Isolation and Identification of Bacterial Etiology and Histopathological Study of Surface Ulcer of Cynoglossus semilaevis

doi:10.13304/j.nykjdb.2021.1108

Abstract

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

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

CLC Number:

S943

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.

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.

Figures/Tables 10

References 24

1	徐晓丽,邵蓬,李贺密,等.半滑舌鳎体表溃疡病病原的分离鉴定[J].中国海洋大学学报(自然科学版),2015,45(11):29-35.
	XU X L, SHAO P, LI H M, et al.. Isolation and identification of pathogen of skin ulcer from Cynoglossus semilaevis and anaiysis of antibiotic sensitivity [J]. Periodical Ocean Univ. China (Nat. Sci.),2015,45(11):29-35.
2	陈政强,姚志贤,林茂,等.半滑舌鳎皮肤溃疡病病原研究[J].水产学报,2012,36(5):764-771.
	CHEN Z Q, YAO Z X, LIN M, et al.. Study on pathogen of skin ulcer disease of half-smooth tongue sole (Cynoglossus semilaevis) [J]. J. Fisheries China,2012,36(5):764-771.
3	罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013,32(9):549-554.
	LUO M, CHEN F X, LIU L L,et al..Progress in disease research of grouper aquaculture in China [J]. Fisheries Sci.,2013,32(9):549-554.
4	崔婧,范雪亭,刘文竹,等.华南地区海水养殖鱼类主要弧菌病原的分离与鉴定[J].海南大学学报(自然科学版),2014,32(3):244-251.
	CUI J, FAN X T, LIU W Z,et al.. Isolation and identification of vibriosis pathogens of marine cultured fishes in Southern China [J]. J. Hainan Univ. (Nat. Sci.), 2014,32(3):244-251.
5	许悦,王印庚,周永灿,等.海南地区养殖石斑鱼皮肤溃疡病病原的分离、鉴定、药敏分析及病理学研究[J].海南大学学报(自然科学版),2018,36(2):153-163.
	XU Y, WANG Y G, ZHOU Y C, et al..Isolation, identification, and pathogen analysis of skin-ulcer disease in cultured groupers in Hainan province [J]. J. Hainan Univ. (Nat. Sci.),2018,36(2):153-163.
6	KARUNASAGAR I, PAI R, MALATHI G R, et al.. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection [J]. Aquaculture,1994,128(3-4): 203-209.
7	KRIEG N R, HOLT. Bergeys manual of systematic bacteriology [J]. Bergeys Manual Syst. Bacteriol., 1984, 38(4):443-491..
8	PUJALTE M J, SITJÀ-BOBADILLA A, MACIÁN M C, et al.. Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass [J]. System. Appl. Microbiol., 2003,26:284-292.
9	夏青,王宝杰,刘梅,等.哈维氏弧菌浸浴后凡纳滨对虾肠道组织病理变化及转录水平的免疫应答[J].水产学报,2015,39(10):1521-1529.
	XIA Q, WANG B J, LIU M,et al..Pathological changes and transcriptional response to immersion infection by Vibrio harveyi in shrimp (Litopenaeus vannamei) gut [J]. J. Fisheries China, 2015,39(10):1521-1529.
10	李洋,李强,张显昱.哈维弧菌及其主要致病因子的研究进展[J].中国农业科技导报,2014,16(4):159-166.
	LI Y, LI Q, ZHANG X Y. Research progress on Vibrio harveyi and its main pathogenic factors [J]. J. Agric. Sci. Techol.,2014,16(4):159-166.
11	WU H, ZHANG L, WANG N, et al.. Analysis of the bioactive components of Sapindus saponins [J]. Ind. Crops Products,2014,61: 422-429.
12	SHAO P, YONG P Z, WANG X Y,et al.. Isolation, identification, and histopathological analysis of Vibrio tubiashii from lined seahorse Hippocampus erectus [J]. Dis. Aquat. Organ.,2019,133(3):195-205.
13	SANTTOS Y, ROMALDE J L, BARJA J L, et al.. Usefulness of the api-20e system for the identification of bacterial fish pathogens [J]. Aquaculture, 1993, 116(2-3):111-120.
14	王燕,韩茵,李筠,等.半滑舌鳎病原菌(发光杆菌杀鱼亚种)的分离与鉴定[J].微生物学报,2007,47(5):763-768.
	WANG Y, HANG Y, LI J, et al.. Isolation of Photobacterium damselae subsp. piscicida from diseased tongue sole (Cynoglossus semilaevis Gunther) in China [J]. Acta Microbiol. Sin.,2007,47(5):763-768.
15	徐晓津,徐斌,王军,等.哈维氏弧菌人工感染大黄鱼的组织病理学研究[J].厦门大学学报(自然科学版),2009,48(2):281-286.
	XU X J, XU B, WANG J, et al.. Histopathological studies on pseudosciaena crocea artificially challenged with Vibrio harveyi [J]. J. Xiamen Univ. (Nat. Sci.),2009,48(2):281-286.
16	姚东瑞,朱明,陈丽,等.基于ToxR基因病原哈氏弧菌PCR检测方法的建立[J].水产科学,2011,30(9):559-562.
	YAO D R, ZHU M, CHEN L, et al.. Development of PCR method for detection of pathogenic baterium vibrio harveyi based on ToxR gene [J]. Fish. Sci., 2011, 30(9):559-562.
17	WAN Q J, WU L Q, YANG Q H, et al.. First identification and pathogenicity study of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata) cultivated in freshwater [J]. Aquac. Res., 2021,53(4):1240-1253.
18	林星.花鲈皮肤溃疡病病原菌的分离与鉴定[J].中国农学通报,2013,29(32):100-104.
	LIN X. Isolation and identification of the pathogens of the skin ulceration disease in Lateolabrax japonicus [J]. Chin. Agric. Sci. Bull., 2013,29(32):100-104.
19	张凤萍,彭志兰,张健,等.鮸鱼弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].微生物学报,2010,50(3):304-309.
	ZHANG F P, PENG Z L, ZHANG J, et al.. Isolation and identification of the pathogenic strain of Vibrio harveyi from Miichthys miiuy [J]. Acta Microbiol. Sin.,2010,50(3):304-309.
20	LIU P C, LIN J Y, CHUANG W H, et al.. Isolation and characterization of pathogenic Vibrio harveyi (V-carchariae) from the farmed marine cobia fish Rachycentron canadum L. with gastroenteritis syndrome [J]. World J. Microbiol. Biotechnol., 2004,20(5):495-499.
21	LIU L, GE M F, ZHENG X Y, et al.. Investigation of Vibrio alginolyticus,V. harveyi, and V. parahaemolyticus in large yellow croaker. Pseudosciaena crocea (Richardson) reared in Xiangshan Bay, China [R]. Aquaculture Reports 3,2016:220-224.
22	XU X D, LIU K F, WANG S F, et al..Identification of pathogenicity, investigation of virulent gene distribution and development of a virulent strain-specific detection PCR method for Vibrio harveyi isolated from Hainan Province and Guangdong Province, China [J]. Aquaculture,2017,468:226-234.
23	谢楚萍,黄思韵,郑仰思,等.养殖浅色黄姑鱼体表溃烂症病原菌的分离与鉴定[J].应用海洋学学报,2013,32(3):411-417.
	XIE C P, HUANG S Y, ZHENG Y S, et al.. Isolation and identification of a bacterial pathogen from Chu’s croaker Nibea coibor [J]. J. Appl. Oceanography,2013,32(3):411-417.
24	杨先乐,郑宗林.我国渔药使用现状、存在的问题及对策[J].上海水产大学学报,2007,16(4):374-380.
	YANG X L, ZHENG Z L. The application status and strategy of fishery medicine in China [J]. J. Shanghai Fish. Univ.,2007,16(4):374-380.

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	+	+	+	+	+