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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2023, Vol. 49 Issue (3): 435-444    DOI: 10.3785/j.issn.1008-9209.2022.04.262
Animal sciences & veterinary medicines     
Isolation, identification, and pathological characteristic analysis of the pathogen of emulsification disease in Chinese mitten crab (Eriocheir sinensis)in Tianjin district
Qun LIU1,2(),Yan SUN1,2(),Jing WANG1,2,Junli WEI1,2,Xuewang DONG1,2,Haonan CHEN1,2
1.Animal Disease Prevention and Control Center of Tianjin, Tianjin 300402, China
2.Tianjin Surveillance Center of Aquatic Animal Infections Disease, Tianjin 300402, China
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Abstract  

In order to explore the pathogen of emulsification disease in Chinese mitten crab (Eriocheir sinensis) in Tianjin district, the epidemiological investigation, pathogen isolation, 18S rDNA gene sequence analysis, artificial infection test, and immunohistochemistry (IHC) analysis were carried out for identification of the pathogen, tissue distribution and temporal variation characteristics in this study. The results showed that the main clinical symptoms of naturally infected crabs included slow or almost no response to external stimuli, emulsify hemolymph accumulation in the cephalothorax cavity, opaque and whitish muscles at cephalothorax and joint membrane at the base of claw joints, and white semi-fluid emulsification of the hepatopancreas. The fungal strain P13, isolated from the diseased crab, was identified as Metschnikowia bicuspidata according to the phylogenetic analysis based on 18S rDNA gene sequences. Artificial infection test results showed that P13 could infect healthy Eriocheir sinensis under the laboratory condition, with cumulative mortality of (55.0±2.4)%, causing clinical symptoms similar to those found in the naturally infected crabs, and the Metschnikowia bicuspidata can be isolated from the experimental crabs again. IHC analysis results indicated that P13 infected various tissues of Eriocheir sinensis, including hepatopancreas, hindgut, gill, heart, and muscle, causing pathological damages in different degrees and temporal variation characteristics in the targeted tissues. In conclusion, Metschnikowia bicuspidata was the pathogen of the emulsification disease in Eriocheir sinensis. This study preliminarily revealed the main target tissues of Metschnikowia bicuspidata and the characteristics of tissue distribution and temporal variation, which provides a theoretical basis for clarifying the pathogenesis of Metschnikowia bicuspidata, and offers technical support for the control and prevention of emulsification disease in Eriocheir sinensis.

Key words： Eriocheir sinensis      emulsification disease      Metschnikowia bicuspidata      artificial infection test      immunohistochemistry     
Received: 26 April 2022      Published: 25 June 2023
CLC:  S917.1  
Corresponding Authors: Yan SUN     E-mail: rhswdzn@126.com;sunyan19850409@aliyun.com
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Qun LIU
Yan SUN
Jing WANG
Junli WEI
Xuewang DONG
Haonan CHEN
Cite this article:

Qun LIU,Yan SUN,Jing WANG,Junli WEI,Xuewang DONG,Haonan CHEN. Isolation, identification, and pathological characteristic analysis of the pathogen of emulsification disease in Chinese mitten crab (Eriocheir sinensis)in Tianjin district. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 435-444.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.04.262     OR     https://www.zjujournals.com/agr/Y2023/V49/I3/435


天津地区1例中华绒螯蟹“牛奶病”病原分离、鉴定及其病理特征分析

为探究天津地区中华绒螯蟹（Eriocheir sinensis）“牛奶病”案例的病原，本研究采用流行病学调查、病原分离、18S rDNA基因序列分析、人工感染试验和免疫组织化学分析来明确病原种类及其组织分布特征、时序变化特征。结果表明：自然发病蟹的主要临床症状包括对外界刺激反应慢或基本不反应，头胸甲腔内蓄积乳白色血淋巴，头胸部及步足基部关节膜连接处肌组织呈不透明乳白色，肝胰腺呈白色半流体糜烂状。基于18S rDNA基因序列的系统发育分析表明，真菌分离株P13被鉴定为二尖梅奇酵母（Metschnikowia bicuspidata）。人工感染试验结果表明，真菌分离株P13能够在实验室条件下感染健康中华绒螯蟹［累积死亡率为（55.0±2.4）%］，导致实验蟹呈现与自然发病蟹相似的临床症状，且能从实验蟹中再分离出二尖梅奇酵母。免疫组织化学分析结果表明，真菌分离株P13能够侵染中华绒螯蟹肝胰腺、后肠、鳃、心脏和肌组织，引起靶组织不同程度的病理损伤并呈现时序变化特征。综上所述，二尖梅奇酵母是天津地区中华绒螯蟹“牛奶病”的病原，本文也初步揭示了该病原的主要靶组织及组织分布特征和时序变化特征，为深入研究二尖梅奇酵母的致病机制提供了理论依据，为中华绒螯蟹“牛奶病”的防控提供了技术支撑。


关键词： 中华绒螯蟹,  牛奶病,  二尖梅奇酵母,  人工感染试验,  免疫组织化学 
Fig. 1 Clinical symptoms of Eriocheir sinensis in the overwintering pondA. Gross signs of naturally infected crabs in the overwintering pond; B. Gross signs of healthy crabs in the overwintering pond.
Fig. 2 Colony morphology of fungal strain P13 on PDA media
Fig. 3 Phylogenetic tree based on 18S rDNA gene sequences
Fig. 4 Cumulative mortality curve of Eriocheir sinensis in the artificial infection test
Fig. 5 Clinical gross signs of experimental crabs in the artificial infection testA-C. Clinical symptoms of experimental crabs after 5 dpi, 20 dpi, and 35 dpi in the injection group, respectively; D. Gross sign of experimental crab in the control group.
Fig. 6 Cell localization and temporal variation characteristics of fungal strain P13 in tissues of artificial infected Eriocheir sinensisIn Figs. A1-A3, red arrows indicate positive signals in basal membrane, interspace between columnar epithelial cells, and hepatopancreatic tubular lumen, and purple arrows indicate empty bubbles in tissue; in Figs. B1-B3, red arrows indicate positive signals in intestinal mucosa, basal membrane, and connective tissue layer, and blue arrows indicate loosely arranged single-layer columnar cells (B2) and karyopyknosis and disintegration (B3), respectively; in Figs. C1-C3, red arrows indicate positive signals, and green arrows indicate thickened gills (C1), wave arched cuticles (C2), and chitin vacuoles (C3), respectively; in Figs. D1-D3, red arrows indicate positive signals in interspace between myolins, and yellow arrows indicate loosely arranged myolins and muscle fibers fracture (D1), and alkaline nuclei between myolins (D2), respectively; in Figs. E1-E3, red arrows indicate positive signals in interspace between myocardial fibers.

组织

Tissue

感染后时间 Time post infection/d
52035
肝胰腺 Hepatopancreas++++++
后肠 Hindgut+++++++
鳃 Gill++++++++
肌 Muscle++++
心脏 Heart++++
Table 1 IHC positive signal distribution intensity in tissues of artificial infected Eriocheir sinensis
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