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

Isolation,identification,and pathological characteristic analysis of the pathogen of emulsification disease in Chinese mitten crab(Eriocheir sinensis)in Tianjin district

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。

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.