based on the number of fish present in the tank at the beginning of the outbreak; the exact mortality value would be 45.8%.
- Line number: 123-135
Comment: Why authors have used European seabass (Dicentrarchuslabrax) instead of meagre? And why only two doses were used?
As stated above, due to strong financial constraints, we were very limited in the number of fish available, and therefore also in the number of doses we could test. The reason for using sea bass instead of meagre was the same. The meagre specimens present in the facilities were intended for other trials and we did not have the capacity to acquire others. Sea bass were used because they were the only specimens we had available.
- Line number: 138-147
Comment: It would be better to add the meagre figures for the manuscript as the etiological agents were having similar characteristics?
A figure with meagre individuals affected by V. tapetis was included.
- Line number: 138-147
Comment: This study lacks the viral, fungal and parasite presence in meagre?
The gills of the affected fish were observed under a microscope to detect parasites (the results were negative). This fact is now included in the text. No tests were performed to detect viruses or fungi.
- Line number: 209-214
Comment: I would be better to put the figures of infected seabass and the control at least to compare both externally and internally. It is better to perform histopathology to observe the tissues architectural changes in infected and control to strengthen and support the research findings
No photographs or histopathological study were taken from the samples of the virulence assays.
- Line number: 233-235
Comment: Is this a serious loss? What about remaining 55 % of meagres?
A mortality of 45% is considerable, although of course there are more deadly pathogens.
- Line number: 280 -295
Comment: There are many experiments to check virulence, it would have given clear idea about significant mortality and the changes in cellular levels. This is one of the important factors to be consider for first evidence against Vibrio tapetis.
We are aware of different procedures to check virulence but, as mentioned before, the option of a direct method (challenge) was selected, since at the experimental facilities of IFAPA at that moment the histopathological method was not running by routine. Such limitations in the present study are mentioned in the manuscript.
Reviewer 2 Report
MS „First evidence of Vibrio tapetis as the causal agent of the disease in meagre (Argyrosomus regius) is a very interesting paper introducing a new putative pathogen in the farming of this interesting marine fish species. It is really interesting that mortalities occurred without any disease symptoms or morphological changes or changes in behaviour other than mortalities. I think that the title should be somehow explained in the discussion chapter. Have you missed anything or maybe some changes in behaviour or changes in tissues have been overlooked? Please, try to analyse the case from the beginning to the experimental infection. It is hard to believe that pathogen bacteria have not caused any symptoms. It means that it was the peracute course of the disease that was not supported by the mortality dynamics. Try to address all these issues in discussion.
Some particular comments:
Line 10 – The first sentence of the summary should be rewritten
Line 50 – „recovery“ is not the best term, use the term isolate and therefore „isolates“ should be replaced with another term; or simply rewrite the sentence
Line 53 – instead of is it should be plural are
Line 56 – from the introduction, it is not clear what kind of facility was where V. tapetis was isolated; either experimental or industrial
Line 59 – why the temperature drop occurred? Please describe the water supply; since you wrote that it is a recirculation system, does it mean that something in the equipment broke? Clarify the environmental conditions of the outbreak
Line 59 – we have an appropriate word for „dying fish“ – moribund fish
Line 61 - It is strange that you start a bacteriological investigation with selective media. Why you did not use TSA or BA supplemented with NaCl or Marine agar?
Line 63 - Does it mean that pure bacterial colonies grew on the FMM agar?
Line 78 - s something missing along the procedure. How did you make an assumption immediately to V, tapetis and compare with known strains? Or when you have suspicion about the V. tapetis after phenotypic characterisation, susceptibility testing and API 20E you used them for Biolog GN2. Explain it as it is not clear
Line 158 - what is clear zone? Susceptibility testing has its own protocols and modality to describe results; the zone of inhibition should be measured and noticed. See; CLSI documents regarding bacteria isolated from the aquatic organism
Author Response
Reviewer 2:
MS „First evidence of Vibrio tapetis as the causal agent of the disease in meagre (Argyrosomus regius) is a very interesting paper introducing a new putative pathogen in the farming of this interesting marine fish species. It is really interesting that mortalities occurred without any disease symptoms or morphological changes or changes in behaviour other than mortalities. I think that the title should be somehow explained in the discussion chapter. Have you missed anything or maybe some changes in behaviour or changes in tissues have been overlooked? Please, try to analyse the case from the beginning to the experimental infection. It is hard to believe that pathogen bacteria have not caused any symptoms. It means that it was the peracute course of the disease that was not supported by the mortality dynamics. Try to address all these issues in discussion.
This lack of gross symptoms could be due to the concurrence of environmental (low temperature), host (handling stress) or pathogen (virulence of the isolates) factors that led the disease to follow an acute or per-acute course. In well-known pathogens, it has been observed that factors such as the virulence of the strain, the age of the fish, the water temperature, etc., can lead to a chronic course of the disease -with the appearance of massive macroscopic lesions before death-, or an acute course, with fulminating infections causing death before macroscopically visible signs appear. It would be interesting to determine the degree of virulence (LD50) of different strains of V. tapetis to see if this was a differentiating factor. Text has been modified in the Discussion section according to reviewer suggestions.
Some particular comments:
Line 10 – The first sentence of the summary should be rewritten
The sentence has been modified.
Line 50 – „recovery“ is not the best term, use the term isolate and therefore „isolates“ should be replaced with another term; or simply rewrite the sentence
The text has been modified.
Line 53 – instead of is it should be plural are
The text has been corrected.
Line 56 – from the introduction, it is not clear what kind of facility was where V. tapetis was isolated; either experimental or industrial
It was an experimental facility. The text has been modified.
Line 59 – why the temperature drop occurred? Please describe the water supply; since you wrote that it is a recirculation system, does it mean that something in the equipment broke? Clarify the environmental conditions of the outbreak
The fish were in one of the water recirculation systems of the facilities, in this case the water temperature control was not very efficient and was greatly influenced by the outside temperature. When the outside temperature experienced a sharp drop, the system was unable to compensate. Water quality parameters at the moment of the disease outbreak have been included in section 2.1.
Line 59 – we have an appropriate word for „dying fish“ – moribund fish
The text has been corrected.
Line 61 - It is strange that you start a bacteriological investigation with selective media. Why you did not use TSA or BA supplemented with NaCl or Marine agar?
Although the FMM was designed for the isolation of Flexibacter maritimus, it is not in fact a selective medium. We routinely use this medium for isolation after running tests with the same samples and different media. In these tests we saw that the variety of marine bacteria we obtained was greater with FMM than with TSA, TSA with NaCl, or TSA made with seawater; with Marine Agar the results were more similar, but also in this case the FMM generally gave better results. For this reason, we adopt it as the standard medium for isolation.
Line 63 - Does it mean that pure bacterial colonies grew on the FMM agar?
Yes, they grew on FMM.
Line 78 - s something missing along the procedure. How did you make an assumption immediately to V, tapetis and compare with known strains? Or when you have suspicion about the V. tapetis after phenotypic characterisation, susceptibility testing and API 20E you used them for Biolog GN2. Explain it as it is not clear
Indeed, at first we did not know that it was V. tapetis. The phenotypic characterization first, and the genotypic characterization later, indicated that it was V. tapetis. As a result, the selected isolates and the type strains of V. tapetis were used simultaneously for the BIOLOG GN2; the rest of the phenotypic characterization was also repeated, using both the meagre isolates and the type strains.
Line 158 - what is clear zone? Susceptibility testing has its own protocols and modality to describe results; the zone of inhibition should be measured and noticed. See; CLSI documentsregarding bacteria isolatedfromtheaquaticorganism
Since for the phenotypic comparison we used basically the compilation made by Buller (2014), in this regard we adopted the criterion used in this book, which considers that a bacterium is sensitive if there are halos of 9mm or more. In the strains tested in this work, the inhibition halos were of 15-21mm. The text has been modified indicating that the criterion used was that indicated by Buller (2014).
Round 2
Reviewer 1 Report
Manuscript has been improved considerably however it is a pity that it is not possible to carry out the trail on the meagre.
Author Response
Thank for the positive comments. We agree with the reviewer on the fact that performing the experimental challenge in meagre would have been beneficial for the study.
Reviewer 2 Report
Dear Authors,
Many thanks for your explanation. However, I have two small comments on the current version:
line 170 - according to Merriam-Webster Medical Dictionary when you mention Gram staining, "G" should be capitalised. On the contrary, in the case of gram-negative bacteria, it should be in lowercase
Figure 1. should be improved. It is not sharp and not acceptable for publication. Use some photo editor to improve the photo
Author Response
Typo errors regarding Gram staining were corrected.
Figure 1 was improved as requested.
