一例海豚源典型异尖线虫形态观察和分子鉴定

doi:10.11843/j.issn.0366-6964.2024.06.031

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (6): 2599-2606.doi: 10.11843/j.issn.0366-6964.2024.06.031

一例海豚源典型异尖线虫形态观察和分子鉴定

朱昊天¹(), 刘路瑶¹, 杨聪山¹, 姜纪¹, 石涵¹, 刘笑笑¹, 陈木新², 徐前明¹^,*()

1. 安徽农业大学动物科技学院, 合肥 230036
2. 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心), 国家卫生健康委员会寄生虫病原与媒介生物学重点实验室(中国疾病预防控制中心寄生虫病预防控制所), 上海 200025

收稿日期:2023-09-20 出版日期:2024-06-23 发布日期:2024-06-28
通讯作者: 徐前明 E-mail:sirius41852@163.com;xuqianming2006@163.com
作者简介:朱昊天(1999-)，男，安徽淮南人，硕士生，主要从事寄生虫与寄生虫病研究，E-mail: sirius41852@163.com
基金资助:
安徽省自然科学基金青年项目(2008085QC136)

Morphological Observation and Molecular Characterization of Anisakis Derived from Dolphin

Haotian ZHU¹(), Luyao LIU¹, Congshan YANG¹, Ji JIANG¹, Han SHI¹, Xiaoxiao LIU¹, Muxin CHEN², Qianming XU¹^,*()

1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2. Key Laboratory of Parasitic Pathogens and Vector Biology of the National Health Commission (Institute of Parasitic Diseases Prevention and Control of the Chinese Center for Disease Control and Prevention), Institute of Parasitic Diseases Prevention and Control of the Chinese Center for Disease Control and Prevention (National Tropical Disease Research Center), Shanghai 200025, China

Received:2023-09-20 Online:2024-06-23 Published:2024-06-28
Contact: Qianming XU E-mail:sirius41852@163.com;xuqianming2006@163.com

摘要/Abstract

摘要：

旨在鉴定由海豚粪便样本中所分离所得线虫种类。首先对所分离虫体进行形态观察，然后提取虫体基因组DNA，并对样本28S rRNA D2-D3基因和ITS基因片段进行PCR扩增，以进行分子生物学鉴定。所得序列经BLAST比对并用邻接法构建系统进化树对所得序列进行分析。结果显示：虫体长120~140 mm，整体呈圆柱形，两端尖细，外观呈乳白色半透明，镜下观察其内部偏黄。可见食道前端细，向后逐渐膨大。PCR结果显示，28S rRNA D2-D3基因与ITS基因扩增长度分别为797和840 bp。BLAST比对结果显示：28S rRNA D2-D3基因(GenBank登录号OR345165)与已报道典型异尖线虫(Anisakis typical, GenBank登录号HF911524.1)的序列一致性为95.36%；ITS基因(GenBank登录号OR345164)与典型异尖线虫(GenBank登录号MF668919.1)一致性为97.11%，其中, ITS-1区域与典型异尖线虫(GenBank登录号MF668919.1)一致性为91.90%，ITS-2区域与典型异尖线虫(GenBank登录号JN968938.1)一致性为94.97%，5.8S rRNA区域与典型异尖线虫(GenBank登录号OQ244221.1)一致性为100.00%。基于ITS基因和28S rRNA D2-D3基因的系统发育分析结果显示，此次研究分离所得寄生虫样本与典型异尖线虫遗传距离最近，且同一分支上。本次分离所得寄生虫样本经过分子鉴定为典型异尖线虫。同源性分析结果显示，其与典型异尖线虫一致性均大于95%；同时，在28S rRNA D2-D3基因序列和ITS基因序列的系统发育分析方面，均与已报道的典型异尖线虫遗传距离最近。

关键词: 异尖线虫, 人畜共患病, 形态学观察, 分子鉴定, 系统进化树

Abstract:

In order to identify the species of nematodes isolated from dolphin fecal samples, the morphology of the isolated nematodes is first identified. The parasite's body length was observed to be 120-140 mm long, with a cylindrical shape and slender ends. Its appearance was milky white and translucent, and its internal structure was observed to be yellowish under the microscope. It can be seen that the front end of the esophagus is thin and gradually expands backwards. We extracted the genomic DNA of the isolated worm and amplified the 28S rRNA D2-D3 gene and ITS gene fragments by PCR for molecular identification. The PCR results showed that the amplification products of the 28S rRNA D2-D3 gene and ITS gene were 797 and 840 bp, respectively. BLAST alignment results showed that the sequence similarity between the 28S rRNA D2-D3 gene (GenBank accession number OR345165) and the Anisakis typical (GenBank accession number HF911524.1) was 95.36%. The sequence similarity between the ITS gene (GenBank accession number OR345164) and the Anisakis typical (GenBank accession number MF668919.1) was 97.11%, with a 91.90% sequence identity between the ITS-1 region and the Anisakis typical (GenBank accession number MF668919.1). There is a 94.97% sequence identity between the ITS-2 region and the Anisakis typical (GenBank accession number JN968938.1) and a 100.00% sequence identity between the 5.8S rRNA region and the Anisakis typical (GenBank accession number OQ244221.1). Phylogenetic analyses based on the ITS gene and the 28S rRNA D2-D3 gene showed that the parasite samples isolated in this study were genetically closest to the Anisakis typical, and on the same branch.In summary, the isolated parasite samples obtained from this study should be the larvae of Anisakis typical based on microscopic examination and molecular analysis.

Key words: Anisakis, zoonosis, morphological observation, molecular identification, phylogenetic tree

中图分类号:

朱昊天, 刘路瑶, 杨聪山, 姜纪, 石涵, 刘笑笑, 陈木新, 徐前明. 一例海豚源典型异尖线虫形态观察和分子鉴定[J]. 畜牧兽医学报, 2024, 55(6): 2599-2606.

Haotian ZHU, Luyao LIU, Congshan YANG, Ji JIANG, Han SHI, Xiaoxiao LIU, Muxin CHEN, Qianming XU. Morphological Observation and Molecular Characterization of Anisakis Derived from Dolphin[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2599-2606.

图/表 7

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	片段长度/bp Fragment length
D2A	CAAGTACCGTGAGGGAAAGTTG	797
D3B	TCGGAAGGAACCAGCTACTA
ITS-F	GGTGAACCTGCGGAAGGATCATT	840
ITS-R	TTAGTTTCTTTTCCTCCGCT

线虫种类 Nematode species	ITS基因长度/bp ITS gene length	GenBank登录号 GenBank Accession No.
Trichuris trichiura	1 404	GQ301555.1
Anisakis simplex	899	OP975693.1
Haemonchus contortus	788	KX534106.1
Rotylenchulus reniformis	1 250	KP018571.1
Heterorhabditis bacteriophora	759	OQ998912.1
Hysterothylacium aduncum	950	JX845127.1
Angiostrongylus cantonensis	833	GU733323.1
Gongylonema pulchrum	1 647	AB495400.1
Oesophagostomum columbianum	732	JX188475.1
Strongyloides stercoralis	384	JX489150.1

线虫种类 Nematode species	ITS基因长度/bp ITS gene length	GenBank登录号 GenBank Accession No.
Anisakis simplex	899	OP975693.1
Anisakis simplex	924	MF959775.1
Anisakis simplex	1 353	JX237374.1
Anisakis simplex	721	MG272328.1
Anisakis typica	697	FJ161074.1
Anisakis typica	915	EU327689.1
Anisakis typica	822	KY524217.1
Anisakis typica	758	KX002242.1
Anisakis pegreffii	907	OP975695.1
Anisakis pegreffii	774	KF512856.1
Anisakis pegreffii	863	OM418764.1
Anisakis pegreffii	1 514	MT820022.1

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一例海豚源典型异尖线虫形态观察和分子鉴定

Morphological Observation and Molecular Characterization of Anisakis Derived from Dolphin

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