| 基于环境DNA的进口凡纳滨对虾携带水传播虾肝肠胞虫风险评估 |
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| 引用本文： | 张娜,刘荭,廖金湾,郑晓聪,郑枢,刘骁,谢艳辉,李家侨,李红权.基于环境DNA的进口凡纳滨对虾携带水传播虾肝肠胞虫风险评估[J].南方农业学报,2022,53(1):219-228. |
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| 作者姓名： | 张娜 刘荭 廖金湾 郑晓聪 郑枢 刘骁 谢艳辉 李家侨 李红权 |
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| 作者单位： | 1 湛江海关技术中心, 广东湛江 524000;2 深圳海关动植物检验检疫技术中心, 广东深圳 518045;3 湛江海关, 广东湛江 524000 |
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| 基金项目： | 海关总署科技项目;湛江海关科技项目 |
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| 摘 要： | 【目的】基于环境DNA（eDNA）分析进口凡纳滨对虾携带水传播和运输虾肝肠胞虫（EHP）的风险性，为环境水中疫病监测和风险评估提供参考依据。【方法】取60批进口亲虾携带水用0.45 μm硝酸纤维素膜过滤并提取eDNA，挑取10个携带水样滤膜eDNA用于凡纳滨对虾物种特异性鉴定，采用实时荧光定量PCR对所有携带水样滤膜进行EHP检测；同时以EHP阳性进口亲虾携带水开展人工感染试验，感染10 d后分别取养殖水样及虾体进行EHP检测；对进口亲虾携带水EHP阳性滤膜进行宏基因组测序分析，采用Krona对物种注释结果进行可视化展示，并将宏基因组测序分析结果与NCBI已公布的EHP氨基酸序列进行BLAST比对分析。【结果】进口亲虾携带水检测结果显示，成功从10个携带水滤膜eDNA扩增出凡纳滨对虾347 bp的特异目的片段，与原始序列片段的相似性达100%；在60批进口亲虾携带水样品中有2批携带水呈EHP阳性。EHP阳性进口亲虾携带水能成功感染虾苗组，且在虾苗肝胰腺组织分离获得成熟的EHP孢子，但亲虾组的虾体EHP检测呈阴性。宏基因组测序得到EHP的物种相对丰度值占真核动物物种的0.7%；与NCBI已公布的EHP氨基酸序列进行BLAST比对，结果获得213个同源EHP氨基酸序列；从进口亲虾携带水样品中分离鉴定获得的EHP与已报道物种黄道蟹肠孢虫（Enterospora canceri）和毕氏肠胞虫（Enterocytozoon bieneusi）的亲缘关系较近，对应的氨基酸序列相似性为别为83%和81%。【结论】进口凡纳滨对虾亲虾携带水具有传播EHP的可能性，即仅针对亲虾虾体进行EHP检测具有不完全性。eDNA检测可用来补充传统的虾体疫病监测方法，作为水环境疫病检测和风险评估的重要手段。
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| 关 键 词： | 凡纳滨对虾 亲虾 携带水 虾肝肠胞虫（EHP） 环境DNA（eDNA） 风险评估 |
| 收稿时间： | 2021-08-23 |
Risk analysis of imported live shrimp carrying water trans-mitted Enterocytozoon hepatopenaei by environmental DNA method |
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| ZHANG Na,LIU Hong,LIAO Jin-wan,ZHENG Xiao-cong,ZHENG Shu,LIU Xiao,XIE Yan-hui,LI Jia-qiao,LI Hong-quan.Risk analysis of imported live shrimp carrying water trans-mitted Enterocytozoon hepatopenaei by environmental DNA method[J].Journal of Southern Agriculture,2022,53(1):219-228. |
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| Authors: | ZHANG Na LIU Hong LIAO Jin-wan ZHENG Xiao-cong ZHENG Shu LIU Xiao XIE Yan-hui LI Jia-qiao LI Hong-quan |
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| Affiliation: | 1 Zhanjiang Customs Technology Center, Zhanjiang, Guangdong 524000, China;2 Animal and Plant Inspection and Quarantine Technical Centre, Shenzhen, Guangdong 518045, China;3 Zhanjiang Customs, Zhanjiang, Guangdong 524000, China |
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| Abstract: | 【Objective】The purpose of this study was to analyze the risks of water-borne Enterocytozoon hepatopenaei （EHP）in imported live shrimp using environmental DNA（eDNA）method，to provide a reference basis for environmental water disease monitoring and risk assessment.【Method】In this study，60 batches of imported parent shrimp carrying water were filtered by 0.45 μm nitrocellulose membrane，eDNA of 10 filter membranes were amplified byspecific primers of Litopenaeus vannamei. All 60 batches of filter membranes were tested for EHP via real-time fluorescence quantitative PCR. At the same time，EHP positive carrying water was used for artificial infection test. After 10 d of infection，environmental water and shrimp body were taken from each group for EHP detection. The metagenome of EHP positive filter membrane was sequenced and analyzed，Krona was used to visualize the annotation results，and the results of metagenome sequencing were compared with the EHP amino acid sequences registered in NCBI by EHP.【Result】The results showed that a 347 bp specific target fragment of L.vannamei was successfully amplified from 10 filter membranes，the sequencing results showed that the homology with the original sequence was 100%. Two batches of EHP were positive in 60 batches of imported prawns. The results of artificial infection test showed that EHP positive carrying water successfully infected the post-larvae group，and mature EHP spores were isolated from the hepatopancreas of shrimps，but the shrimps in parent group body were negative in EHP detection. The species relative abundance of EHP accounted for 0.7% of eukaryotic，the results compared with the amino acid sequences registered in NCBI，213 homologous EHP amino acid sequences were obtained，the amino acid sequences of EHP obtained were similar to those of other reported evolutionary trees:Enterospora canceri（amino acid similarity 83%），Enterocytozoon bieneusi（amino acid similarity 81%）.【Conclusion】It is possible for imported animals to carry water to spread EHP. EHP detection on parent shrimp only is incomplete. eDNA method can be used to supplement the traditional shrimp disease monitoring method as an important means of water disease detection and risk assessment. |
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