半滑舌鳎体表溃疡症细菌性病原的分离鉴定及组织病理学研究

doi:10.13304/j.nykjdb.2021.1108

摘要/Abstract

摘要：

为了探究引起某养殖场半滑舌鳎体表溃疡并大量死亡的原因，从发病鱼体的肝脏、脾脏、肾脏等组织分离细菌，分别从脾脏和肾脏中分离出优势菌株8-0634和8-0635。对这2株菌分别进行人工感染试验，结果显示，只有菌株8-0635能够引起半滑舌鳎的死亡，死亡鱼体表现为鳞片脱落、腹水、肠道充血向外翻出，脾脏、肾脏等器官萎缩充血。病理组织切片HE染色观察显示，发病鱼体内脏组织间隙出现大量炎细胞浸润。革兰氏染色及生理生化试验结果显示菌株8-0635为哈维氏弧菌，利用管家基因及16S rRNA构建系统发育树进行序列分析，进一步鉴定该致病菌为哈维氏弧菌。药敏试验结果显示，菌株8-0635对头孢哌酮、氟苯尼考、氯霉素、氨苄西林等多种药物高度敏感。研究结果为半滑舌鳎体表溃疡症的防治提供了重要参考。

关键词: 半滑舌鳎, 体表溃疡症, 组织病理, 哈维氏弧菌, 分离鉴定, 管家基因

Abstract:

To investigate the causes of ulcers on the surface and a large number of deaths of the Cynoglossus semilaevis in a certain farm， bacterial isolation was performed from the liver， spleen and kidney of the diseased fish， and dominant strains 8-0634 and 8-0635 were obtained from the spleen and kidney， respectively. The strains were used for artificial infection experiments， and the results showed that only strain 8-0635 could cause the death of the Cynoglossus semilaevis and the symptoms were scale off， ascites， intestinal congestion to the outward， spleen， kidney and other organs atrophy congestion. A large amount of inflammatory cell infiltration occured between tissues. According to the results of Gram staining and physiological and biochemical experiments， the strain was initially identified as Vibrio harveyi， and then the phylogenetic tree was constructed based on the housekeeping gene and16S rRNA sequence to further identify the pathogenic bacteria as Vibrio harveyi. The pathological description and drug susceptibility test were performed. The results of drug susceptibility test showed that the pathogen was high sensitivity to various drugs such as cefoperazone， florfenicol， chloramphenicol and ampicillin. This study provided a theoretical basis for disease prevention and control of fish infected with Vibrio harveyi.

Key words: Cynoglossus semilaevis, surface ulcer, histopathology, Vibrio harveyi, isolation and identification, housekeeping gene

中图分类号:

S943

吴雅婷, 汪笑宇, 汤青平, 武尊, 高浩峰, 何学欣, 杨张磊, 贾旭颖, 邵蓬. 半滑舌鳎体表溃疡症细菌性病原的分离鉴定及组织病理学研究[J]. 中国农业科技导报, 2023, 25(6): 135-143.

Yating WU, Xiaoyu WANG, Qingping TANG, Zun WU, Haofeng GAO, Xuexin HE, Zhanglei YANG, Xuying JIA, Peng SHAO. Isolation and Identification of Bacterial Etiology and Histopathological Study of Surface Ulcer of Cynoglossus semilaevis[J]. Journal of Agricultural Science and Technology, 2023, 25(6): 135-143.

图/表 10

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基因名称 Gene name	引物序列 Primer sequence（3’-5’）	长度 Sequence size/bp
16S rRNA	27F：AGAGTTTGATCCTGGCTCAG 1492R：ACGGCTACCTTGTTACGACTT	1 422
gyrB	280F：ATCATGACGGTACTGCAYGC 1486R：ACGTCTGCGTCGGTCATGAT	1 158
ftsZ	75F：GCTGTTGAACACATGGTDCG 700R：ATCATGGCGTGACCCATYTC	609
gapA	150F：AACTCACGGTCGTTTCAAC 899R：CGTTGTCGTACCAAGATAC	805

基因名称 Gene name	引物序列 Primer sequence（3’-5’）	长度 Sequence size/bp
16S rRNA	27F：AGAGTTTGATCCTGGCTCAG 1492R：ACGGCTACCTTGTTACGACTT	1 422
gyrB	280F：ATCATGACGGTACTGCAYGC 1486R：ACGTCTGCGTCGGTCATGAT	1 158
ftsZ	75F：GCTGTTGAACACATGGTDCG 700R：ATCATGGCGTGACCCATYTC	609
gapA	150F：AACTCACGGTCGTTTCAAC 899R：CGTTGTCGTACCAAGATAC	805

组别 Group	菌株浓度 Concentration/ （CFU·mL^-1）	感染死亡鱼数 Number of infected dead fish							死亡数 Death number	死亡率 Mortality/%
组别 Group	菌株浓度 Concentration/ （CFU·mL^-1）	1 d	2 d	3 d	4 d	5 d	6 d	7 d	死亡数 Death number	死亡率 Mortality/%
试验组Test	1×10⁶	2	2	1	1	2	1	1	10	100
	1×10⁵	1	1	0	0	2	1	0	5	50
	1×10⁴	0	0	1	0	1	0	1	3	30
	1×10³	0	0	0	0	1	0	0	1	10
	1×10²	0	0	0	0	0	0	0	0	0
对照组Control	0.85%NaCl	0	0	0	0	0	0	0	0	0

组别 Group	菌株浓度 Concentration/ （CFU·mL^-1）	感染死亡鱼数 Number of infected dead fish							死亡数 Death number	死亡率 Mortality/%
组别 Group	菌株浓度 Concentration/ （CFU·mL^-1）	1 d	2 d	3 d	4 d	5 d	6 d	7 d	死亡数 Death number	死亡率 Mortality/%
试验组Test	1×10⁶	2	2	1	1	2	1	1	10	100
	1×10⁵	1	1	0	0	2	1	0	5	50
	1×10⁴	0	0	1	0	1	0	1	3	30
	1×10³	0	0	0	0	1	0	0	1	10
	1×10²	0	0	0	0	0	0	0	0	0
对照组Control	0.85%NaCl	0	0	0	0	0	0	0	0	0

生理生化指标 Physiological and biochemical indexes	菌株8-0635 Strain 8-0635		标准菌株 Standard strain
生理生化指标 Physiological and biochemical indexes	8.5‰ NaCl	20.0‰ NaCl	ATCC 14126	ATCC 14129	ATCC 25919
运动 Motion	+	+	+	+	+
氧化酶 Oxidase	+	+	+	+	+
β-半乳糖苷酶 β-galactosidase	-	-	+	-	+
精氨酸水解酶 Arginine hydrolase	-	-	-	-	-
鸟氨酸脱羧酶 Ornithine decarboxylase	-	-	-	+	+
柠檬酸盐利用 Citrate utilization	+	-	-	+	+
色氨酸脱羧酶 Tryptophan decarboxylase	-	-	-	-	-
H₂S 产气 H₂S gas production	-	-	+	-	-
尿酶 Urinary enzyme	-	-	-	-	-
吲哚实验 Indole experiment	+	+	-	+	+
VP	-	-	-	-	-
明胶 Gelatin	+	-	-	+	+
葡萄糖 Glucose	+	+	+	+	+
甘露醇 Mannitol	+	+	-	+	+
肌醇 Inositol	-	-	+	-	-
山梨糖 Sorbose	+	+	+	-	-
鼠李糖 Rhamnose	-	-	+	-	+
蔗糖 Sucrose	+	+	+	+	-
蜜二糖 Honey disaccharide	-	-	-	-	-
苦杏仁苷 Amygdalin	+	+	-	+	+
阿拉伯糖 Arabinose	-	-	+	-	+
OF	+	+	+	+	+
发酵 Fermentation	+	+	+	+	+