Isolation, Identification and Virulence Gene Analysis of an Aeromonas hydrophila from Diseased Pelodiscus sinensis

doi:10.11924/j.issn.1000-6850.casb2020-0484

Abstract

Abstract:

The study aims to determine the pathogen causing disease in a Chinese soft-shelled turtle farm in Anhui Province, and provide reference for effective prevention and control of the disease in future production. The pathogenic bacteria were isolated from the moribund turtle with typical symptoms under aseptic condition, and then were identified by the morphological examination, biochemical analysis, and sequence analysis of bacterial 16S rDNA. The drug susceptibility test was conducted by Kirby-Bauer agar diffusion method, and the virulence genes of the pathogen were analyzed. The results showed that the pathogen was identified as Aeromonas hydrophila, called AM-1. Artificial infection showed that the typical symptoms of the disease could be replicated as the ones naturally occurred and this bacterium was of high pathogenicity to Chinese soft-shelled turtle. The drug resistance tests showed that the isolated pathogen was highly sensitive to enrofloxacin and levofloxacin, while extremely resistant to cotrimoxazole, ampicillin and tetracycline. The amplification results of 6 virulence genes showed that the isolated pathogen carried 5 virulence genes including aerolysin, heat-labile enterotoxin, heat-stable enterotoxin, hemolysin and elastase. This study indicates that Aeromonas hydrophila AM-1 is the pathogenic bacteria which could cause the deadly disease outbreak of the Chinese turtle. Enrofloxacin and levofloxacin can be used for treatment.

Key words: Pelodiscus sinensis, Aeromonas hydrophila, virulence gene, pathogenicity

CLC Number:

S941

Xue Mingyang, Zhou Yong, Liang Hongwei, Li Xiang, Fan Yuding, Zeng Lingbing, Qu Chunjuan, Meng Yan. Isolation, Identification and Virulence Gene Analysis of an Aeromonas hydrophila from Diseased Pelodiscus sinensis[J]. Chinese Agricultural Science Bulletin, 2021, 37(20): 152-159.

Figures/Tables 9

References 29

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目的基因	引物序列(5’-3’)	退火温度/℃	长度/bp
STIV	F: GACTTGGCCACTTATGAC	52	531
STIV	R: GTCTCTGGAGAAGAAGAA	52	531
16S rDNA	F: AGAGTTTGATCCTGGCTCAG	55	1500
16S rDNA	R: GGTTACCTTGTTACGACTT	55	1500
aero	F: CAAGAACAAGTTCAAGTGGCCA	57	309
aero	R: ACGAAGGTGTGGTTCCAGT	57	309
hly	F: TGACAGGCAAGTAGAATAACGC	58	1815
hly	R: TGTCCGCTTTCCACTCCC	58	1815
ahp	F: GTTAGCGTTGGCAATCTCG	57	874
ahp	R: CGCTGGAGTAGGAGGAACG	57	874
alt	F: TGACCCAGTCCTGGCACGGC	57	442
alt	R: GGTGATCGATCACCACCAGC	57	442
ast	F: ATCGTCAGCGACAGCTTCTT	58	504
ast	R: CTCATCCCTTGGCTTGTTGT	58	504
act	F: TACCACCACCTCCCTGTCGC	60	249
act	R: ATGCTGCTCGCCTTGTGGTT	60	249

目的基因	引物序列(5’-3’)	退火温度/℃	长度/bp
STIV	F: GACTTGGCCACTTATGAC	52	531
STIV	R: GTCTCTGGAGAAGAAGAA	52	531
16S rDNA	F: AGAGTTTGATCCTGGCTCAG	55	1500
16S rDNA	R: GGTTACCTTGTTACGACTT	55	1500
aero	F: CAAGAACAAGTTCAAGTGGCCA	57	309
aero	R: ACGAAGGTGTGGTTCCAGT	57	309
hly	F: TGACAGGCAAGTAGAATAACGC	58	1815
hly	R: TGTCCGCTTTCCACTCCC	58	1815
ahp	F: GTTAGCGTTGGCAATCTCG	57	874
ahp	R: CGCTGGAGTAGGAGGAACG	57	874
alt	F: TGACCCAGTCCTGGCACGGC	57	442
alt	R: GGTGATCGATCACCACCAGC	57	442
ast	F: ATCGTCAGCGACAGCTTCTT	58	504
ast	R: CTCATCCCTTGGCTTGTTGT	58	504
act	F: TACCACCACCTCCCTGTCGC	60	249
act	R: ATGCTGCTCGCCTTGTGGTT	60	249

检测项目	AM-1	嗜水气单胞菌	检测项目	AM-1	嗜水气单胞菌
葡聚糖 Glucan	+	+	明胶 Gelatin	+	+
D-麦芽糖 D-Maltose	+	+	异硫氰酸胍 GuSCN	+	+
蔗糖 Sucrose	+	+	果胶 Pectin	+	+
α-D-乳糖α-D-Lactose	+	+	粘酸 Mucic Acid	+	+
D-甘露糖 D-Mannose	+	+	奎宁酸 Quinic Acid	+	+
D-果糖 D-Fuctose	+	+	D-苹果酸 D-Malic Acid	–	+
夫西地酸 Fusidic Acid	–	–	L-苹果酸L-Malic Acid	–	–
D-丝氨酸 D-Serine	+	+	氯化锂Lithium Chloride	+	+
D-甘露醇 D-Mannitol	+	+	萘啶酮酸Nalidixic Acid	+	+
D-水杨苷 D-Salicin	d	–	D-葡萄糖 D-Glucose	d	–
丙三醇 Glycerol	+	+	γ-氨基丁酸 γ-ABA	+	+
肌醇Myo-lnositol	+	+	α-羟基丁酸α-HBA	–	–

检测项目	AM-1	嗜水气单胞菌	检测项目	AM-1	嗜水气单胞菌
葡聚糖 Glucan	+	+	明胶 Gelatin	+	+
D-麦芽糖 D-Maltose	+	+	异硫氰酸胍 GuSCN	+	+
蔗糖 Sucrose	+	+	果胶 Pectin	+	+
α-D-乳糖α-D-Lactose	+	+	粘酸 Mucic Acid	+	+
D-甘露糖 D-Mannose	+	+	奎宁酸 Quinic Acid	+	+
D-果糖 D-Fuctose	+	+	D-苹果酸 D-Malic Acid	–	+
夫西地酸 Fusidic Acid	–	–	L-苹果酸L-Malic Acid	–	–
D-丝氨酸 D-Serine	+	+	氯化锂Lithium Chloride	+	+
D-甘露醇 D-Mannitol	+	+	萘啶酮酸Nalidixic Acid	+	+
D-水杨苷 D-Salicin	d	–	D-葡萄糖 D-Glucose	d	–
丙三醇 Glycerol	+	+	γ-氨基丁酸 γ-ABA	+	+
肌醇Myo-lnositol	+	+	α-羟基丁酸α-HBA	–	–

菌液浓度/(CFU/mL)	数量	死亡数量								合计	死亡率/%
菌液浓度/(CFU/mL)	数量	1 d	2 d	3 d	4 d	5 d	6 d	7 d	8 d	合计	死亡率/%
1×10⁶	8	0	0	2	2	2	0	0	0	6	75
1×10⁷	8	0	1	3	2	1	0	0	0	7	87.5
1×10⁸	8	0	1	4	2	0	0	0	0	7	87.5
1×10⁹	8	0	3	3	2	0	0	0	0	8	100
1×10¹⁰	8	0	4	3	1	0	0	0	0	8	100
0	8	0	0	0	0	0	1	0	0	1	12.5