杂交鲟出血病病原的分离鉴定与组织病理学观察

doi:10.3969/j.issn.1004-1524.2021.12.07

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (12): 2275-2285.DOI: 10.3969/j.issn.1004-1524.2021.12.07

杂交鲟出血病病原的分离鉴定与组织病理学观察

杨成年(), 李芳, 朱成科, 唐征县, 易子琳, 韩璐璐, 阳龙江, 彭小倩, 贺蝶, 李杨, 任朝颖, 吕光俊^*()

西南大学水产学院/淡水鱼类资源与生殖发育教育部重点实验室,重庆 402460

收稿日期:2021-04-29 出版日期:2021-12-25 发布日期:2022-01-10
通讯作者: 吕光俊
作者简介:* 吕光俊,E-mail: 295591639@qq.com
杨成年(1993—),男,甘肃兰州人,硕士研究生,研究方向为水产动物病害防治。E-mail: ycn12103@email.swu.edu.cn
基金资助:
重庆市技术创新与应用发展项目(cstc2020jscx-lyjsAX0011);科技部“科技助力经济2020”重点专项;2021年度信豚-西南大学创新基金;西南大学大学生创新创业训练计划

Identification and pathological observation of Aeromonas veronii from hybrid sturgeon (Acipenser baerii♀×Acipenser schrenckii♂)

YANG Chengnian(), LI Fang, ZHU Chengke, TANG Zhengxian, YI Zilin, HAN Lulu, YANG Longjiang, PENG Xiaoqian, HE Die, LI Yang, REN Chaoying, LYU Guangjun^*()

Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education/College of Fisheries, Southwest University,Chongqing 402460, China

Received:2021-04-29 Online:2021-12-25 Published:2022-01-10
Contact: LYU Guangjun

摘要/Abstract

摘要：

为确定重庆彭水地区杂交鲟患病死亡的原因,从患病鲟肝中分离到一株优势菌CQST-2,对其进行形态学观察、人工感染、理化特性测定、分子生物学鉴定、毒力基因检测与药敏试验,进一步对患病组织进行病理观察。结果表明,该菌为革兰氏阴性短杆菌,感染健康杂交鲟可复现与自然患病鱼类似病症;16S rRNA、gyrB、rpoB和cpn60基因与维氏气单胞菌的同源性分别达99.7%、99.9%、99.4%和99.8%,构建的系统发育树与维氏气单胞菌聚为一支;在水温为(22±1)℃时,对杂交鲟的半数致死量LD₅₀为2.0×10⁵ CFU·g^-1;毒力基因检测发现,该菌携带ompA、act、aer、hlyA和alt等5种基因;病理组织观察发现,患病鱼肝细胞结构模糊,肝血窦扩张充血,肠绒毛坏死脱落,肌肉层充血,鳃小片弯曲,上皮细胞增生。药敏试验表明,菌株CQST-2对氧氟沙星、氟苯尼考等18种药物高度敏感,对麦迪霉素、吉他霉素、青霉素等9种抗菌药物耐药。研究结果表明,菌株CQST-2是杂交鲟的致病菌,该菌对杂交鲟具有较强的致病性,可引起多个器官发生病变。

关键词: 杂交鲟, 维氏气单胞菌, 分离鉴定, 组织病理, 药敏试验

Abstract:

In order to find out the reason that caused death of hybrid sturgeon in Pengshui, Chongqing, a dominant strain named CQST-2 was obtained from liver of diseased hybrid sturgeon. Pathogen morphological observation, challenge test, physical and chemical characteristics of isolated bacteria, sequence analysis of 16S rRNA,gyrB, rpoB and cpn60 genes, drug test and pathological were carried out and investigated. The result indicated that the isolated strain was a gram-negative bacterium and challenge test symptoms infected with Aeromonas veronii were similar to the natural symptoms. Phylogenetic trees constructed by 16S rRNA, gyrB, rpoB and cpn60 gene sequences were the same as A. veronii, with similarity of 99.7%, 99.9%, 99.4% and 99.8%. The challenge test showed that the value of LD₅₀ was 2.0×10⁵ CFU·g^-1 fish weight by intraperitoneal injection at (22±1)℃. Five virulent genes, including ompA, act, aer, hlyA and alt genes were screened by PCR assay. Pathological observation showed that the structure of liver cells was fuzzy, hepatic sinuses were dilated and congested. Most of intestinal villi were necrotic and exfoliated, and muscle layer was congested.Gills of infected fish were curved and epithelial cells were hyperplasia. Antibiotic resistances showed that the strain was sensitive to 18 drugs, including ofloxacin, polymyxinB, ciprofloxacin, enrofloxacin, and resistance to 9 drugs, including medications, guitamycin, penicillin, et al.This study provided that disease of hybrid sturgeon caused by A. veronii, which had strong pathogenicity and could cause multiple tissue lesions of hybrid sturgeon.

Key words: hybrid sturgeon (Acipenser. baerii♀×Acipenser. schrenckii♂), Aeromonas veronii, isolation and identifiation, histopathology, antibiotics test

中图分类号:

S941.1

杨成年, 李芳, 朱成科, 唐征县, 易子琳, 韩璐璐, 阳龙江, 彭小倩, 贺蝶, 李杨, 任朝颖, 吕光俊. 杂交鲟出血病病原的分离鉴定与组织病理学观察[J]. 浙江农业学报, 2021, 33(12): 2275-2285.

YANG Chengnian, LI Fang, ZHU Chengke, TANG Zhengxian, YI Zilin, HAN Lulu, YANG Longjiang, PENG Xiaoqian, HE Die, LI Yang, REN Chaoying, LYU Guangjun. Identification and pathological observation of Aeromonas veronii from hybrid sturgeon (Acipenser baerii♀×Acipenser schrenckii♂)[J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2275-2285.

图/表 11

参考文献 32

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基因 Gene	上游引物 Forward primer	下游引物 Reverse primer	产物大小 Product size/bp	退火温度 Annealing temperature/℃
16S rRNA	AGAGTTTGATCCTGGCTCAG	TACGGCTACCTTGTTACGACTT	1 413	56
gyrB	TCCGGCGGTCTGCACGGCGT	TTGTCCGGGTTGTACTCGTC	1 084	56
rpoB	GCAGTGAAAGARTTCTTTGGTTC	GTTGCATGTTNGNACCCAT	540	56
cpn60	GAAATYGAACTGGAAGACAA	GTYGCTTTTTCCAGCTCC	743	56
ompA	GCTATCCCGGCTCTGTTCGCATCT	CAGCAGGGTTTCGTCAAGCAGGTC	903	56
act	AGAAGGTGACCACCACCAAGAACA	AACTGACATCGGCCTTGAACTC	232	55
aer	CCTATGGCCTGAGCGAGAAG	CCAGTTCCAGTCCCACCACT	431	52
hlyA	GGCCGGTGGCCCGAAGATACGGG	GGCGGCGCCGGACGAGACGGG	592	62
alt	AAAGCGTCTGACAGCGAAGT	AGCGCATAGGCGTTCTCTT	320	62

基因 Gene	上游引物 Forward primer	下游引物 Reverse primer	产物大小 Product size/bp	退火温度 Annealing temperature/℃
16S rRNA	AGAGTTTGATCCTGGCTCAG	TACGGCTACCTTGTTACGACTT	1 413	56
gyrB	TCCGGCGGTCTGCACGGCGT	TTGTCCGGGTTGTACTCGTC	1 084	56
rpoB	GCAGTGAAAGARTTCTTTGGTTC	GTTGCATGTTNGNACCCAT	540	56
cpn60	GAAATYGAACTGGAAGACAA	GTYGCTTTTTCCAGCTCC	743	56
ompA	GCTATCCCGGCTCTGTTCGCATCT	CAGCAGGGTTTCGTCAAGCAGGTC	903	56
act	AGAAGGTGACCACCACCAAGAACA	AACTGACATCGGCCTTGAACTC	232	55
aer	CCTATGGCCTGAGCGAGAAG	CCAGTTCCAGTCCCACCACT	431	52
hlyA	GGCCGGTGGCCCGAAGATACGGG	GGCGGCGCCGGACGAGACGGG	592	62
alt	AAAGCGTCTGACAGCGAAGT	AGCGCATAGGCGTTCTCTT	320	62

菌液浓度 Concentration of bacterium/(CFU·mL^-1)	试验鱼数量 Number of fish samples(n)	感染时间 Infection time/d							死亡率 Mortality rate/%
菌液浓度 Concentration of bacterium/(CFU·mL^-1)	试验鱼数量 Number of fish samples(n)	1	2	3	4	5	6	7	死亡率 Mortality rate/%
1.3×10⁹	20	15	5	0	0	0	0	0	100
1.3×10⁸	20	8	5	2	1	0	0	0	80
1.3×10⁷	20	3	2	0	0	1	1	0	35
1.3×10⁶	20	0	1	0	1	0	0	0	10
0	20	0	0	0	0	0	0	0	0

菌液浓度 Concentration of bacterium/(CFU·mL^-1)	试验鱼数量 Number of fish samples(n)	感染时间 Infection time/d							死亡率 Mortality rate/%
菌液浓度 Concentration of bacterium/(CFU·mL^-1)	试验鱼数量 Number of fish samples(n)	1	2	3	4	5	6	7	死亡率 Mortality rate/%
1.3×10⁹	20	15	5	0	0	0	0	0	100
1.3×10⁸	20	8	5	2	1	0	0	0	80
1.3×10⁷	20	3	2	0	0	1	1	0	35
1.3×10⁶	20	0	1	0	1	0	0	0	10
0	20	0	0	0	0	0	0	0	0

项目 Items	CQST-2	再分菌株 Re-isolated strain	标准菌株^[12] Standard strain
氧化酶Oxidase	+	+	+
接触酶Catalase	+	+	+
赖氨酸脱羧酶	+	+	+
Lys decarboxylase
精氨酸双水解酶	-	-	-
Arg dihydrolase
鸟氨酸脱羧酶	-	-	-
Orn decarboxylase
淀粉Starch	+	+	/
麦芽糖Maltose	+	+	+
半乳糖Galactose	+	+	+
阿拉伯糖Arabinose	-	-	-
鼠李糖Rhamnose	-	-	-
D-葡萄糖D-mannose	+	+	+
D-甘露醇D-mannitol	+	+	+
纤维二糖Cellobiose	-	-	+
山梨醇Sorbitol	-	-	/
肌醇Inositol	-	-	+
吲哚Indole production	+	+	+
明胶Gelatinase	+	+	+
H₂S	-	-	-
V-P	+	+	+
柠檬酸Citrate	+	+	+
D-葡萄糖产酸	+	+	+
D-glucose produces acid
D-葡萄糖产气	+	+	+
D-glucose produces gas

杂交鲟出血病病原的分离鉴定与组织病理学观察

Identification and pathological observation of Aeromonas veronii from hybrid sturgeon (Acipenser baerii♀×Acipenser schrenckii♂)

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药物名称 Drug name	判断标准Assessment criteria			直径 Diameter/mm	敏感性 Sensitivity
药物名称 Drug name	R	I	S	直径 Diameter/mm	敏感性 Sensitivity
麦迪霉素Midecamycin	≤13	14~22	≥23	11	R
吉他霉素Kitasamycin	≤21	22~30	≥31	14	R
头孢他啶Ceftazidime	≤14	15~17	≥18	30	S
头孢哌酮Cefoperazone	≤15	16~20	≥21	34	S
氨曲南Aztreonam	≤15	16~21	≥22	30	S
青霉素Peniciillin	≤19	20~27	≥28	6	R
羧苄西林Carbenicillin	≤19	20~22	≥23	6	R
氨苄西林Ampicillin	≤13	14~16	≥17	9	R
阿莫西林Amoxicillin	≤18	19~25	≥26	6	R
米诺环素Minocycline	≤14	15~17	≥18	22	S
复方新诺明Cotrimoxazole	≤23	24~32	≥33	20	R
磺胺甲基异恶唑Benzenesulfonamide	≤14	15~23	≥24	21	I
妥布霉素Tobramycin	≤12	13~14	≥15	20	S
新霉素Neomycin	≤12	13~16	≥17	25	S
吡哌酸Pipemidic acid	≤21	22~28	≥29	32	S
氧氟沙星Ofloxacin	≤12	13~15	≥16	46	S
氟苯尼考Florfenicol	≤12	13~17	≥18	25	S
多黏霉素B Polymyxin B	≤8	9~11	≥12	15	S
杆菌肽Bacitracin	≤8	9~12	≥13	6	R
头孢唑林Cefazolin	≤14	15~17	≥18	25	S
丁胺卡那Amikacin	≤14	15~16	≥17	23	S
环丙沙星Ciprofloxacin	≤15	16~20	≥21	48	S
链霉素Streptomycin	≤11	12~14	≥15	20	S
呋喃妥因Nitrofurantoin	≤14	15~16	≥17	21	S
呋喃唑酮furazolidone	≤14	15~16	≥17	17	S
杆菌肽Bacitracin	≤8	9~12	≥13	6	R
多西环素Doxycycline	≤12	13~15	≥16	19	S
恩诺沙星Enoxacin	≤12	13~15	≥16	42	S
红霉素Erythromycin	≤13	14~22	≥23	14	I
庆大霉素Gentamicin	≤12	13~14	≥15	14	I

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