Isolation, Identification and Characteristics of Aeromonas caviae from Diseased Largemouth Bass (Micropterus salmoides)

Round 1

Reviewer 1 Report

The manuscript „Isolation, identification and characteristics of Aeromonas caviae from diseased largemouth bass (Micropterus salmoides)” includes important knowledge about bacteria Aeromonas isolated form largemouth bass in China. The authors used several methods to examine and characterize bacteria.

Comments

Introduction

Introduction not include all relevant references and sufficient background.

Reference 1 and 2 (Line 29 and 30) do not apply directly to the text. References on the importance of largemouth bass in China should be included.

Reference 2 and 3 (Line 36) do not apply directly to the text. Appropriate reference should be added.

Moreover, reference 5 should be moved from lines 38 to 36.

Line 36-38 contains information about bacteria caused outbrakes in largemouth bass. The author mentions such bacteria as Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii, Edwardsiella piscicida, therefore the appropriate references should be added for example:

-Camus at. al.: A spontaneous outbreak of systemic edwardsiella piscicida infection in largemouth bass micropterus salmoides (Lacépède, 1802) in California, USA J. Fish Dis., 42 (2019), pp. 759-763,
-Reference  26,27.

In addition, bacteria such as Edwarsiella tarda and Flavobacterium columnare can cause lesions in these fish and should be added to the text according to Buller N.B. 2015: Bacteria and Fungi from Fish and Other Aquatic Animals: A Practical Identification Manual, p.19.

Reference 10 (Line 44) focused on the isolation of bacteria A. caviae from the common carp, this fish species should be added to the text.

Line 44: The references which describe the isolation of bacteria A. caviae from rainbow trout (Oncorhynchus mykiss), Penaeus vannamei, crayfish (Procambarus clarkia) and grass carp (Ctenopharyngodon idellus) should be added.

Materials and Methods

Fish specimens

Line 66: The size of aquarium, information about fish feeding and quarantine should be added.

Pathogen detection and identification

Line 73: The  samples were analyzed for virological and parasitological. The used methods should be described.

Line 75: The name and dose of the anesthetic should be added.

16S ribosomal DNA sequencing analysis, Screening of virulence genes

Line 86: If the optimal annealing temperature with universal primers was 55⁰C (Table 1), why 56⁰C was used?

Table 1: The primer for lipase (lip) virulence genes should be added to the                   table.  
            The references for appropriate primers should be added to the                      table.

Antibiotic susceptibility test

Line 130: The interpretation of antibiotic resistance was done according to "The National Committee for Clinical Laboratory Standards". The criteria for sensitive/intermediate or resistant should be clarified. 
According to current knowledge, the criteria for only one bacteria isolated from fish - Aeromonas salmonicida were specified (Clinical and Laboratory Standards Institute: VET03/VET04-S2 Performance standards for antimicrobial susceptibility testing of bacteria isolated from aquatic animals; Second Informational Supplement. Vol. 34, No. 15, 2014).

Results

Bacterial 16S rDNA sequence analysis

Which tool was the phylogenetic analysis carried out? The software and method should be added.

Has the 16S rDNA sequence of WH21406 submit to database such as GenBank? Accession number should be added to the text or Figure 3.

Line 172: The similarity at the level of 99% is not sufficient for the species classification of Aeromonas. To confirm the identification to the species level, for example the gyrB or rpoB genes or whole genome sequensing should be sequenced. If it is not possible, the name Aeromonas caviae like bacteria should be used.

Pathogenicity assays

Figure 6. is indistinct.

The experiment of pathogenicity was repeated twice. Were there differences in mortality and symptoms?

Discussion

Line 224: The reference 24 is not strictly about infections cause by Aeromonas species, but bacteriophages. Additional references should be added.
Line 226: The reference directly related to Aeromonas caviae should be added.

Conclusions

Line 275: Term "prevention"  should be clarified, because this aspect is not studied in current research.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The present manuscript by Xue et al. investigated and reported the infection of Aeromonas caviae in large mouth bass. The manuscript is of potential interest to researchers working on fish diseases and clinical diagnostic. Aeromonas is extremely important bacterial disease in aquaculture. The identification of A. caviae in LMB is timely and need to be addressed to mitigate further problems of A. caviae in aquaculture.

Introduction should highlight the virulence gene in Aeromonas

Page 2 line 49-Objectives should be precisely mentioned

Section 2.1- Authors must provide the details of farming practices and clinical signs observed from the farm.

Section 3.1. external and internal clinical must be highlighted.

Section 3.4- Authors must discuss the absence of act, ahp, alt and lip genes in A. caviae

Discussion should be made on the pathogenicity of A. caviae during artificial infection.

The authors have isolated specific strain of AC and characterized biochemically and evaluated some virulence gene and tested antibiotic susceptible test and proved its pathogenesis in laboratory conditions. Overall the manuscript is well written and could be consider for publication in this journal.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The new version of the manuscript can be accepted for publication.