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. 2019 Nov 26:10:2742.
doi: 10.3389/fmicb.2019.02742. eCollection 2019.

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio)

Feng Chen  1 , Jingfeng Sun  1 , Zhuoran Han  1 , Xijun Yang  1 , Jian-An Xian  2 , Aijun Lv  1 , Xiucai Hu  1 , Hongyue Shi  1
Affiliations

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio)

Feng Chen et al. Front Microbiol. .

Abstract

Aeromonas species often cause disease in farmed fish. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). Based on this, a bacterial isolate was tentatively named CFJY-623. This isolate was identified as Aeromonas veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Six virulence genes related to pathogenicity including aerolysin, cytotonic enterotoxins, elastase, glycerophospholipid: cholesterol acyltransferase, lipase, and serine protease were identified in this A. veronii isolate. The median lethal dosage (LD50) of the CFJY-623 isolate for crucian carp was determined as 1.31 × 107 CFU/mL. Artificial experimental infection showed that the CFJY-623 isolate caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Drug sensitivity testing showed that the isolate was susceptible to aminoglycosides, carbapenemes, and nitrofurans. Exploring its growing features showed that this isolate exhibited a high level of environmental adaptability. These results provided a scientific basis for the identification of A. veronii and treatment for fish infected by this pathogen.

Keywords: 16S rRNA gene; Aeromonas veronii; Carassius auratus gibelio; growing characteristics; gyrB gene; virulence genes.

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Figures

FIGURE 1
FIGURE 1
Clinical symptoms in Carassius auratus gibelio. (A) Abdominal distension (short arrow) and congestion of fin base (long arrow). (B) Substantial fluid in the abdominal cavity and intestine (arrow). (C) Branchial ischemia (arrow).
FIGURE 2
FIGURE 2
Agarose gel electrophoresis of PCR products of the 16S rRNA and gyrB gene of CFJY-623.
FIGURE 3
FIGURE 3
Unrooted neighbor-joining phylogenetic trees based on 16S rRNA gene (A) and gyrB gene (B) sequences of Aeromonas strains. Evolutionary history was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together based on 1,000 bootstrap replicates are shown adjacent to branches. Sequence accession numbers are in parentheses.
FIGURE 4
FIGURE 4
Histological lesions of diseased fish. (A) Inflammatory infiltration (thick arrow) and tissue loosening (thin arrow), bar = 200 μm. (B) Hemosiderin stratification in spleen tissue (thick arrow), with vacuolar degeneration (thin arrow), bar = 100 μm. (C) Islet cell necrosis and vacuolar degeneration (thick arrow), bar = 20 μm. (D) Severe intravascular congestion (thick arrow), and severe swelling of liver cells, karyolysis, cell necrosis (thin arrow), bar = 200 μm. (E) Renal tubular (thick arrow) necrosis, hematopoietic and inflammatory cell infiltration, interstitial congestion (thin arrow), bar = 200 μm. (F) Hyperplasia (arrow) in gill lamellae, with inflammatory cells infiltration, bar = 200 μm.
FIGURE 5
FIGURE 5
Agarose gel electrophoresis of the amplification products of virulent genes. Six virulent genes (aer, alt, ahyB, gcaT, lip, and ser) were present in CFJY-623.
FIGURE 6
FIGURE 6
Growing characteristics of CFJY-623. Growth at pH 2-11 (A) and temperature 27–42°C (B). The growth curve of CFJY-623 at pH 7.0 and 37°C exhibited four distinct phases of bacterial growth (C).
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